U.S. patent application number 16/159542 was filed with the patent office on 2019-02-07 for methods and compositions for improving plant traits.
The applicant listed for this patent is Pivot Bio, Inc.. Invention is credited to Sarah Bloch, Rosemary Clark, Austin Davis-Richardson, Kevin Hammill, Douglas Higgins, Alvin Tamsir, Karsten Temme, Emily Tung.
Application Number | 20190039964 16/159542 |
Document ID | / |
Family ID | 62839550 |
Filed Date | 2019-02-07 |
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United States Patent
Application |
20190039964 |
Kind Code |
A1 |
Temme; Karsten ; et
al. |
February 7, 2019 |
METHODS AND COMPOSITIONS FOR IMPROVING PLANT TRAITS
Abstract
Methods and systems are provided for generating and utilizing a
bacterial composition that comprises at least one genetically
engineered bacterial strain that fixes atmospheric nitrogen in an
agricultural system that has been fertilized with more than 20 lbs
of Nitrogen per acre.
Inventors: |
Temme; Karsten; (Oakland,
CA) ; Tamsir; Alvin; (San Francisco, CA) ;
Bloch; Sarah; (Emeryville, CA) ; Clark; Rosemary;
(El Cerrito, CA) ; Tung; Emily; (Milbrae, CA)
; Hammill; Kevin; (Danville, CA) ; Higgins;
Douglas; (Berkeley, CA) ; Davis-Richardson;
Austin; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pivot Bio, Inc. |
Berkeley |
CA |
US |
|
|
Family ID: |
62839550 |
Appl. No.: |
16/159542 |
Filed: |
October 12, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2018/013671 |
Jan 12, 2018 |
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16159542 |
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62577147 |
Oct 25, 2017 |
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62566199 |
Sep 29, 2017 |
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62467032 |
Mar 3, 2017 |
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62447889 |
Jan 18, 2017 |
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62445557 |
Jan 12, 2017 |
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62445570 |
Jan 12, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/70 20130101;
C12N 15/743 20130101; C12R 1/22 20130101; C05F 11/08 20130101; C12R
1/07 20130101; C12N 15/52 20130101; A01N 63/10 20200101; C12N
15/111 20130101; A01H 6/4684 20180501; A01N 63/00 20130101; C12N
1/20 20130101; A01H 3/00 20130101; C12N 1/04 20130101; A01H 5/06
20130101; C12R 1/065 20130101; C12R 1/01 20130101; C12R 1/025
20130101 |
International
Class: |
C05F 11/08 20060101
C05F011/08; A01H 6/46 20060101 A01H006/46; A01H 5/06 20060101
A01H005/06; C12R 1/22 20060101 C12R001/22; C12R 1/025 20060101
C12R001/025; C12R 1/065 20060101 C12R001/065; C12R 1/07 20060101
C12R001/07; C12N 1/04 20060101 C12N001/04; C12N 15/74 20060101
C12N015/74; C12N 15/11 20060101 C12N015/11; C12N 15/52 20060101
C12N015/52 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with the support of the United
States government under SBIR grant 1520545 awarded by the National
Science Foundation. The government has certain rights in the
disclosed subject matter.
Claims
1. A method of increasing nitrogen fixation in a non-leguminous
plant, comprising: applying to the plant a plurality of
non-intergeneric bacteria, said plurality comprising
non-intergeneric bacteria that: i. have an average colonization
ability per unit of plant root tissue of at least about
1.0.times.10.sup.4 bacterial cells per gram of fresh weight of
plant root tissue; and ii. produce fixed N of at least about
1.times.10.sup.-17 mmol N per bacterial cell per hour, and wherein
the plurality of non-intergeneric bacteria, in planta, produce 1%
or more of the fixed nitrogen in the plant, and wherein each member
of the plurality of non-intergeneric bacteria comprises at least
one genetic variation introduced into at least one gene, or
non-coding polynucleotide, of the nitrogen fixation or assimilation
genetic regulatory network, such that the non-intergeneric bacteria
are capable of fixing atmospheric nitrogen in the presence of
exogenous nitrogen.
2. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria comprise bacteria that: have an average
colonization ability per unit of plant root tissue of at least
about 1.0.times.10.sup.4 bacterial cells per gram of fresh weight
of plant root tissue.
3. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria comprise bacteria that: produce fixed N
of at least about 1.times.10.sup.-17 mmol N per bacterial cell per
hour.
4. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria comprise bacteria that: have an average
colonization ability per unit of plant root tissue of at least
about 1.0.times.10.sup.4 bacterial cells per gram of fresh weight
of plant root tissue and produce fixed N of at least about
1.times.10.sup.-17 mmol N per bacterial cell per hour.
5. The method according to claim 1, wherein the at least one
genetic variation comprises an introduced control sequence operably
linked to the at least one gene of the nitrogen fixation or
assimilation genetic regulatory network.
6. The method according to claim 1, wherein the at least one
genetic variation comprises a promoter operably linked to the at
least one gene of the nitrogen fixation or assimilation genetic
regulatory network.
7. The method according to claim 1, wherein the at least one
genetic variation comprises an inducible promoter operably linked
to the at least one gene of the nitrogen fixation or assimilation
genetic regulatory network.
8. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria do not comprise a constitutive promoter
operably linked to a gene of the nitrogen fixation or assimilation
genetic regulatory network.
9. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria do not comprise a constitutive promoter
operably linked to a gene in the nif gene cluster.
10. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria, in planta, excrete nitrogen-containing
products of nitrogen fixation.
11. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria applied to the plant do not stimulate an
increase in the uptake of exogenous non-atmospheric nitrogen.
12. The method according to claim 1, wherein the plant is grown in
soil from a field which has been administered at least about 50 lbs
of nitrogen-containing fertilizer per acre, and wherein the
nitrogen-containing fertilizer comprises at least about 5% nitrogen
by weight.
13. The method according to claim 1, wherein the plant is grown in
soil from a field which has been administered at least about 50 lbs
of nitrogen-containing fertilizer per acre, and wherein the
nitrogen-containing fertilizer comprises at least about 5% nitrogen
by weight, and wherein the nitrogen-containing fertilizer comprises
ammonium or an ammonium containing molecule.
14. The method according to claim 1, wherein the exogenous nitrogen
is selected from fertilizer comprising one or more of: glutamine,
ammonia, ammonium, urea, nitrate, nitrite, ammonium-containing
molecules, nitrate-containing molecules, and nitrite-containing
molecules.
15. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria, in planta, produce 5% or more of the
fixed nitrogen in the plant.
16. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria, in planta, produce 10% or more of the
fixed nitrogen in the plant.
17. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria, in planta, fix atmospheric nitrogen in
non-nitrogen-limiting conditions.
18. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria, in planta, excrete nitrogen-containing
products of nitrogen fixation.
19. The method according to claim 1, wherein the fixed nitrogen
produced by the plurality of non-intergeneric bacteria is measured
through dilution of enriched fertilizer by atmospheric N.sub.2 gas
in plant tissue.
20. The method according to claim 1, wherein the at least one gene,
or non-coding polynucleotide, of the nitrogen fixation or
assimilation genetic regulatory network are selected from the group
consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding
glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide
encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY,
nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ,
and a gene associated with biosynthesis of a nitrogenase
enzyme.
21. The method according to claim 1, wherein the at least one
genetic variation is a mutation that results in one or more of:
increased expression or activity of NifA or glutaminase; decreased
expression or activity of NifL, NtrB, glutamine synthetase, GlnB,
GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or
decreased uridylyl-removing activity of GlnD.
22. The method according to claim 1, wherein the at least one
genetic variation is selected from: (A) a knock-out mutation; (B)
alters or abolishes a regulatory sequence of a target gene; (C)
comprises the insertion of a heterologous regulatory sequence; or
(D) a domain deletion.
23. The method according to claim 1, wherein the at least one
genetic variation is a mutated nifL gene that has been altered to
comprise a heterologous promoter inserted into said nifL gene.
24. The method according to claim 1, wherein the at least one
genetic variation is a mutated glnE gene that results in a
truncated GlnE protein lacking an adenylyl-removing (AR)
domain.
25. The method according to claim 1, wherein the at least one
genetic variation is a mutated amtB gene that results in the lack
of expression of said amtB gene.
26. The method according to claim 1, wherein the at least one
genetic variation is selected from: a mutated nifL gene that has
been altered to comprise a heterologous promoter inserted into said
nifL gene; a mutated glnE gene that results in a truncated GlnE
protein lacking an adenylyl-removing (AR) domain; a mutated amtB
gene that results in the lack of expression of said amtB gene; and
combinations thereof.
27. The method according to claim 1, wherein the at least one
genetic variation is a mutated nifL gene that has been altered to
comprise a heterologous promoter inserted into said nifL gene and a
mutated glnE gene that results in a truncated GlnE protein lacking
an adenylyl-removing (AR) domain.
28. The method according to claim 1, wherein the at least one
genetic variation is a mutated nifL gene that has been altered to
comprise a heterologous promoter inserted into said nifL gene and a
mutated glnE gene that results in a truncated GlnE protein lacking
an adenylyl-removing (AR) domain and a mutated amtB gene that
results in the lack of expression of said amtB gene.
29. The method according to claim 1, wherein the plant comprises
the seed, stalk, flower, fruit, leaves, or rhizome.
30. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria are formulated into a composition.
31. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria are formulated into a composition
comprising an agriculturally acceptable carrier.
32. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria are applied into furrows in which seeds
of said plant are planted.
33. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria are formulated into a liquid in-furrow
composition comprising bacteria at a concentration of about
1.times.10.sup.7 to about 1.times.10.sup.10 cfu per milliliter.
34. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria are applied onto a seed of said
plant.
35. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria are formulated into a seed coating and
are applied onto a seed of said plant.
36. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria are formulated into a seed coating and
are applied onto a seed of said plant, at a concentration of about
1.times.10.sup.5 to about 1.times.10.sup.7 cfu per seed.
37. The method according to claim 1, wherein the plant is a cereal
crop.
38. The method according to claim 1, wherein the plant is selected
from the group consisting of: corn, rice, wheat, barley, sorghum,
millet, oat, rye, and triticale.
39. The method according to claim 1, wherein the plant is corn.
40. The method according to claim 1, wherein the plant is an
agricultural crop plant.
41. The method according to claim 1, wherein the plant is a
genetically modified organism.
42. The method according to claim 1, wherein the plant is not a
genetically modified organism.
43. The method according to claim 1, wherein the plant has been
genetically engineered or bred for efficient nitrogen use.
44. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria comprise at least two different species
of bacteria.
45. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria comprise at least two different strains
of the same species of bacteria.
46. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria comprise bacteria selected from: Rahnella
aquatilis, Klebsiella variicola, Achromobacter spiritinus,
Achromobacter marplatensis, Microbacterium murale, Kluyvera
intermedia, Kosakonia pseudosacchari, Enterobacter sp.,
Azospirillum lipoferum, Kosakonia sacchari, and combinations
thereof.
47. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria are endophytic, epiphytic, or
rhizospheric.
48. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria comprise bacteria selected from: a
bacteria deposited as PTA-122293, a bacteria deposited as
PTA-122294, a bacteria deposited as NCMA 201701002, a bacteria
deposited as NCMA 201708004, a bacteria deposited as NCMA
201708003, a bacteria deposited as NCMA 201708002, a bacteria
deposited as NCMA 201712001, a bacteria deposited as NCMA
201712002, and combinations thereof.
49. The method according to claim 1, wherein the bacterium produces
fixed N of at least about 1.times.10.sup.-17 mmol N per bacterial
cell per hour when in the presence of exogenous nitrogen.
50. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria, in planta, produce 5% or more of the
fixed nitrogen in the plant.
51. The method accordingly to claim 1, wherein the plurality of
non-intergeneric bacteria, in planta, produce 10% or more of the
fixed nitrogen in the plant.
52. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria, in planta, produce 15% or more of the
fixed nitrogen in the plant.
53. The method according to claim 1, wherein the plurality of
non-intergeneric bacteria, in planta, produce 20% or more of the
fixed nitrogen in the plant.
54. The method according to claim 1, wherein the product of (i) the
average colonization ability per unit of plant root tissue and (ii)
produced fixed N per bacterial cell per hour, is at least about
2.0.times.10.sup.-7 mmol N per gram of fresh weight of plant root
tissue per hour.
55. The method according to claim 1, wherein the product of (i) the
average colonization ability per unit of plant root tissue and (ii)
produced fixed N per bacterial cell per hour, is at least about
2.0.times.10.sup.-6 mmol N per gram of fresh weight of plant root
tissue per hour.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/445,570, filed Jan. 12, 2017; U.S. Provisional
Application No. 62/445,557, filed Jan. 12, 2017; U.S. Provisional
Application No. 62/447,889, filed Jan. 18, 2017; U.S. Provisional
Application No. 62/467,032, filed Mar. 3, 2017; U.S. Provisional
Application No. 62/566,199, filed Sep. 29, 2017; and U.S.
Provisional Application No. 62/577,147, filed Oct. 25, 2017, which
applications are incorporated herein by reference.
STATEMENT REGARDING SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 3, 2018, is named 47736-707_601_SL.txt and is .apprxeq.599
kb in size.
BACKGROUND OF THE INVENTION
[0004] Plants are linked to the microbiome via a shared metabolome.
A multidimensional relationship between a particular crop trait and
the underlying metabolome is characterized by a landscape with
numerous local maxima. Optimizing from an inferior local maximum to
another representing a better trait by altering the influence of
the microbiome on the metabolome may be desirable for a variety of
reasons, such as for crop optimization. Economically-,
environmentally-, and socially-sustainable approaches to
agriculture and food production are required to meet the needs of a
growing global population. By 2050 the United Nations' Food and
Agriculture Organization projects that total food production must
increase by 70% to meet the needs of the growing population, a
challenge that is exacerbated by numerous factors, including
diminishing freshwater resources, increasing competition for arable
land, rising energy prices, increasing input costs, and the likely
need for crops to adapt to the pressures of a drier, hotter, and
more extreme global climate.
[0005] One area of interest is in the improvement of nitrogen
fixation. Nitrogen gas (N.sub.2) is a major component of the
atmosphere of Earth. In addition, elemental nitrogen (N) is an
important component of many chemical compounds which make up living
organisms. However, many organisms cannot use N.sub.2 directly to
synthesize the chemicals used in physiological processes, such as
growth and reproduction. In order to utilize the N.sub.2, the
N.sub.2 must be combined with hydrogen. The combining of hydrogen
with N.sub.2 is referred to as nitrogen fixation. Nitrogen
fixation, whether accomplished chemically or biologically, requires
an investment of large amounts of energy. In biological systems, an
enzyme known as nitrogenase catalyzes the reaction which results in
nitrogen fixation. An important goal of nitrogen fixation research
is the extension of this phenotype to non-leguminous plants,
particularly to important agronomic grasses such as wheat, rice,
and maize. Despite enormous progress in understanding the
development of the nitrogen-fixing symbiosis between rhizobia and
legumes, the path to use that knowledge to induce nitrogen-fixing
nodules on non-leguminous crops is still not clear. Meanwhile, the
challenge of providing sufficient supplemental sources of nitrogen,
such as in fertilizer, will continue to increase with the growing
need for increased food production.
SUMMARY OF THE INVENTION
[0006] An aspect of the invention provides a bacterial composition
that comprises at least one genetically engineered bacterial strain
that fixes atmospheric nitrogen in an agricultural system that has
been fertilized with more than 20 lbs of Nitrogen per acre.
[0007] Another aspect of the invention provides a bacterial
composition that comprises at least one bacterial strain that has
been bred to fix atmospheric nitrogen in an agricultural system
that has been fertilized with more than 20 lbs of Nitrogen per
acre.
[0008] An additional aspect of the invention provides a bacterial
composition that comprises at least one genetically engineered
bacterial strain that fixes atmospheric nitrogen, the at least one
genetically engineered bacterial strain comprising exogenously
added DNA wherein said exogenously added DNA shares at least 80%
identity to a corresponding native bacterial strain.
[0009] A further aspect of the invention provides a seed
composition comprising a seed of a plant that is inoculated with a
bacterial composition.
[0010] Another aspect of the invention provides a method of growing
a crop using a plurality of seeds having a seed composition that is
inoculated with a bacterial composition.
[0011] An additional aspect of the invention provides a method of
applying a bacterial composition to a field.
[0012] A further aspect of the invention provides a fertilizer
composition comprising a bacterial composition.
[0013] Another aspect of the invention provides a method of
maintaining soil nitrogen levels. The method comprises planting, in
soil of a field, a crop inoculated by a genetically engineered
bacterium that fixes atmospheric nitrogen. The method also
comprises harvesting said crop, wherein no more than 90% of a
nitrogen dose required for producing said crop is administered to
said soil of said field between planting and harvesting.
[0014] An additional aspect of the invention provides a method of
delivering a probiotic supplement to a crop plant. The method
comprises coating a crop seed with a seed coating, seed treatment,
or seed dressing. Said seed coating, seed dressing, or seed
treatment comprises living representatives of said probiotic.
Additionally, the method comprises applying, in soil of a field,
said crop seeds.
[0015] In a further aspect of the invention, the genetically
engineered bacterial strain is a genetically engineered
Gram-positive bacterial strain. In some cases, the genetically
engineered Gram-positive bacterial strain has an altered expression
level of a regulator of a Nif cluster. In some cases, the
genetically engineered Gram-positive bacterial strain expresses a
decreased amount of a negative regulator of a Nif cluster. In some
cases, the genetically engineered bacterial strain expresses a
decreased amount of GlnR. In some cases, the genome of the
genetically engineered bacterial strain encodes a polypeptide with
at least 75% identity to a sequence from the Zehr lab NifH
database. In some cases, the genome of the genetically engineered
bacterial strain encodes a polypeptide with at least 85% identity
to a sequence from the Zehr lab NifH database. In some cases, the
genome of the genetically engineered bacterial strain encodes a
polypeptide with at least 75% identity to a sequence from the
Buckley lab NifH database. In some cases, the genome of the
genetically engineered bacterial strain encodes a polypeptide with
at least 85% identity to a sequence from the Buckley lab NifH
database.
[0016] Another aspect of the invention provides a method of
increasing nitrogen fixation in a non-leguminous plant. The method
comprises applying to the plant a plurality of non-intergeneric
bacteria, said plurality comprising non-intergeneric bacteria that
(i) have an average colonization ability per unit of plant root
tissue of at least about 1.0.times.10.sup.4 bacterial cells per
gram of fresh weight of plant root tissue and (ii) produce fixed N
of at least about 1.times.10.sup.-17 mmol N per bacterial cell per
hour. Additionally, the plurality of non-intergeneric bacteria, in
planta, produce 1% or more of the fixed nitrogen in the plant.
Further, each member of the plurality of non-intergeneric bacteria
comprises at least one genetic variation introduced into at least
one gene, or non-coding polynucleotide, of the nitrogen fixation or
assimilation genetic regulatory network, such that the
non-intergeneric bacteria are capable of fixing atmospheric
nitrogen in the presence of exogenous nitrogen.
[0017] An additional aspect of the invention provides a method of
increasing nitrogen fixation in a non-leguminous plant. The method
comprises applying to the plant a plurality of non-intergeneric
bacteria, said plurality comprising non-intergeneric bacteria that
(i) have an average colonization ability per unit of plant root
tissue of at least about 1.0.times.10.sup.4 bacterial cells per
gram of fresh weight of plant root tissue and/or (ii) produce fixed
N of at least about 1.times.10.sup.-17 mmol N per bacterial cell
per hour. Additionally, the plurality of non-intergeneric bacteria,
in planta, produce 1% or more of the fixed nitrogen in the plant.
Further, each member of the plurality of non-intergeneric bacteria
comprises at least one genetic variation introduced into at least
one gene, or non-coding polynucleotide, of the nitrogen fixation or
assimilation genetic regulatory network, such that the
non-intergeneric bacteria are capable of fixing atmospheric
nitrogen in the presence of exogenous nitrogen.
[0018] A further aspect of the invention provides a method of
breeding microbial strains to improve specific traits of agronomic
relevance. The method comprises providing a plurality of microbial
strains that have the ability to colonize a desired crop. The
method also comprises improving regulatory networks influencing the
trait through intragenomic rearrangement. Further, the method
comprises assessing microbial strains within the plurality of
microbial strains to determine a measure of the trait.
Additionally, the method comprises selecting one or more microbial
strains of the plurality of microbial strains that generate an
improvement in the trait in the presence of the desired crop.
[0019] Another aspect of the invention provides a method of
breeding microbial strains to improve specific traits of agronomic
relevance. The method comprises providing a plurality of microbial
strains that have the ability to colonize a desired crop. The
method also comprises introducing genetic diversity into the
plurality of microbial strains. Additionally, the method comprises
assessing microbial strains within the plurality of microbial
strains to determine a measure of the trait. Further, the method
comprises selecting one or more microbial strains of the plurality
of microbial strains that generate an improvement in the trait in
die presence of the desired crop.
[0020] Another aspect of the invention provides a method of
increasing the amount of atmospheric derived nitrogen in a
non-leguminous plant. The method comprises exposing said
non-leguminous plant to engineered non-intergeneric microbes, said
engineered non-intergeneric microbes comprising at least one
genetic variation introduced into a nitrogen fixation genetic
regulatory network or at least one genetic variation introduced
into a nitrogen assimilation genetic regulatory network.
[0021] A further aspect of the invention provides a method of
increasing an amount of atmospheric derived nitrogen in a corn
plant. The method comprises exposing said corn plant to engineered
non-intergeneric microbes comprising engineered genetic variations
within at least two genes selected from the group consisting of
nifL, glnB, glnE, and amtB.
[0022] Another aspect of the invention provides a method of
increasing an amount of atmospheric derived nitrogen in a corn
plant. The method comprises exposing said corn plant to engineered
non-intergeneric microbes comprising at least one genetic variation
introduced into a nitrogen fixation genetic regulatory network and
at least one genetic variation introduced into a nitrogen
assimilation genetic regulatory network, wherein said engineered
non-intergeneric microbes, in planta, produce at least 5% of fixed
nitrogen in said corn plant as measured by dilution of 15N in crops
grown in fields treated with fertilizer containing 1.2% 15N.
[0023] An additional aspect of the invention provides a method of
increasing nitrogen fixation in a non-leguminous plant. The method
comprises applying to the plant a plurality of non-intergeneric
bacteria, said plurality comprising non-intergeneric bacteria that
(i) have an average colonization ability per unit of plant root
tissue of at least about 1.0.times.10.sup.4 bacterial cells per
gram of fresh weight of plant root tissue and (ii) produce fixed N
of at least about 1.times.10.sup.-17 mmol N per bacterial cell per
hour. Additionally, the product of (i) the average colonization
ability per unit of plant root tissue and (ii) produced fixed N per
bacterial cell per hour, is at least about 2.5.times.10.sup.-8 mmol
N per grain of fresh weight of plant root tissue per hour. Further,
the plurality of non-intergeneric bacteria, in planta, produce 1%
or more of the fixed nitrogen in the plant. Additionally, each
member of the plurality of non-intergeneric bacteria comprises at
least one genetic variation introduced into at least one gene, or
non-coding polynucleotide, of the nitrogen fixation or assimilation
genetic regulatory network, such that the non-intergeneric bacteria
are capable of fixing atmospheric nitrogen in the presence of
exogenous nitrogen.
[0024] Another aspect of the invention provides a method of
increasing nitrogen fixation in a non-leguminous plant. The method
comprises applying to the plant a plurality of bacteria, said
plurality comprising bacteria that (i) have an average colonization
ability per unit of plant root tissue of at least about
1.0.times.10.sup.4 bacterial cells per gram of fresh weight of
plant root tissue and/or (ii) produce fixed N of at least about
1.times.10.sup.-17 mmol N per bacterial cell per hour.
Additionally, the plurality of bacteria, in planta, produce 1% or
more of the fixed nitrogen in the plant.
[0025] An additional aspect of the invention provides a
non-intergeneric bacterial population capable of increasing
nitrogen fixation in a non-leguminous plant, comprising a plurality
of non-intergeneric bacteria that (i) have an average colonization
ability per unit of plant root tissue of at least about
1.0.times.10.sup.4 bacterial cells per gram of fresh weight of
plant root tissue and/or (ii) produce fixed N of at least about
1.times.10.sup.-17 mmol N per bacterial cell per hour.
Additionally, the plurality of non-intergeneric bacteria, in
planta, produce 1% or more of the fixed nitrogen in a plant grown
in the presence of the plurality of non-intergeneric bacteria.
Further, each member of the plurality of non-intergeneric bacteria
comprises at least one genetic variation introduced into at least
one gene, or non-coding polynucleotide, of the nitrogen fixation or
assimilation genetic regulatory network, such that the
non-intergeneric bacteria are capable of fixing atmospheric
nitrogen in die presence of exogenous nitrogen.
[0026] A further aspect of the invention provides a bacterial
population capable of increasing nitrogen fixation in a
non-leguminous plant, the bacterial population comprising a
plurality of bacteria that (i) have an average colonization ability
per unit of plant root tissue of at least about 1.0.times.10.sup.4
bacterial cells per gram of fresh weight of plant root tissue;
and/or (ii) produce fixed N of at least about 1.times.10.sup.-17
mmol N per bacterial cell per hour. Additionally, the plurality of
bacteria, in planta, produce 1% or more of the fixed nitrogen in
the plant.
[0027] In another aspect of the invention, a bacterium is provided
that (i) has an average colonization ability per unit of plant root
tissue of at least about 1.0.times.10.sup.4 bacterial cells per
gram of fresh weight of plant root tissue and/or (ii) produces
fixed N of at least about 1.times.10.sup.-17 mmol N per bacterial
cell per hour.
[0028] In a further aspect of the invention, a non-intergeneric
bacterium is provided that comprises at least one genetic variation
introduced into at least one gene, or non-coding polynucleotide, of
the nitrogen fixation or assimilation genetic regulatory network,
such that the non-intergeneric bacterium is capable of fixing
atmospheric nitrogen in the presence of exogenous nitrogen, and
wherein said bacterium (i) has an average colonization ability per
unit of plant root tissue of at least about 1.0.times.10.sup.4
bacterial cells per gram of fresh weight of plant root tissue
and/or (ii) produces fixed N of at least about 1.times.10.sup.-17
mmol N per bacterial cell per hour.
[0029] In an additional aspect of the invention provides a method
for increasing nitrogen fixation in a plant, comprising
administering to the plant an effective amount of a composition
that comprises a purified population of bacteria that comprises
bacteria with a 16S nucleic acid sequence that is at least about
97% identical to a nucleic acid sequence selected from SEQ ID NOs:
85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269,
277-283; a purified population of bacteria that comprises bacteria
with a nucleic acid sequence that is at least about 90% identical
to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110,
112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268,
270-274, 275, 276, 284-295; and/or a purified population of
bacteria that comprises bacteria with a nucleic acid sequence that
is at least about 90% identical to a nucleic acid sequence selected
from SEQ ID NOs: 177-260, 296-303; and wherein the plant
administered the effective amount of the composition exhibits an
increase in nitrogen fixation, as compared to a plant not having
been administered the composition.
[0030] A further aspect of the invention provides an isolated
bacteria comprising a 16S nucleic acid sequence that is at least
about 97% identical to a nucleic acid sequence selected from SEQ ID
NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262,
269, 277-283; a nucleic acid sequence that is at least about 90%
identical to a nucleic acid sequence selected from SEQ ID NOs:
86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166,
168-176, 263-268, 270-274, 275, 276, 284-295; and/or a nucleic acid
sequence that is at least about 90% identical to a nucleic acid
sequence selected from SEQ ID NOs: 177-260, 296-303.
[0031] Another aspect of the invention provides a method of
detecting a non-native junction sequence, comprising: amplifying a
nucleotide sequence that shares at least about 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to SEQ ID NOs: 372-405.
[0032] An additional aspect of the invention provides a method of
detecting a non-native junction sequence, comprising: amplifying a
nucleotide sequence that shares at least about 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
100% sequence identity to at least a 10 contiguous base pair
fragment contained in SEQ ID NOs: 372-405, said contiguous base
pair fragment being comprised of nucleotides at the intersection
of: an upstream sequence comprising SEQ ID NOs: 304-337 and
downstream sequence comprising SEQ ID NOs: 338-371.
[0033] A further aspect of the invention provides a non-native
junction sequence comprising a nucleotide sequence that shares at
least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 884/0, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID
NOs: 372-405.
[0034] An additional aspect of the invention provides a non-native
junction sequence comprising a nucleotide sequence that shares at
least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least
a 10 contiguous base pair fragment contained in SEQ ID NOs:
372-405, said contiguous base pair fragment being comprised of
nucleotides at the intersection of: an upstream sequence comprising
SEQ ID NOs: 304-337 and downstream sequence comprising SEQ ED NOs:
338-371.
[0035] A further aspect of the invention provides a bacterial
composition comprising at least one remodeled bacterial strain that
fixes atmospheric nitrogen, the at least one remodeled bacterial
strain comprising exogenously added DNA wherein said exogenously
added DNA shares at least 80% identity to a corresponding native
bacterial strain.
[0036] An additional aspect of the invention provides a method of
maintaining soil nitrogen levels. The method comprises planting, in
soil of a field, a crop inoculated by a remodeled bacterium that
fixes atmospheric nitrogen. The method also comprises harvesting
said crop, wherein no more than 90% of a nitrogen dose required for
producing said crop is administered to said soil of said field
between planting and harvesting.
[0037] Another aspect of the invention provides a method of
delivering a probiotic supplement to a crop plant. The method
comprises coating a crop seed with a seed coating, seed treatment,
or seed dressing, wherein said seed coating, seed dressing, or seed
treatment comprise living representatives of said probiotic. The
method also comprises applying said crop seeds in soil of a
field.
[0038] An additional aspect of the invention provides a method of
increasing the amount of atmospheric derived nitrogen in a
non-leguminous plant. The method comprises exposing said
non-leguminous plant to remodeled non-intergeneric microbes, said
remodeled non-intergeneric microbes comprising at least one genetic
variation introduced into a nitrogen fixation genetic regulatory
network or at least one genetic variation introduced into a
nitrogen assimilation genetic regulatory network.
[0039] A further aspect of the invention provides a method of
increasing an amount of atmospheric derived nitrogen in a corn
plant. The method comprises exposing said corn plant to remodeled
non-intergeneric microbes comprising remodeled genetic variations
within at least two genes selected from the group consisting of
nifL, glnB, glnE, and amtB.
[0040] Another aspect of the invention provides a method of
increasing an amount of atmospheric derived nitrogen in a corn
plant. The method comprises exposing said corn plant to remodeled
non-intergeneric microbes comprising at least one genetic variation
introduced into a nitrogen fixation genetic regulatory network and
at least one genetic variation introduced into a nitrogen
assimilation genetic regulatory network, wherein said remodeled
non-intergeneric microbes, in planta, produce at least 5% of fixed
nitrogen in said corn plant as measured by dilution of 15N in crops
grown in fields treated with fertilizer containing 1.2% 15N.
[0041] Additional aspects of the invention provide genus of
microbes that are evolved and optimized for in planta nitrogen
fixation in non-leguminous crops. In particular, methods of
increasing nitrogen fixation in a non-leguminous plant are
disclosed. The methods can comprise exposing the plant to a
plurality of bacteria. Each member of the plurality comprises one
or more genetic variations introduced into one or more genes of
non-coding polynucleotides of the bacteria's nitrogen fixation or
assimilation genetic regulatory network, such that the bacteria are
capable of fixing atmospheric nitrogen in the presence of exogenous
nitrogen. The bacteria are not intergeneric microorganisms.
Additionally, the bacteria, in planta, produce 1% or more of the
fixed nitrogen in the plant.
[0042] Further aspects of the invention provide beneficial isolated
microbes and microbial compositions. In particular, isolated and
biologically pure microorganisms that have applications, inter
alia, in increasing nitrogen fixation in a crop are provided. The
disclosed microorganism can be utilized in their isolated and
biologically pure states, as well as being formulated into
compositions. Furthermore, the disclosure provides microbial
compositions containing at least two members of the disclosed
microorganisms, as well as methods of utilizing said microbial
compositions.
INCORPORATION BY REFERENCE
[0043] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0045] FIGS. 1A-B depict enrichment and isolation of nitrogen
fixing bacteria. (A) Nfb agar plate was used to isolate single
colonies of nitrogen fixing bacteria. (B) Semi-solid Nfb agar
casted in Balch tube. The arrow points to pellicle of enriched
nitrogen fixing bacteria.
[0046] FIG. 2 depicts a representative nifH PCR screen. Positive
bands were observed at .about.350 bp for two colonies in this
screen. Lower bands represent primer-dimers.
[0047] FIG. 3 depicts an example of a PCR screen of colonies from
CRISPR-Cas-selected mutagenesis. CI006 colonies were screened with
primers specific for the nifL locus. The wild type PCR product is
expected at .about.2.2 kb, whereas the mutant is expected at
.about.1.1 kb. Seven of ten colonies screened unambiguously show
the desired deletion.
[0048] FIGS. 4A-D depict in vitro phenotypes of various strains.
The Acetylene Reduction Assay (ARA) activities of mutants of strain
CI010 (FIG. 4A) and mutants of strain CI006 (FIG. 4B) grown in
nitrogen fixation media supplemented with 0 to 10 mM glutamine. ARA
activities of additional strains are shown in FIG. 4C, and the
ammonium excretion profile across time of two strains is shown in
FIG. 4D.
[0049] FIG. 5 depicts in culture expression profile of 9 different
genes in strains CI006 involved in diazaotrophic nitrogen fixation.
Numbers represent counts of each transcript. Various conditions (0,
1, 10 mM Glutamine and 0%, 10%, 20% atmospheric air in N2) are
indicated.
[0050] FIG. 6 depicts CI006 colonization of corn roots. Corn
seedlings were inoculated with CI006 harboring an RFP expression
plasmid. After two weeks of growth and plasmid maintenance through
watering with the appropriate antibiotic, roots were harvested and
imaged through fluorescence microscopy. Colonization of the root
intercellular space is observed.
[0051] FIG. 7 depicts nitrogen derived from microbe level in WT
(CI050) and optimized (CM002) strain.
[0052] FIG. 8 shows an experimental setup for a Micro-Tom fruiting
mass assay.
[0053] FIG. 9 shows a screen of 10 strains for increase in
Micro-Tom plant fruit mass. Results for six replicates are
presented. For column 3, p=0.07. For column 7, p=0.05.
[0054] FIGS. 10A-C depict additional results for ARA activities of
candidate microbes and counterpart candidate mutants grown in
nitrogen fixation media supplemented with 0 to 10 mM glutamine.
[0055] FIG. 11 depicts a double mutant that exhibits higher ammonia
excretion than the single mutant from which it was derived.
[0056] FIG. 12 depicts NDFA obtained from 15N Gas Uptake experiment
(extrapolated back using days exposed) to measure NDFA in Corn
plants in fertilized condition.
[0057] FIG. 13 depicts NDFA value obtained from 15N Gas Uptake
experiment (extrapolated back using days exposed) to measure NDFA
in Setaria plants in fertilized condition.
[0058] FIG. 14A depicts rate of incorporation of 15N gas. Plants
inoculated with evolved strain showed increase in 151 gas
incorporation compared to uninoculated plants.
[0059] FIG. 14B depicts 4 weeks after planting, up to 7% of the
nitrogen in plants inoculated with an evolved strain is derived
from microbially fixed nitrogen.
[0060] FIG. 14C depicts leaf area (and other biomass measurement,
data not shown) is increased in plants inoculated with an evolved
strain when compared to uninoculated or wild type inoculated
plants.
[0061] FIG. 15A depicts evolved strains that show significantly
higher nifH production in the root tissue, as measured by in planta
transcriptomic study.
[0062] FIG. 15B depicts that rate of fixed nitrogen found in plant
tissue is correlated with the rate in which that particular plant
is colonized by HoME optimized strain.
[0063] FIG. 16A depicts a soil texture map of various field soils
tested for colonization. Soils in which a few microbes were
originally source from are indicated as stars.
[0064] FIG. 16B depicts the colonization rate of Strain 1 and
Strain 5 that are tested across four different soil types
(circles). Both strains showed relatively robust colonization
profile across diverse soil types.
[0065] FIG. 16C depicts colonization of Strain 1 as tested in a
field trial over the span of a growing season. Strain 1 persists in
the corn tissue up to week 12 after planting and starts to show
decline in colonization after that time.
[0066] FIG. 17A depicts a schematic of microbe breeding, in
accordance with embodiments.
[0067] FIG. 17B depicts an expanded view of the measurement of
microbiome composition as shown in FIG. 17A.
[0068] FIG. 17C depicts sampling of roots utilized in Example
7.
[0069] FIG. 18 depicts the lineage of modified strains that were
derived from strain CI006.
[0070] FIG. 19 depicts the lineage of modified strains that were
derived from strain CI019.
[0071] FIG. 20 depicts a heatmap of the pounds of nitrogen
delivered per acre-season by microbes of the present disclosure
recorded as a function of microbes per g-fresh weight by mmol of
nitrogen/microbe-hr. Below the thin line that transects the larger
image are the microbes that deliver less than one pound of nitrogen
per acre-season, and above the line are the microbes that deliver
greater than one pound of nitrogen per acre-season. The table below
the heatmap gives the precise value of mmol N produced per microbe
per hour (mmol N/Microbe hr) along with the precise CFU per gram of
fresh weight (CFU/g fw) for each microbe shown in the heatmap. The
microbes utilized in the heatmap were assayed for N production in
corn. For the WT strains CI006 and CI019, corn root colonization
data was taken from a single field site. For the remaining strains,
colonization was assumed to be the same as the % VT field level.
N-fixation activity was determined using an in vitro ARA assay at 5
mM glutamine.
[0072] FIG. 21 depicts the plant yield of plants having been
exposed to strain CI006. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0073] FIG. 22 depicts the plant yield of plants having been
exposed to strain CM029. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0074] FIG. 23 depicts the plant yield of plants having been
exposed to strain CM038. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0075] FIG. 24 depicts the plant yield of plants having been
exposed to strain CI019. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0076] FIG. 25 depicts die plant yield of plants having been
exposed to strain CM081. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0077] FIG. 26 depicts the plant yield of plants having been
exposed to strains CM029 and CM081. The area of the circles
corresponds to the relative yield, while the shading corresponds to
the particular MRTN treatment. The x-axis is the p value and the
y-axis is the win rate.
[0078] FIG. 27 depicts the plant yield of plants as the aggregated
bushel gain/loss. The area of the circles corresponds to the
relative yield, while the shading corresponds to the particular
MRTN treatment. The x-axis is the p value and the y-axis is the win
rate.
[0079] FIGS. 28A-28E illustrate derivative microbes that fix and
excrete nitrogen in vitro under conditions similar to high nitrate
agricultural soils. FIG. 28A illustrates the regulatory network
controlling nitrogen fixation and assimilation in PBC6.1 is shown,
including the key nodes NifL, NifA, GS, GlnE depicted as the
two-domain ATase-AR enzyme, and AmtB. FIG. 28B illustrates the
genome of Kosakonia sacchari isolate PBC6.1 is shown. The three
tracks circumscribing the genome convey transcription data from
PBC6.1, PBC6.38, and the differential expression between the
strains respectively. FIG. 28C illustrates the nitrogen fixation
gene cluster and transcription data is expanded for finer detail.
FIG. 28D illustrates nitrogenase activity under varying
concentrations of exogenous nitrogen is measured with the acetylene
reduction assay. The wild type strain exhibits repression of
nitrogenase activity as glutamine concentrations increase, while
derivative strains show varying degrees of robustness. Error bars
represent standard error of the mean of at least three biological
replicates. FIG. 28E illustrates temporal excretion of ammonia by
derivative strains is observed at mM concentrations. Wild type
strains are not observed to excrete fixed nitrogen, and negligible
ammonia accumulates in the media. Error bars represent standard
error of the mean.
[0080] FIGS. 29A-29C illustrate greenhouse experiments demonstrate
microbial nitrogen fixation in corn. FIG. 29A illustrates microbe
colonization six weeks after inoculation of corn plants by PBC6.1
derivative strains. Error bars show standard error of the mean of
at least eight biological replicates. FIG. 29B illustrates in
planta transcription of nifH measured by extraction of total RNA
from roots and subsequent Nanostring analysis. Only derivative
strains show WIN transcription in the root environment. Error bars
show standard error of the mean of at least 3 biological
replicates. FIG. 29C illustrates microbial nitrogen fixation
measured by the dilution of isotopic tracer in plant tissues.
Derivative microbes exhibit substantial transfer of fixed nitrogen
to the plant. Error bars show standard error of the mean of at
least ten biological replicates.
[0081] FIG. 30 illustrates PBC6.1 colonization to nearly 21%
abundance of the root-associated microbiota in corn roots.
Abundance data is based on 16S amplicon sequencing of the
rhizosphere and endosphere of corn plants inoculated with PBC6.1
and grown in greenhouse conditions.
[0082] FIG. 31 illustrates transcriptional rates of nifA in
derivative strains of PBC6.1 correlated with acetylene reduction
rates. An ARA assay was performed as described in the Methods,
after which cultures were sampled and subjected to qPCR analysis to
determine nifA transcript levels. Error bars show standard error of
the mean of at least three biological replicates in each
measure.
[0083] FIG. 32 illustrates results from a summer 2017 field testing
experiment. The yield results obtained demonstrate that the
microbes of the disclosure can serve as a potential fertilizer
replacement. For instance, the utilization of a microbe of the
disclosure (i.e. 6-403) resulted in a higher yield than the wild
type strain (WT) and a higher yield than the untreated control
(UTC). The "-25 lbs N" treatment utilizes 25 lbs less N per acre
than standard agricultural practices of the region. The "100% N"
UTC treatment is meant to depict standard agricultural practices of
the region, in which 100% of the standard utilization of N is
deployed by the farmer. The microbe "6-403" was deposited as NCMA
201708004 and can be found in Table A. This is a mutant Kosakonia
sacchari (also called CM037) and is a progeny mutant strain from
CI006 WT.
[0084] FIG. 33 illustrates results from a summer 2017 field testing
experiment. The yield results obtained demonstrate that the
microbes of the disclosure perform consistently across locations.
Furthermore, the yield results demonstrate that the microbes of the
disclosure perform well in both a nitrogen stressed environment, as
well as an environment that has sufficient supplies of nitrogen.
The microbe "6-881" (also known as CM094, PBC6.94), and which is a
progeny mutant Kosakonia sacchari strain from CI006 WT, was
deposited as NCMA 201708002 and can be found in Table A. The
microbe "137-1034," which is a progeny mutant Klebsiella variicola
strain from CI137 WT, was deposited as NCMA 201712001 and can be
found in Table A. The microbe "137-1036," which is a progeny mutant
Klebsiella variicola strain from CI137 WT, was deposited as NCMA
201712002 and can be found in Table A. The microbe "6-404" (also
known as CM38, PBC6.38), and which is a progeny mutant Kosakonia
sacchari strain from CI006 WT, was deposited as NCMA 201708003 and
can be found in Table A. The "Nutrient Stress" condition
corresponds to the 0% nitrogen regime. The "Sufficient Fertilizer"
condition corresponds to the 100% nitrogen regime.
[0085] FIG. 34 depicts the lineage of modified strains that were
derived from strain CI006 (also termed "6", Kosakonia sacchari
WT).
[0086] FIG. 35 depicts the lineage of modified strains that were
derived from strain CI019 (also termed "19", Rahnella aquatilis
WT).
[0087] FIG. 36 depicts the lineage of modified strains that were
derived from strain CI137 (also termed ("137", Klebsiella variicola
WT).
[0088] FIG. 37 depicts the lineage of modified strains that were
derived from strain 1021 (Kosakonia pseudosacchari WT).
[0089] FIG. 38 depicts the lineage of modified strains that were
derived from strain 910 (Kluyvera intermedia WT).
[0090] FIG. 39 depicts the lineage of modified strains that were
derived from strain 63 (Rahnella aquatilis WT).
[0091] FIG. 40 depicts a heatmap of the pounds of nitrogen
delivered per acre-season by microbes of the present disclosure
recorded as a function of microbes per g-fresh weight by mmol of
nitrogen/microbe-hr. Below the thin line that transects the larger
image are the microbes that deliver less than one pound of nitrogen
per acre-season, and above the line are the microbes that deliver
greater than one pound of nitrogen per acre-season. The Table C in
Example 12 gives the precise value of mmol N produced per microbe
per hour (mmol N/Microbe hr) along with the precise CFU per gram of
fresh weight (CFU/g fw) for each microbe shown in the heatmap. The
data in FIG. 40 is derived from microbial strains assayed for N
production in corn in field conditions. Each point represents 1b
N/acre produced by a microbe using corn root colonization data from
a single field site. N-fixation activity was determined using in
vitro ARA assay at 5 mM N in the form of glutamine or ammonium
phosphate.
[0092] FIG. 41 depicts a heatmap of the pounds of nitrogen
delivered per acre-season by microbes of the present disclosure
recorded as a function of microbes per g-fresh weight by mmol of
nitrogen/microbe-hr. Below the thin line that transects the larger
image are the microbes that deliver less than one pound of nitrogen
per acre-season, and above the line are the microbes that deliver
greater than one pound of nitrogen per acre-season. The Table Din
Example 12 gives the precise value of mmol N produced per microbe
per hour (mmol N/Microbe hr) along with the precise CFU per gram of
fresh weight (CFU/g fw) for each microbe shown in the heatmap. The
data in FIG. 41 is derived from microbial strains assayed for N
production in corn in laboratory and greenhouse conditions. Each
point represents 1b N/acre produced by a single strain. White
points represent strains in which corn root colonization data was
gathered in greenhouse conditions. Black points represent mutant
strains for which corn root colonization levels are derived from
average field corn root colonization levels of the wild-type parent
strain. Hatched points represent the wild type parent strains at
their average field corn root colonization levels. In all cases,
N-fixation activity was determined by in vitro ARA assay at 5 mM N
in the form of glutamine or ammonium phosphate.
DETAILED DESCRIPTION OF THE INVENTION
[0093] While various embodiments of the invention have been shown
and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions may occur to those
skilled in the art without departing from the invention. It should
be understood that various alternatives to the embodiments of the
invention described herein may be employed.
[0094] Increased fertilizer utilization brings with it
environmental concerns and is also likely not possible for many
economically stressed regions of the globe. Furthermore, many
industry players in the microbial arena are focused on creating
intergeneric microbes. However, there is a heavy regulatory burden
placed on engineered microbes that are characterized/classified as
intergeneric. These intergeneric microbes face not only a higher
regulatory burden, which makes widespread adoption and
implementation difficult, but they also face a great deal of public
perception scrutiny.
[0095] Currently, there are no engineered microbes on the market
that are non-intergeneric and that are capable of increasing
nitrogen fixation in non-leguminous crops. This dearth of such a
microbe is a missing element in helping to usher in a truly
environmentally friendly and more sustainable 21.sup.st century
agricultural system.
[0096] The present disclosure solves the aforementioned problems
and provides a non-intergeneric microbe that has been engineered to
readily fix nitrogen in crops. These microbes are not
characterized/classified as intergeneric microbes and thus will not
face the steep regulatory burdens of such. Further, the taught
non-intergeneric microbes will serve to help 21.sup.st century
farmers become less dependent upon utilizing ever increasing
amounts of exogenous nitrogen fertilizer.
Definitions
[0097] The terms "polynucleotide", "nucleotide", "nucleotide
sequence", "nucleic acid" and "oligonucleotide" are used
interchangeably. They refer to a polymeric form of nucleotides of
any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. Polynucleotides may have any three dimensional
structure, and may perform any function, known or unknown. The
following are non-limiting examples of polynucleotides: coding or
non-coding regions of a gene or gene fragment, loci (locus) defined
from linkage analysis, exons, introns, messenger RNA (mRNA),
transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA
(siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes,
cDNA, recombinant polynucleotides, branched polynucleotides,
plasmids, vectors, isolated DNA of any sequence, isolated RNA of
any sequence, nucleic acid probes, and primers. A polynucleotide
may comprise one or more modified nucleotides, such as methylated
nucleotides and nucleotide analogs. If present, modifications to
the nucleotide structure may be imparted before or after assembly
of the polymer. The sequence of nucleotides may be interrupted by
non-nucleotide components. A polynucleotide may be further modified
after polymerization, such as by conjugation with a labeling
component.
[0098] "Hybridization" refers to a reaction in which one or more
polynucleotides react to form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
hydrogen bonding may occur by Watson Crick base pairing, Hoogstein
binding, or in any other sequence specific manner according to base
complementarity. The complex may comprise two strands forming a
duplex structure, three or more strands forming a multi stranded
complex, a single self-hybridizing strand, or any combination of
these. A hybridization reaction may constitute a step in a more
extensive process, such as the initiation of PCR, or the enzymatic
cleavage of a polynucleotide by an endonuclease. A second sequence
that is complementary to a first sequence is referred to as the
"complement" of the first sequence. The term "hybridizable" as
applied to a polynucleotide refers to the ability of the
polynucleotide to form a complex that is stabilized via hydrogen
bonding between the bases of the nucleotide residues in a
hybridization reaction.
[0099] "Complementarity" refers to the ability of a nucleic acid to
form hydrogen bond(s) with another nucleic acid sequence by either
traditional Watson-Crick or other non-traditional types. A percent
complementarity indicates the percentage of residues in a nucleic
acid molecule which can form hydrogen bonds (e.g., Watson-Crick
base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7,
8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%
complementary, respectively). "Perfectly complementary" means that
all the contiguous residues of a nucleic acid sequence will
hydrogen bond with the same number of contiguous residues in a
second nucleic acid sequence. "Substantially complementary" as used
herein refers to a degree of complementarity that is at least 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a
region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to
two nucleic acids that hybridize under stringent conditions.
Sequence identity, such as for the purpose of assessing percent
complementarity, may be measured by any suitable alignment
algorithm, including but not limited to the Needleman-Wunsch
algorithm (see e.g. the EMBOSS Needle aligner available at
www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally
with default settings), the BLAST algorithm (see e.g. the BLAST
alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi,
optionally with default settings), or the Smith-Waterman algorithm
(see e.g. the EMBOSS Water aligner available at
www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html, optionally
with default settings). Optimal alignment may be assessed using any
suitable parameters of a chosen algorithm, including default
parameters.
[0100] In general, "stringent conditions" for hybridization refer
to conditions under which a nucleic acid having complementarity to
a target sequence predominantly hybridizes with a target sequence,
and substantially does not hybridize to non-target sequences.
Stringent conditions are generally sequence-dependent and vary
depending on a number of factors. In general, the longer the
sequence, the higher the temperature at which the sequence
specifically hybridizes to its target sequence. Non-limiting
examples of stringent conditions are described in detail in Tijssen
(1993), Laboratory Techniques In Biochemistry And Molecular
Biology-Hybridization With Nucleic Acid Probes Part I, Second
Chapter "Overview of principles of hybridization and the strategy
of nucleic acid probe assay", Elsevier, N.Y.
[0101] As used herein, "expression" refers to the process by which
a polynucleotide is transcribed from a DNA template (such as into
and mRNA or other RNA transcript) and/or the process by which a
transcribed mRNA is subsequently translated into peptides,
polypeptides, or proteins. Transcripts and encoded polypeptides may
be collectively referred to as "gene product." If the
polynucleotide is derived from genomic DNA, expression may include
splicing of the mRNA in a eukaryotic cell.
[0102] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified; for example, disulfide bond formation, glycosylation,
lipidation, acetylation, phosphorylation, or any other
manipulation, such as conjugation with a labeling component. As
used herein the term "amino acid" includes natural and/or unnatural
or synthetic amino acids, including glycine and both the D or L
optical isomers, and amino acid analogs and peptidomimetics.
[0103] As used herein, the term "about" is used synonymously with
the term "approximately." Illustratively, the use of the term
"about" with regard to an amount indicates that values slightly
outside the cited values, e.g., plus or minus 0.1% to 10%.
[0104] The term "biologically pure culture" or "substantially pure
culture" refers to a culture of a bacterial species described
herein containing no other bacterial species in quantities
sufficient to interfere with the replication of the culture or be
detected by normal bacteriological techniques.
[0105] "Plant productivity" refers generally to any aspect of
growth or development of a plant that is a reason for which the
plant is grown. For food crops, such as grains or vegetables,
"plant productivity" can refer to the yield of grain or fruit
harvested from a particular crop. As used herein, improved plant
productivity refers broadly to improvements in yield of grain,
fruit, flowers, or other plant parts harvested for various
purposes, improvements in growth of plant parts, including stems,
leaves and roots, promotion of plant growth, maintenance of high
chlorophyll content in leaves, increasing fruit or seed numbers,
increasing fruit or seed unit weight, reducing NO.sub.2 emission
due to reduced nitrogen fertilizer usage and similar improvements
of the growth and development of plants.
[0106] Microbes in and around food crops can influence the traits
of those crops. Plant traits that may be influenced by microbes
include: yield (e.g., grain production, biomass generation, fruit
development, flower set); nutrition (e.g., nitrogen, phosphorus,
potassium, iron, micronutrient acquisition); abiotic stress
management (e.g., drought tolerance, salt tolerance, heat
tolerance); and biotic stress management (e.g., pest, weeds,
insects, fungi, and bacteria). Strategies for altering crop traits
include: increasing key metabolite concentrations; changing
temporal dynamics of microbe influence on key metabolites; linking
microbial metabolite production/degradation to new environmental
cues; reducing negative metabolites; and improving the balance of
metabolites or underlying proteins.
[0107] As used herein, a "control sequence" refers to an operator,
promoter, silencer, or terminator.
[0108] As used herein, "in planta" refers to in the plant, and
wherein the plant further comprises plant parts, tissue, leaves,
roots, stems, seed, ovules, pollen, flowers, fruit, etc.
[0109] In some embodiments, native or endogenous control sequences
of genes of the present disclosure are replaced with one or more
intrageneric control sequences.
[0110] As used herein, "introduced" refers to the introduction by
means of modern biotechnology, and not a naturally occurring
introduction.
[0111] In some embodiments, the bacteria of the present disclosure
have been modified such that they are not naturally occurring
bacteria.
[0112] In some embodiments, the bacteria of the present disclosure
are present in the plant in an amount of at least 10.sup.3 cfu,
10.sup.4 cfu, 10.sup.5 cfu, 10.sup.6 cfu, 10.sup.7 cfu, 10.sup.8
cfu, 10.sup.9 cfu, 10.sup.10 cfu, 10.sup.11 cfu, or 10.sup.12 cfu
per gram of fresh or dry weight of the plant. In some embodiments,
the bacteria of the present disclosure are present in the plant in
an amount of at least about 10.sup.3 cfu, about 10.sup.4 cfu, about
10.sup.5 cfu, about 10.sup.6 cfu, about 10.sup.7 cfu, about
10.sup.8 cfu, about 10.sup.9 cfu, about 10.sup.10 cfu, about
10.sup.11 du, or about 10.sup.12 cfu per gram of fresh or dry
weight of the plant. In some embodiments, the bacteria of the
present disclosure are present in the plant in an amount of at
least 10.sup.3 to 10.sup.9, 10.sup.3 to 10.sup.7, 10.sup.3 to
10.sup.5, 10.sup.5 to 10.sup.9, 10.sup.5 to 10.sup.7, 10.sup.6 to
10.sup.10, 10.sup.6 to 10.sup.7 cfu per gram of fresh or dry weight
of the plant.
[0113] Fertilizers and exogenous nitrogen of the present disclosure
may comprise the following nitrogen-containing molecules: ammonium,
nitrate, nitrite, ammonia, glutamine, etc. Nitrogen sources of the
present disclosure may include anhydrous ammonia, ammonia sulfate,
urea, diammonium phosphate, urea-form, monoammonium phosphate,
ammonium nitrate, nitrogen solutions, calcium nitrate, potassium
nitrate, sodium nitrate, etc.
[0114] As used herein, "exogenous nitrogen" refers to
non-atmospheric nitrogen readily available in the soil, field, or
growth medium that is present under non-nitrogen limiting
conditions, including ammonia, ammonium, nitrate, nitrite, urea,
uric acid, ammonium acids, etc.
[0115] As used herein, "non-nitrogen limiting conditions" refers to
non-atmospheric nitrogen available in the soil, field, media at
concentrations greater than about 4 mM nitrogen, as disclosed by
Kant et al. (2010. J. Exp. Biol. 62(4):1499-1509), which is
incorporated herein by reference.
[0116] As used herein, an "intergeneric microorganism" is a
microorganism that is formed by the deliberate combination of
genetic material originally isolated from organisms of different
taxonomic genera. An "intergeneric mutant" can be used
interchangeably with "intergeneric microorganism". An exemplary
"intergeneric microorganism" includes a microorganism containing a
mobile genetic element which was first identified in a
microorganism in a genus different from the recipient
microorganism. Further explanation can be found, inter alia, in 40
C.F.R. .sctn. 725.3.
[0117] In aspects, microbes taught herein are "non-intergeneric,"
which means that the microbes are not intergeneric.
[0118] As used herein, an "intrageneric microorganism" is a
microorganism that is formed by the deliberate combination of
genetic material originally isolated from organisms of the same
taxonomic genera. An "intrageneric mutant" can be used
interchangeably with "intrageneric microorganism".
[0119] As used herein, "introduced genetic material" means genetic
material that is added to, and remains as a component of, die
genome of the recipient.
[0120] In some embodiments, the nitrogen fixation and assimilation
genetic regulatory network comprises polynucleotides encoding genes
and non-coding sequences that direct, modulate, and/or regulate
microbial nitrogen fixation and/or assimilation and can comprise
polynucleotide sequences of the nif cluster (e.g., nifA, niFB,
nifC, . . . , nifZ), polynucleotides encoding nitrogen regulatory
protein C, polynucleotides encoding nitrogen regulatory protein B,
polynucleotide sequences of the gin cluster (e.g. glnA and glnD),
draT, and ammonia transporters/permeases. In some cases, the Nif
cluster may comprise NifB, NifH, NifD, NifK, NifE, NifN, NifX,
hesa, and NifV. In some cases, the Nif cluster may comprise a
subset of NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa, and
NifV.
[0121] In some embodiments, fertilizer of the present disclosure
comprises at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,
28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,
41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,
54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 854/0, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% nitrogen by weight.
[0122] In some embodiments, fertilizer of the present disclosure
comprises at least about 5%, about 6%, about 7%, about 8%, about
9%, about 10%, about 11%, about 12%, about 13%, about 14%, about
15%, about 16%, about 17%, about 18%, about 19%, about 20%, about
21%, about 22%, about 23%, about 24%, about 25%, about 26%, about
27%, about 28%, about 29%, about 30%, about 31%, about 32%, about
33%, about 34%, about 35%, about 36%, about 37%, about 38%, about
39%, about 40%, about 41%, about 42%, about 43%, about 44%, about
45%, about 46%, about 47%, about 48%, about 49%, about 50%, about
51%, about 52%, about 53%, about 54%, about 55%, about 56%, about
57%, about 58%, about 59%, about 60%, about 61%, about 62%, about
63%, about 64%, about 65%, about 66%, about 67%, about 68%, about
69%, about 70%, about 71%, about 72%, about 73%, about 74%, about
75%, about 76%, about 77%, about 78%, about 79%, about 80%, about
81%, about 82%, about 83%, about 84%, about 85%, about 86%, about
87%, about 88%, about 89%, about 90%, about 91%, about 92%, about
93%, about 94%, about 95%, about 96%, about 97%, about 98%, or
about 99% nitrogen by weight.
[0123] In some embodiments, fertilizer of the present disclosure
comprises about 5% to 50%, about 5% to 75%, about 10% to 50%, about
10% to 75%, about 15% to 50%, about 15% to 75%, about 20% to 50%,
about 20% to 75%, about 25% to 50%, about 25% to 75%, about 30% to
50%, about 30% to 75%, about 35% to 50%, about 35% to 75%, about
40% to 50%, about 40% to 75%, about 45% to 50%, about 45% to 75%,
or about 50% to 75% nitrogen by weight.
[0124] In some embodiments, the increase of nitrogen fixation
and/or the production of 1% or more of the nitrogen in the plant
are measured relative to control plants, which have not been
exposed to the bacteria of the present disclosure. All increases or
decreases in bacteria are measured relative to control bacteria.
All increases or decreases in plants are measured relative to
control plants.
[0125] As used herein, a "constitutive promoter" is a promoter,
which is active under most conditions and/or during most
development stages. There are several advantages to using
constitutive promoters in expression vectors used in biotechnology,
such as: high level of production of proteins used to select
transgenic cells or organisms; high level of expression of reporter
proteins or storable markers, allowing easy detection and
quantification; high level of production of a transcription factor
that is part of a regulatory transcription system; production of
compounds that requires ubiquitous activity in the organism; and
production of compounds that are required during all stages of
development. Non-limiting exemplary constitutive promoters include,
CaMV 35S promoter, opine promoters, ubiquitin promoter, alcohol
dehydrogenase promoter, etc.
[0126] As used herein, a "non-constitutive promoter" is a promoter
which is active under certain conditions, in certain types of
cells, and/or during certain development stages. For example,
tissue specific, tissue preferred, cell type specific, cell type
preferred, inducible promoters, and promoters under development
control are non-constitutive promoters. Examples of promoters under
developmental control include promoters that preferentially
initiate transcription in certain tissues.
[0127] As used herein, "inducible" or "repressible" promoter is a
promoter which is under chemical or environmental factors control.
Examples of environmental conditions that may affect transcription
by inducible promoters include anaerobic conditions, certain
chemicals, the presence of light, acidic or basic conditions,
etc.
[0128] As used herein, a "tissue specific" promoter is a promoter
that initiates transcription only in certain tissues. Unlike
constitutive expression of genes, tissue-specific expression is the
result of several interacting levels of gene regulation. As such,
in the art sometimes it is preferable to use promoters from
homologous or closely related species to achieve efficient and
reliable expression of transgenes in particular tissues. This is
one of the main reasons for the large amount of tissue-specific
promoters isolated from particular tissues found in both scientific
and patent literature.
[0129] As used herein, the term "operably linked" refers to the
association of nucleic acid sequences on a single nucleic acid
fragment so that the function of one is regulated by the other. For
example, a promoter is operably linked with a coding sequence when
it is capable of regulating the expression of that coding sequence
(i.e., that the coding sequence is under the transcriptional
control of the promoter). Coding sequences can be operably linked
to regulatory sequences in a sense or antisense orientation. In
another example, the complementary RNA regions of the disclosure
can be operably linked, either directly or indirectly, 5' to the
target mRNA, or 3' to the target mRNA, or within the target mRNA,
or a first complementary region is 5' and its complement is 3' to
the target mRNA.
[0130] In aspects, "applying to the plant a plurality of
non-intergeneric bacteria," includes any means by which the plant
(including plant parts such as a seed, root, stem, tissue, etc.) is
made to come into contact (i.e. exposed) with said bacteria at any
stage of the plant's life cycle. Consequently, "applying to the
plant a plurality of non-intergeneric bacteria," includes any of
the following means of exposing the plant (including plant parts
such as a seed, root, stem, tissue, etc.) to said bacteria:
spraying onto plant, dripping onto plant, applying as a seed coat,
applying to a field that will then be planted with seed, applying
to a field already planted with seed, applying to a field with
adult plants, etc.
[0131] As used herein "MRTN" is an acronym for maximum return to
nitrogen and is utilized as an experimental treatment in the
Examples. MRTN was developed by Iowa State University and
information can be found at: http://cnrc.agron.iastate.edu/ The
MRTN is the nitrogen rate where the economic net return to nitrogen
application is maximized. The approach to calculating the MRTN is a
regional approach for developing corn nitrogen rate guidelines in
individual states. The nitrogen rate trial data was evaluated for
Illinois, Iowa, Michigan, Minnesota, Ohio, and Wisconsin where an
adequate number of research trials were available for corn
plantings following soybean and corn plantings following corn. The
trials were conducted with spring, sidedress, or split
preplant/sidedress applied nitrogen, and sites were not irrigated
except for those that were indicated for irrigated sands in
Wisconsin. MRTN was developed by Iowa State University due to
apparent differences in methods for determining suggested nitrogen
rates required for corn production, misperceptions pertaining to
nitrogen rate guidelines, and concerns about application rates. By
calculating the MRTN, practitioners can determine the following:
(1) the nitrogen rate where the economic net return to nitrogen
application is maximized, (2) the economic optimum nitrogen rate,
which is the point where the last increment of nitrogen returns a
yield increase large enough to pay for the additional nitrogen, (3)
the value of corn grain increase attributed to nitrogen
application, and the maximum yield, which is the yield where
application of more nitrogen does not result in a corn yield
increase. Thus the MRTN calculations provide practitioners with the
means to maximize corn crops in different regions while maximizing
financial gains from nitrogen applications.
[0132] The term mmol is an abbreviation for millimole, which is a
thousandth (10.sup.-3) of a mole, abbreviated herein as mol.
[0133] As used herein the terms "microorganism" or "microbe" should
be taken broadly. These terms, used interchangeably, include but
are not limited to, the two prokaryotic domains, Bacteria and
Archaea. The term may also encompass eukaryotic fungi and
protists.
[0134] The term "microbial consortia" or "microbial consortium"
refers to a subset of a microbial community of individual microbial
species, or strains of a species, which can be described as
carrying out a common function, or can be described as
participating in, or leading to, or correlating with, a
recognizable parameter, such as a phenotypic trait of interest.
[0135] The term "microbial community" means a group of microbes
comprising two or more species or strains. Unlike microbial
consortia, a microbial community does not have to be carrying out a
common function, or does not have to be participating in, or
leading to, or correlating with, a recognizable parameter, such as
a phenotypic trait of interest.
[0136] As used herein, "isolate," "isolated," "isolated microbe,"
and like terms, are intended to mean that the one or more
microorganisms has been separated from at least one of the
materials with which it is associated in a particular environment
(for example soil, water, plant tissue, etc.). Thus, an "isolated
microbe" does not exist in its naturally occurring environment;
rather, it is through the various techniques described herein that
the microbe has been removed from its natural setting and placed
into a non-naturally occurring state of existence. Thus, the
isolated strain or isolated microbe may exist as, for example, a
biologically pure culture, or as spores (or other forms of the
strain). In aspects, the isolated microbe may be in association
with an acceptable carrier, which may be an agriculturally
acceptable carrier.
[0137] In certain aspects of the disclosure, the isolated microbes
exist as "isolated and biologically pure cultures." It will be
appreciated by one of skill in the art, that an isolated and
biologically pure culture of a particular microbe, denotes that
said culture is substantially free of other living organisms and
contains only the individual microbe in question. The culture can
contain varying concentrations of said microbe. The present
disclosure notes that isolated and biologically pure microbes often
"necessarily differ from less pure or impure materials." See, e.g.
In re Bergstrom, 427 F.2d 1394, (CCPA 1970)(discussing purified
prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA
1979)(discussing purified microbes), see also, Parke-Davis &
Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned
Hand discussing purified adrenaline), aff'd in part, rev'd in part,
196 F. 496 (2d Cir. 1912), each of which are incorporated herein by
reference. Furthermore, in some aspects, the disclosure provides
for certain quantitative measures of the concentration, or purity
limitations, that must be found within an isolated and biologically
pure microbial culture. The presence of these purity values, in
certain embodiments, is a further attribute that distinguishes the
presently disclosed microbes from those microbes existing in a
natural state. See, e.g., Merck & Co. v. Olin Mathieson
Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity
limitations for vitamin B12 produced by microbes), incorporated
herein by reference.
[0138] As used herein, "individual isolates" should be taken to
mean a composition, or culture, comprising a predominance of a
single genera, species, or strain, of microorganism, following
separation from one or more other microorganisms.
[0139] Microbes of the present disclosure may include spores and/or
vegetative cells. In some embodiments, microbes of the present
disclosure include microbes in a viable but non-culturable (VBNC)
state. As used herein, "spore" or "spores" refer to structures
produced by bacteria and fungi that are adapted for survival and
dispersal. Spores are generally characterized as dormant
structures; however, spores are capable of differentiation through
the process of germination. Germination is the differentiation of
spores into vegetative cells that are capable of metabolic
activity, growth, and reproduction. The germination of a single
spore results in a single fungal or bacterial vegetative cell.
Fungal spores are units of asexual reproduction, and in some cases
are necessary structures in fungal life cycles. Bacterial spores
are structures for surviving conditions that may ordinarily be
nonconducive to the survival or growth of vegetative cells.
[0140] As used herein, "microbial composition" refers to a
composition comprising one or more microbes of the present
disclosure. In some embodiments, a microbial composition is
administered to plants (including various plant parts) and/or in
agricultural fields.
[0141] As used herein, "carrier," "acceptable carrier," or
"agriculturally acceptable carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the microbe can be administered,
which does not detrimentally effect the microbe.
Regulation of Nitrogen Fixation
[0142] In some cases, nitrogen fixation pathway may act as a target
for genetic engineering and optimization. One trait that may be
targeted for regulation by the methods described herein is nitrogen
fixation. Nitrogen fertilizer is the largest operational expense on
a farm and the biggest driver of higher yields in row crops like
corn and wheat. Described herein are microbial products that can
deliver renewable forms of nitrogen in non-leguminous crops. While
some endophytes have the genetics necessary for fixing nitrogen in
pure culture, the fundamental technical challenge is that wild-type
endophytes of cereals and grasses stop fixing nitrogen in
fertilized fields. The application of chemical fertilizers and
residual nitrogen levels in field soils signal the microbe to shut
down the biochemical pathway for nitrogen fixation.
[0143] Changes to the transcriptional and post-translational levels
of components of the nitrogen fixation regulatory network may be
beneficial to the development of a microbe capable of fixing and
transferring nitrogen to corn in the presence of fertilizer. To
that end, described herein is Host-Microbe Evolution (HoME)
technology to precisely evolve regulatory networks and elicit novel
phenotypes. Also described herein are unique, proprietary libraries
of nitrogen-fixing endophytes isolated from corn, paired with
extensive omics data surrounding the interaction of microbes and
host plant under different environmental conditions like nitrogen
stress and excess. In some embodiments, this technology enables
precision evolution of the genetic regulatory network of endophytes
to produce microbes that actively fix nitrogen even in the presence
of fertilizer in the field. Also described herein are evaluations
of the technical potential of evolving microbes that colonize corn
root tissues and produce nitrogen for fertilized plants and
evaluations of the compatibility of endophytes with standard
formulation practices and diverse soils to determine feasibility of
integrating the microbes into modern nitrogen management
strategies.
[0144] In order to utilize elemental nitrogen (N) for chemical
synthesis, life forms combine nitrogen gas (N.sub.2) available in
the atmosphere with hydrogen in a process known as nitrogen
fixation. Because of the energy-intensive nature of biological
nitrogen fixation, diazotrophs (bacteria and archaea that fix
atmospheric nitrogen gas) have evolved sophisticated and fight
regulation of the nif gene cluster in response to environmental
oxygen and available nitrogen. Nif genes encode enzymes involved in
nitrogen fixation (such as the nitrogenase complex) and proteins
that regulate nitrogen fixation. Shamseldin (2013. Global J.
Biotechnol. Biochem. 8(4):84-94) discloses detailed descriptions of
nif genes and their products, and is incorporated herein by
reference. Described herein are methods of producing a plant with
an improved trait comprising isolating bacteria from a first plant,
introducing a genetic variation into a gene of the isolated
bacteria to increase nitrogen fixation, exposing a second plant to
the variant bacteria, isolating bacteria from the second plant
having an improved trait relative to the first plant, and repeating
the steps with bacteria isolated from the second plant.
[0145] In Proteobacteria, regulation of nitrogen fixation centers
around the .sigma..sub.54-dependent enhancer-binding protein NifA,
the positive transcriptional regulator of the nif cluster.
Intracellular levels of active NifA are controlled by two key
factors: transcription of the nifLA operon, and inhibition of NifA
activity by protein-protein interaction with NifL. Both of these
processes are responsive to intraceullar glutamine levels via the
PII protein signaling cascade. This cascade is mediated by GlnD,
which directly senses glutamine and catalyzes the uridylylation or
deuridylylation of two PII regulatory proteins--GlnB and GlnK--in
response the absence or presence, respectively, of bound glutamine.
Under conditions of nitrogen excess, unmodified GlnB signals the
deactivation of the nifLA promoter. However, under conditions of
nitrogen limitation, GlnB is post-translationally modified, which
inhibits its activity and Leads to transcription of the nifLA
operon. In this way, nifLA transcription is tightly controlled in
response to environmental nitrogen via the PII protein signaling
cascade. On the post-translational level of NifA regulation, GlnK
inhibits the NifL/NifA interaction in a matter dependent on the
overall level of free GlnK within the cell.
[0146] NifA is transcribed from the nifLA operon, whose promoter is
activated by phosphorylated NtrC, another .sigma..sub.54-dependent
regulator. The phosphorylation state of NtrC is mediated by the
histidine kinase NtrB, which interacts with deuridylylated GlnB but
not undylylated GlnB. Under conditions of nitrogen excess, a high
intracellular level of glutamine leads to deuridylylation of GlnB,
which then interacts with NtrB to deactivate its phosphorylation
activity and activate its phosphatase activity, resulting in
dephosphorylation of NtrC and the deactivation of the nifLA
promoter. However, under conditions of nitrogen limitation, a low
level of intracellular glutamine results in uridylylation of GlnB,
which inhibits its interaction with NtrB and allows the
phosphorylation of NtrC and transcription of the nifLA operon. In
this way, nifLA expression is tightly controlled in response to
environmental nitrogen via the PII protein signaling cascade. nifA,
ntrB, ntrC, and glnB, are all genes that can be mutated in the
methods described herein. These processes may also be responsive to
intracellular or extracellular levels of ammonia, urea or
nitrates.
[0147] The activity of NifA is also regulated post-translationally
in response to environmental nitrogen, most typically through
Nil-mediated inhibition of NifA activity. In general, the
interaction of NifL and NifA is influenced by the PII protein
signaling cascade via GlnK, although the nature of the interactions
between GlnK and NifL/NifA varies significantly between
diazotrophs. In Klebsiella pneumoniae, both forms of GlnK inhibit
the NifL/NifA interaction, and the interaction between GlnK and
NifL/NifA is determined by the overall level of free GlnK within
the cell. Under nitrogen-excess conditions, deuridylylated GlnK
interacts with the ammonium transporter AmtB, which serves to both
block ammonium uptake by AmtB and sequester GlnK to the membrane,
allowing inhibition of NifA by NifL. On the other hand, in
Azotobacter vinelandii, interaction with deuridylylated GlnK is
required for the NifL/NifA interaction and NifA inhibition, while
uridylylation of GlnK inhibits its interaction with Nil. In
diazotrophs lacking the nifL gene, there is evidence that NifA
activity is inhibited directly by interaction with the
deuridylylated forms of both GlnK and GlnB under nitrogen-excess
conditions. In some bacteria the Nif cluster may be regulated by
glnR, and further in some cases this may comprise negative
regulation. Regardless of the mechanism, post-translational
inhibition of NifA is an important regulator of the nif cluster in
most known diazotrophs. Additionally, nifL, amtB, glnK, and glnR
are genes that can be mutated in the methods described herein.
[0148] In addition to regulating the transcription of the nif gene
cluster, many diazotrophs have evolved a mechanism for the direct
post-translational modification and inhibition of the nitrogenase
enzyme itself, known as nitrogenase shutoff. This is mediated by
ADP-ribosylation of the Fe protein (NifH) under nitrogen-excess
conditions, which disrupts its interaction with the MoFe protein
complex (NifDK) and abolishes nitrogenase activity. DraT catalyzes
the ADP-ribosylation of the Fe protein and shutoff of nitrogenase,
while DraG catalyzes the removal of ADP-ribose and reactivation of
nitrogenase. As with nifLA transcription and NifA inhibition,
nitrogenase shutoff is also regulated via the PII protein signaling
cascade. Under nitrogen-excess conditions, deuridylylated GlnB
interacts with and activates DraT, while deuridylylated GlnK
interacts with both DraG and AmtB to form a complex, sequestering
DraG to the membrane. Under nitrogen-limiting conditions, the
uridylylated forms of GlnB and GlnK do not interact with DraT and
DraG, respectively, leading to the inactivation of DraT and the
diffusion of DraG to the Fe protein, where it removes the
ADP-ribose and activates nitrogenase. The methods described herein
also contemplate introducing genetic variation into the nifH, nifD,
nifK, and draT genes.
[0149] Although some endophytes have the ability to fix nitrogen in
vitro, often the genetics are silenced in the field by high levels
of exogenous chemical fertilizers. One can decouple the sensing of
exogenous nitrogen from expression of the nitrogenase enzyme to
facilitate field-based nitrogen fixation. Improving the integral of
nitrogenase activity across time further serves to augment the
production of nitrogen for utilization by the crop. Specific
targets for genetic variation to facilitate field-based nitrogen
fixation using the methods described herein include one or more
genes selected from the group consisting of nifA, ntrB, ntrC, glnA,
glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY,
nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and
nifQ.
[0150] An additional target for genetic variation to facilitate
field-based nitrogen fixation using the methods described herein is
the NifA protein. The NifA protein is typically the activator for
expression of nitrogen fixation genes. Increasing the production of
NifA (either constitutively or during high ammonia condition)
circumvents the native ammonia-sensing pathway. In addition,
reducing the production of NifL proteins, a known inhibitor of
NifA, also leads to an increased level of freely active NifA. In
addition, increasing the transcription level of the nifAL operon
(either constitutively or during high ammonia condition) also leads
to an overall higher level of NifA proteins. Elevated level of
nifAL expression is achieved by altering the promoter itself or by
reducing the expression of NtrB (part of ntrB and ntrC signaling
cascade that originally would result in the shutoff of nifAL operon
during high nitrogen condition). High level of NifA achieved by
these or any other methods described herein increases the nitrogen
fixation activity of the endophytes.
[0151] Another target for genetic variation to facilitate
field-based nitrogen fixation using the methods described herein is
the glnD/GlnB/GlnK PII signaling cascade. The intracellular
glutamine level is sensed through the GlnD/GlnB/GlnK PII signaling
cascade. Active site mutations in GlnD that abolish the
uridylyl-removing activity of GlnD disrupt the nitrogen-sensing
cascade. In addition, reduction of the GlnB concentration short
circuits the glutamine-sensing cascade. These mutations "trick" the
cells into perceiving a nitrogen-limited state, thereby increasing
the nitrogen fixation level activity. These processes may also be
responsive to intracellular or extracellular levels of ammonia,
urea or nitrates.
[0152] The amtB protein is also a target for genetic variation to
facilitate field-based nitrogen fixation using the methods
described herein. Ammonia uptake from the environment can be
reduced by decreasing the expression level of amtB protein. Without
intracellular ammonia, the endophyte is not able to sense the high
level of ammonia, preventing the down-regulation of nitrogen
fixation genes. Any ammonia that manages to get into the
intracellular compartment is converted into glutamine.
Intracellular glutamine level is the major currency of nitrogen
sensing. Decreasing the intracellular glutamine level prevents the
cells from sensing high ammonium levels in the environment. This
effect can be achieved by increasing the expression level of
glutaminase, an enzyme that converts glutamine into glutamate. In
addition, intracellular glutamine can also be reduced by decreasing
glutamine synthase (an enzyme that converts ammonia into
glutamine). In diazotrophs, fixed ammonia is quickly assimilated
into glutamine and glutamate to be used for cellular processes.
Disruptions to ammonia assimilation may enable diversion of fixed
nitrogen to be exported from the cell as ammonia. The fixed ammonia
is predominantly assimilated into glutamine by glutamine synthetase
(GS), encoded by glnA, and subsequently into glutamine by glutamine
oxoglutarate aminotransferase (GOGAT). In some examples, glnS
encodes a glutamine synthetase. GS is regulated
post-translationally by GS adenylyl transferase (GlnE), a
bi-functional enzyme encoded by glnE that catalyzes both the
adenylylation and deadenylylation of GS through activity of its
adenylyl-transferase (AT) and adenylyl-removing (AR) domains,
respectively. Under nitrogen limiting conditions, glnA is
expressed, and GlnE's AR domain de-adynylylates GS, allowing it to
be active. Under conditions of nitrogen excess, glnA expression is
turned off, and GlnE's AT domain is activated allosterically by
glutamine, causing the adenylylation and deactivation of GS.
[0153] Furthermore, the draT gene may also be a target for genetic
variation to facilitate field-based nitrogen fixation using the
methods described herein. Once nitrogen fixing enzymes are produced
by the cell, nitrogenase shut-off represents another level in which
cell downregulates fixation activity in high nitrogen condition.
This shut-off could be removed by decreasing the expression level
of DraT.
[0154] Methods for imparting new microbial phenotypes can be
performed at the transcriptional, translational, and
post-translational levels. The transcriptional level includes
changes at the promoter (such as changing sigma factor affinity or
binding sites for transcription factors, including deletion of all
or a portion of the promoter) or changing transcription terminators
and attenuators. The translational level includes changes at the
ribosome binding sites and changing mRNA degradation signals. The
post-translational level includes mutating an enzyme's active site
and changing protein-protein interactions. These changes can be
achieved in a multitude of ways. Reduction of expression level (or
complete abolishment) can be achieved by swapping the native
ribosome binding site (RBS) or promoter with another with lower
strength/efficiency. ATG start sites can be swapped to a GTG, TTG,
or CTG start codon, which results in reduction in translational
activity of the coding region. Complete abolishment of expression
can be done by knocking out (deleting) the coding region of a gene.
Frameshifting the open reading frame (ORF) likely will result in a
premature stop codon along the ORF, thereby creating a
non-functional truncated product. Insertion of in-frame stop codons
will also similarly create a non-functional truncated product.
Addition of a degradation tag at the N or C terminal can also be
done to reduce the effective concentration of a particular
gene.
[0155] Conversely, expression level of the genes described herein
can be achieved by using a stronger promoter. To ensure high
promoter activity during high nitrogen level condition (or any
other condition), a transcription profile of the whole genome in a
high nitrogen level condition could be obtained and active
promoters with a desired transcription level can be chosen from
that dataset to replace the weak promoter. Weak start codons can be
swapped out with an ATG start codon for better translation
initiation efficiency. Weak ribosomal binding sites (RBS) can also
be swapped out with a different RBS with higher translation
initiation efficiency. In addition, site specific mutagenesis can
also be performed to alter the activity of an enzyme.
[0156] Increasing the level of nitrogen fixation that occurs in a
plain can lead to a reduction in the amount of chemical fertilizer
needed for crop production and reduce greenhouse gas emissions
(e.g., nitrous oxide).
Generation of Bacterial Populations
Isolation of Bacteria
[0157] Microbes useful in methods and compositions disclosed herein
can be obtained by extracting microbes from surfaces or tissues of
native plants. Microbes can be obtained by grinding seeds to
isolate microbes. Microbes can be obtained by planting seeds in
diverse soil samples and recovering microbes from tissues.
Additionally, microbes can be obtained by inoculating plants with
exogenous microbes and determining which microbes appear in plant
tissues. Non-limiting examples of plant tissues may include a seed,
seedling, leaf cutting, plant, bulb, or tuber.
[0158] A method of obtaining microbes may be through the isolation
of bacteria from soils. Bacteria may be collected from various soil
types. In some example, the soil can be characterized by traits
such as high or low fertility, levels of moisture, levels of
minerals, and various cropping practices. For example, the soil may
be involved in a crop rotation where different crops are planted in
the same soil in successive planting seasons. The sequential growth
of different crops on the same soil may prevent disproportionate
depletion of certain minerals. The bacteria can be isolated from
the plants growing in the selected soils. The seedling plants can
be harvested at 2-6 weeks of growth. For example, at least 400
isolates can be collected in a round of harvest. Soil and plant
types reveal the plant phenotype as well as the conditions, which
allow for the downstream enrichment of certain phenotypes.
[0159] Microbes can be isolated from plant tissues to assess
microbial traits. The parameters for processing tissue samples may
be varied to isolate different types of associative microbes, such
as rhizopheric bacteria, epiphytes, or endophytes. The isolates can
be cultured in nitrogen-free media to enrich for bacteria that
perform nitrogen fixation. Alternatively, microbes can be obtained
from global strain banks.
[0160] In planta analytics are performed to assess microbial
traits. In some embodiments, the plant tissue can be processed for
screening by high throughput processing for DNA and RNA.
Additionally, non-invasive measurements can be used to assess plant
characteristics, such as colonization. Measurements on wild
microbes can be obtained on a plant-by-plant basis. Measurements on
wild microbes can also be obtained in the field using medium
throughput methods. Measurements can be done successively over
time. Model plant system can be used including, but not limited to,
Setaria.
[0161] Microbes in a plant system can be screened via
transcriptional profiling of a microbe in a plant system. Examples
of screening through transcriptional profiling are using methods of
quantitative polymerase chain reaction (qPCR), molecular barcodes
for transcript detection, Next Generation Sequencing, and microbe
tagging with fluorescent markers. Impact factors can be measured to
assess colonization in the greenhouse including, but not limited
to, microbiome, abiotic factors, soil conditions, oxygen, moisture,
temperature, inoculum conditions, and root localization. Nitrogen
fixation can be assessed in bacteria by measuring 15N
gas/fertilizer (dilution) with IRMS or NanoSIMS as described herein
NanoSIMS is high-resolution secondary ion mass spectrometry. The
NanoSIMS technique is a way to investigate chemical activity from
biological samples. The catalysis of reduction of oxidation
reactions that drive the metabolism of microorganisms can be
investigated at the cellular, subcellular, molecular and elemental
level. NanoSIMS can provide high spatial resolution of greater than
0.1 .mu.m. NanoSIMS can detect the use of isotope tracers such as
.sup.13C, .sup.15N, and .sup.18O. Therefore, NanoSIMS can be used
to the chemical activity nitrogen in the cell.
[0162] Automated greenhouses can be used for planta analytics.
Plant metrics in response to microbial exposure include, but are
not limited to, biomass, chloroplast analysis, CCD camera,
volumetric tomography measurements.
[0163] One way of enriching a microbe population is according to
genotype. For example, a polymerase chain reaction (PCR) assay with
a targeted primer or specific primer. Primers designed for the nifH
gene can be used to identity diazotrophs because diazotrophs
express the nifH gene in the process of nitrogen fixation. A
microbial population can also be enriched via single-cell
culture-independent approaches and chemotaxis-guided isolation
approaches. Alternatively, targeted isolation of microbes can be
performed by culturing the microbes on selection media.
Premeditated approaches to enriching microbial populations for
desired traits can be guided by bioinformatics data and are
described herein.
Enriching for Microbes with Nitrogen Fixation Capabilities Using
Bioinformatics
[0164] Bioinformatic tools can be used to identify and isolate
plant growth promoting rhizobacteria (PGPRs), which are selected
based on their ability to perform nitrogen fixation. Microbes with
high nitrogen fixing ability can promote favorable traits in
plants. Bioinformatic modes of analysis for the identification of
PGPRs include, but are not limited to, genomics, metagenomics,
targeted isolation, gene sequencing, transcriptome sequencing, and
modeling.
[0165] Genomics analysis can be used to identify PGPRs and confirm
the presence of mutations with methods of Next Generation
Sequencing as described herein and microbe version control.
[0166] Metagenomics can be used to identify and isolate PGPR using
a prediction algorithm for colonization. Metadata can also be used
to identify the presence of an engineered strain in environmental
and greenhouse samples.
[0167] Transcriptomic sequencing can be used to predict genotypes
leading to PGPR phenotypes. Additionally, transcriptomic data is
used to identify promoters for altering gene expression.
Transcriptomic data can be analyzed in conjunction with the Whole
Genome Sequence (WGS) to generate models of metabolism and gene
regulatory networks.
Domestication of Microbes
[0168] Microbes isolated from nature can undergo a domestication
process wherein the microbes are converted to a form that is
genetically trackable and identifiable. One way to domesticate a
microbe is to engineer it with antibiotic resistance. The process
of engineering antibiotic resistance can begin by determining the
antibiotic sensitivity in the wild type microbial strain. If the
bacteria are sensitive to the antibiotic, then the antibiotic can
be a good candidate for antibiotic resistance engineering.
Subsequently, an antibiotic resistant gene or a counterselectable
suicide vector can be incorporated into the genome of a microbe
using recombineering methods. A counterselectable suicide vector
may consist of a deletion of the gene of interest, a selectable
marker, and the counterselectable marker sacB. Counterselection can
be used to exchange native microbial DNA sequences with antibiotic
resistant genes. A medium throughput method can be used to evaluate
multiple microbes simultaneously allowing for parallel
domestication. Alternative methods of domestication include the use
of homing nucleases to prevent the suicide vector sequences from
looping out or from obtaining intervening vector sequences.
[0169] DNA vectors can be introduced into bacteria via several
methods including electroporation and chemical transformations. A
standard library of vectors can be used for transformations. An
example of a method of gene editing is CRISPR preceded by Cas9
testing to ensure activity of Cas9 in the microbes.
Non-Transgenic Engineering of Microbes
[0170] A microbial population with favorable traits can be obtained
via directed evolution. Direct evolution is an approach wherein the
process of natural selection is mimicked to evolve proteins or
nucleic acids towards a user-defined goal. An example of direct
evolution is when random mutations are introduced into a microbial
population, the microbes with the most favorable traits are
selected, and the growth of the selected microbes is continued. The
most favorable traits in growth promoting rhizobacteria (PGPRs) may
be in nitrogen fixation. The method of directed evolution may be
iterative and adaptive based on the selection process after each
iteration.
[0171] Plant growth promoting rhizobacteria (PGPRs) with high
capability of nitrogen fixation can be generated. The evolution of
PGPRs can be carried out via the introduction of genetic variation.
Genetic variation can be introduced via polymerase chain reaction
mutagenesis, oligonucleotide-directed mutagenesis, saturation
mutagenesis, fragment shuffling mutagenesis, homologous
recombination, CRISPR/Cas9 systems, chemical mutagenesis, and
combinations thereof. These approaches can introduce random
mutations into the microbial population. For example, mutants can
be generated using synthetic DNA or RNA via
oligonucleotide-directed mutagenesis. Mutants can be generated
using tools contained on plasmids, which are later cured. Genes of
interest can be identified using libraries from other species with
improved traits including, but not limited to, improved PGPR
properties, improved colonization of cereals, increased oxygen
sensitivity, increased nitrogen fixation, and increased ammonia
excretion. Intrageneric genes can be designed based on these
libraries using software such as Geneious or Platypus design
software. Mutations can be designed with the aid of machine
learning, Mutations can be designed with the aid of a metabolic
model. Automated design of the mutation can be done using a la
Platypus and will guide RNAs for Cas-directed mutagenesis.
[0172] The intra-generic genes can be transferred into the host
microbe. Additionally, reporter systems can also be transferred to
the microbe. The reporter systems characterize promoters, determine
the transformation success, screen mutants, and act as negative
screening tools.
[0173] The microbes carrying the mutation can be cultured via
serial passaging. A microbial colony contains a single variant of
the microbe. Microbial colonies are screened with the aid of an
automated colony picker and liquid handler. Mutants with gene
duplication and increased copy number express a higher genotype of
the desired trait.
Selection of Plant Growth Promoting Microbes Based on Nitrogen
Fixation
[0174] The microbial colonies can be screened using various assays
to assess nitrogen fixation. One way to measure nitrogen fixation
is via a single fermentative assay, which measures nitrogen
excretion. An alternative method is the acetylene reduction assay
(ARA) with in-line sampling over time. ARA can be performed in high
throughput plates of microtube arrays. ARA can be performed with
live plants and plant tissues. The media formulation and media
oxygen concentration can be varied in ARA assays. Another method of
screening microbial variants is by using biosensors. The use of
NanoSIMS and Raman microspectroscopy can be used to investigate the
activity of the microbes. In some cases, bacteria can also be
cultured and expanded using methods of fermentation in bioreactors.
The bioreactors are designed to improve robustness of bacteria
growth and to decrease the sensitivity of bacteria to oxygen.
Medium to high TP plate-based microfermentors are used to evaluate
oxygen sensitivity, nutritional needs, nitrogen fixation, and
nitrogen excretion. The bacteria can also be co-cultured with
competitive or beneficial microbes to elucidate cryptic pathways.
Flow cytometry can be used to screen for bacteria that produce high
levels of nitrogen using chemical, colorimetric, or fluorescent
indicators. The bacteria may be cultured in the presence or absence
of a nitrogen source. For example, the bacteria may be cultured
with glutamine, ammonia, urea or nitrates.
Microbe Breeding
[0175] Microbe breeding is a method to systematically identify and
improve the role of species within the crop microbiome. The method
comprises three steps: 1) selection of candidate species by mapping
plant-microbe interactions and predicting regulatory networks
linked to a particular phenotype, 2) pragmatic and predictable
improvement of microbial phenotypes through intra-species crossing
of regulatory networks and gene clusters, and 3) screening and
selection of new microbial genotypes that produce desired crop
phenotypes. To systematically assess the improvement of strains, a
model is created that links colonization dynamics of the microbial
community to genetic activity by key species. The model is used to
predict genetic targets breeding and improve the frequency of
selecting improvements in microbiome-encoded traits of agronomic
relevance. See, FIG. 17A for a graphical representation of an
embodiment of the process. In particular, FIG. 17A depicts a
schematic of microbe breeding, in accordance with embodiments. As
illustrated in FIG. 17A, rational improvement of the crop
microbiome may be used to increase soil biodiversity, tune impact
of keystone species, and/or alter timing and expression of
important metabolic pathways. To this end, the inventors have
developed a microbe breeding pipeline to identify and improve the
role of strains within the crop microbiome. The method comprises
three steps: 1) selection of candidate species by mapping
plant-microbe interactions and predicting regulatory networks
linked to a particular phenotype, 2) pragmatic and predictable
improvement of microbial phenotypes through intragenomic crossing
of gene regulatory networks and gene clusters, and 3) screening and
selection of new microbial genotypes that produce desired crop
phenotypes. To systematically assess the improvement of strains,
the inventors employ a model that links colonization dynamics of
the microbial community to genetic activity by key species. This
process represents a methodology for breeding and selecting
improvements in microbiome-encoded traits of agronomic
relevance.
[0176] Production of bacteria to improve plant traits (e.g.,
nitrogen fixation) can be achieved through serial passage. The
production of this bacteria can be done by selecting plants, which
have a particular improved trait that is influenced by the
microbial flora, in addition to identifying bacteria and/or
compositions that are capable of imparting one or more improved
traits to one or more plants. One method of producing a bacteria to
improve a plant trait includes the steps of: (a) isolating bacteria
from tissue or soil of a first plant; (b) introducing a genetic
variation into one or more of the bacteria to produce one or more
variant bacteria; (c) exposing a plurality of plants to the variant
bacteria; (d) isolating bacteria from tissue or soil of one of the
plurality of plants, wherein the plant from which the bacteria is
isolated has an improved trait relative to other plants in the
plurality of plants; and (e) repeating steps (b) to (d) with
bacteria isolated from the plant with an improved trait (step (d)).
Steps (b) to (d) can be repeated any number of times (e.g., once,
twice, three times, four times, five times, ten times, or more)
until the improved trait in a plant reaches a desired level.
Further, the plurality of plants can be more than two plants, such
as 10 to 20 plants, or 20 or more, 50 or more, 100 or more, 300 or
more, 500 or more, or 1000 or more plants.
[0177] In addition to obtaining a plant with an improved trait, a
bacterial population comprising bacteria comprising one or more
genetic variations introduced into one or more genes (e.g., genes
regulating nitrogen fixation) is obtained. By repeating the steps
described above, a population of bacteria can be obtained that
include the most appropriate members of the population that
correlate with a plant trait of interest. The bacteria in this
population can be identified and their beneficial properties
determined, such as by genetic and/or phenotypic analysis. Genetic
analysis may occur of isolated bacteria in step (a). Phenotypic
and/or genotypic information may be obtained using techniques
including: high through-put screening of chemical components of
plant origin, sequencing techniques including high throughput
sequencing of genetic material, differential display techniques
(including DDRT-PCR, and DD-PCR), nucleic acid microarray
techniques, RNA-sequencing (Whole Transcriptome Shotgun
Sequencing), and qRT-PCR (quantitative real time PCR). Information
gained can be used to obtain community profiling information on the
identity and activity of bacteria present, such as phylogenetic
analysis or microarray-based screening of nucleic acids coding for
components of rRNA operons or other taxonomically informative loci.
Examples of taxonomically informative loci include 16S rRNA gene,
23S rRNA gene, 5S rRNA gene, 5.85 rRNA gene, 12S rRNA gene, 18S
rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA
gene, coxl gene, nifD gene. Example processes of taxonomic
profiling to determine taxa present in a population are described
in US20140155283. Bacterial identification may comprise
characterizing activity of one or more genes or one or more
signaling pathways, such as genes associated with the nitrogen
fixation pathway. Synergistic interactions (where two components,
by virtue of their combination, increase a desired effect by more
than an additive amount) between different bacterial species may
also be present in the bacterial populations.
Genetic Variation--Locations and Sources of Genomic Alteration
[0178] The genetic variation may be a gene selected from the group
consisting of: nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT,
amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU,
nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ. The genetic
variation may be a variation in a gene encoding a protein with
functionality selected from the group consisting of: glutamine
synthetase, glutaminase, glutamine synthetase adenylyltransferase,
transcriptional activator, anti-transcriptional activator, pyruvate
flavodoxin oxidoreductase, flavodoxin, or NAD+-dinitrogen-reductase
aDP-D-ribosyltransferase. The genetic variation may be a mutation
that results in one or more of: increased expression or activity of
NifA or glutaminase; decreased expression or activity of NifL,
NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased
adenylyl-removing activity of GlnE; or decreased uridylyl-removing
activity of GlnD. Introducing a genetic variation may comprise
insertion and/or deletion of one or more nucleotides at a target
site, such as 1, 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or more
nucleotides. The genetic variation introduced into one or more
bacteria of the methods disclosed herein may be a knock-out
mutation (e.g. deletion of a promoter, insertion or deletion to
produce a premature stop codon, deletion of an entire gene), or it
may be elimination or abolishment of activity of a protein domain
(e.g. point mutation affecting an active site, or deletion of a
portion of a gene encoding the relevant portion of the protein
product), or it may alter or abolish a regulatory sequence of a
target gene. One or more regulatory sequences may also be inserted,
including heterologous regulatory sequences and regulatory
sequences found within a genome of a bacterial species or genus
corresponding to the bacteria into which the genetic variation is
introduced. Moreover, regulatory sequences may be selected based on
the expression level of a gene in a bacterial culture or within a
plant tissue. The genetic variation may be a pre-determined genetic
variation that is specifically introduced to a target site. The
genetic variation may be a random mutation within the target site.
The genetic variation may be an insertion or deletion of one or
more nucleotides. In some cases, a plurality of different genetic
variations (e.g. 2, 3, 4, 5, 10, or more) are introduced into one
or more of the isolated bacteria before exposing the bacteria to
plants for assessing trait improvement. The plurality of genetic
variations can be any of the above types, the same or different
types, and in any combination. In some cases, a plurality of
different genetic variations are introduced serially, introducing a
first genetic variation after a first isolation step, a second
genetic variation after a second isolation step, and so forth so as
to accumulate a plurality of genetic variations in bacteria
imparting progressively improved traits on the associated
plants.
Genetic Variation--Methods of Introducing Genomic Alteration
[0179] In general, the term "genetic variation" refers to any
change introduced into a polynucleotide sequence relative to a
reference polynucleotide, such as a reference genome or portion
thereof, or reference gene or portion thereof. A genetic variation
may be referred to as a "mutation," and a sequence or organism
comprising a genetic variation may be referred to as a "genetic
variant" or "mutant". Genetic variations can have any number of
effects, such as the increase or decrease of some biological
activity, including gene expression, metabolism, and cell
signaling. Genetic variations can be specifically introduced to a
target site, or introduced randomly. A variety of molecular tools
and methods are available for introducing genetic variation. For
example, genetic variation can be introduced via polymerase chain
reaction mutagenesis, oligonucleotide-directed mutagenesis,
saturation mutagenesis, fragment shuffling mutagenesis, homologous
recombination, recombineering, lambda red mediated recombination,
CRISPR/Cas9 systems, chemical mutagenesis, and combinations
thereof. Chemical methods of introducing genetic variation include
exposure of DNA to a chemical mutagen, e.g., ethyl methanesulfonate
(EMS), methyl methanesulfonate (MMS), N-nitrosourea (EN U),
N-methyl-N-nitro-N'-nitrosoguanidine, 4-nitroquinoline N-oxide,
diethylsulfate, benzopyrene, cyclophosphamide, bleomycin,
trimethylmelamine, acrylamide monomer, nitrogen mustard,
vincristine, diepoxyalkanes (for example, diepoxybutane), ICR-170,
formaldehyde, procarbazine hydrochloride, ethylene oxide,
dimethylnitrosamine, 7,12 dimethylbenz(a)anthracene, chlorambucil,
hexamethylphosphoramide, bisulfan, and the like. Radiation
mutation-inducing agents include ultraviolet radiation,
.gamma.-irradiation, X-rays, and fast neutron bombardment. Genetic
variation can also be introduced into a nucleic acid using, e.g.,
trimethylpsoralen with ultraviolet light. Random or targeted
insertion of a mobile DNA element, e.g., a transposable element, is
another suitable method for generating genetic variation. Genetic
variations can be introduced into a nucleic acid during
amplification in a cell-free in vitro system, e.g., using a
polymerase chain reaction (PCR) technique such as error-prone PCR.
Genetic variations can be introduced into a nucleic acid in vitro
using DNA shuffling techniques (e.g., exon shuffling, domain
swapping, and the like). Genetic variations can also be introduced
into a nucleic acid as a result of a deficiency in a DNA repair
enzyme in a cell, e.g., the presence in a cell of a mutant gene
encoding a mutant DNA repair enzyme is expected to generate a high
frequency of mutations (i.e., about 1 mutation/100 genes-1
mutation/10,000 genes) in the genome of the cell. Examples of genes
encoding DNA repair enzymes include but are not limited to Mut H,
Mut S, Mut L, and Mut U, and the homologs thereof in other species
(e.g., MSH 1 6, PMS 1 2, MLH 1, GTBP, ERCC-1, and the like).
Example descriptions of various methods for introducing genetic
variations are provided in e.g., Stemple (2004) Nature 5:1-7;
Chiang et al. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994)
Proc. Natl. Acad. Sci. USA 91:10747-10751; and U.S. Pat. Nos.
6,033,861, and 6,773,900.
[0180] Genetic variations introduced into microbes may be
classified as transgenic, cisgenic, intragenomic, intrageneric,
intergeneric, synthetic, evolved, rearranged, or SNPs.
[0181] Genetic variation may be introduced into numerous metabolic
pathways within microbes to elicit improvements in the traits
described above. Representative pathways include sulfur uptake
pathways, glycogen biosynthesis, the glutamine regulation pathway,
the molybdenum uptake pathway, the nitrogen fixation pathway,
ammonia assimilation, ammonia excretion or secretion, nNitrogen
uptake, glutamine biosynthesis, annamox, phosphate solubilization,
organic acid transport, organic acid production, agglutinins
production, reactive oxygen radical scavenging genes, Indole Acetic
Acid biosynthesis, trehalose biosynthesis, plant cell wall
degrading enzymes or pathways, root attachment genes,
exopolysaccharide secretion, glutamate synthase pathway, iron
uptake pathways, siderophore pathway, chitinase pathway, ACC
deaminase, glutathione biosynthesis, phosphorous signaling genes,
quorum quenching pathway, cytochrome pathways, hemoglobin pathway,
bacterial hemoglobin-like pathway, small RNA rsmZ, rhizobitoxine
biosynthesis, lapA adhesion protein, AHL quorum sensing pathway,
phenazine biosynthesis, cyclic lipopeptide biosynthesis, and
antibiotic production.
[0182] CRISPR/Cas9 (Clustered regularly interspaced short
palindromic repeats)/CRISPR-associated (Cas) systems can be used to
introduce desired mutations. CRISPR/Cas9 provide bacteria and
archaea with adaptive immunity against viruses and plasmids by
using CRISPR RNAs (crRNAs) to guide the silencing of invading
nucleic acids. The Cas9 protein (or functional equivalent and/or
variant thereof, i.e., Cas9-like protein) naturally contains DNA
endonuclease activity that depends on the association of the
protein with two naturally occurring or synthetic RNA molecules
called crRNA and tracrRNA (also called guide RNAs). In some cases,
the two molecules are covalently link to form a single molecule
(also called a single guide RNA ("sgRNA"). Thus, the Cas9 or
Cas9-like protein associates with a DNA-targeting RNA (which term
encompasses both the two-molecule guide RNA configuration and the
single-molecule guide RNA configuration), which activates the Cas9
or Cas9-like protein and guides the protein to a target nucleic
acid sequence. If the Cas9 or Cas9-like protein retains its natural
enzymatic function, it will cleave target DNA to create a
double-stranded break, which can lead to genome alteration (i.e.,
editing: deletion, insertion (when a donor polynucleotide is
present), replacement, etc.), thereby altering gene expression.
Some variants of Cas9 (which variants are encompassed by the term
Cas9-like) have been altered such that they have a decreased DNA
cleaving activity (in some cases, they cleave a single strand
instead of both strands of the target DNA, while in other cases,
they have severely reduced to no DNA cleavage activity). Further
exemplary descriptions of CRISPR systems for introducing genetic
variation can be found in, e.g. U.S. Pat. No. 8,795,965.
[0183] As a cyclic amplification technique, polymerase chain
reaction (PCR) mutagenesis uses mutagenic primers to introduce
desired mutations. PCR is performed by cycles of denaturation,
annealing, and extension. After amplification by PCR, selection of
mutated DNA and removal of parental plasmid DNA can be accomplished
by: 1) replacement of dCTP by hydroxymethylated-dCTP during PCR,
followed by digestion with restriction enzymes to remove
non-hydroxymethylated parent DNA only; 2) simultaneous mutagenesis
of both an antibiotic resistance gene and the studied gene changing
the plasmid to a different antibiotic resistance, the new
antibiotic resistance facilitating the selection of the desired
mutation thereafter; 3) after introducing a desired mutation,
digestion of the parent methylated template DNA by restriction
enzyme Dpnl which cleaves only methylated DNA, by which the
mutagenized unmethylated chains are recovered; or 4)
circularization of the mutated PCR products in an additional
ligation reaction to increase the transformation efficiency of
mutated DNA. Further description of exemplary methods can be found
in e.g. U.S. Pat. No. 7,132,265, U.S. Pat. No. 6,713,285, U.S. Pat.
No. 6,673,610, U.S. Pat. No. 6,391,548, U.S. Pat. No. 5,789,166,
U.S. Pat. No. 5,780,270, U.S. Pat. No. 5,354,670, U.S. Pat. No.
5,071,743, and US20100267147.
[0184] Oligonucleotide-directed mutagenesis, also called
site-directed mutagenesis, typically utilizes a synthetic DNA
primer. This synthetic primer contains the desired mutation and is
complementary to the template DNA around the mutation site so that
it can hybridize with the DNA in the gene of interest. The mutation
may be a single base change (a point mutation), multiple base
changes, deletion, or insertion, or a combination of these. The
single-strand primer is then extended using a DNA polymerase, which
copies the rest of the gene. The gene thus copied contains the
mutated site, and may then be introduced into a host cell as a
vector and cloned. Finally, mutants can be selected by DNA
sequencing to check that they contain the desired mutation.
[0185] Genetic variations can be introduced using error-prone PCR.
In this technique the gene of interest is amplified using a DNA
polymerase under conditions that are deficient in the fidelity of
replication of sequence. The result is that the amplification
products contain at least one error in the sequence. When a gene is
amplified and the resulting product(s) of the reaction contain one
or more alterations in sequence when compared to the template
molecule, the resulting products are mutagenized as compared to the
template. Another means of introducing random mutations is exposing
cells to a chemical mutagen, such as nitrosoguanidine or ethyl
methanesulfonate (Nestmann, Mutat Res 1975 June; 28(3):323-30), and
the vector containing the gene is then isolated from the host.
[0186] Saturation mutagenesis is another form of random
mutagenesis, in which one tries to generate all or nearly all
possible mutations at a specific site, or narrow region of a gene.
In a general sense, saturation mutagenesis is comprised of
mutagenizing a complete set of mutagenic cassettes (wherein each
cassette is, for example, 1-500 bases in length) in defined
polynucleotide sequence to be mutagenized (wherein the sequence to
be mutagenized is, for example, from 15 to 100, 000 bases in
length). Therefore, a group of mutations (e.g. ranging from 1 to
100 mutations) is introduced into each cassette to be mutagenized.
A grouping of mutations to be introduced into one cassette can be
different or the same from a second grouping of mutations to be
introduced into a second cassette during the application of one
round of saturation mutagenesis. Such groupings are exemplified by
deletions, additions, groupings of particular codons, and groupings
of particular nucleotide cassettes.
[0187] Fragment shuffling mutagenesis, also called DNA shuffling,
is a way to rapidly propagate beneficial mutations. In an example
of a shuffling process, DNAse is used to fragment a set of parent
genes into pieces of e.g. about 50-100 bp in length. This is then
followed by a polymerase chain reaction (PCR) without primers--DNA
fragments with sufficient overlapping homologous sequence will
anneal to each other and are then be extended by DNA polymerase.
Several rounds of this PCR extension are allowed to occur, after
some of the DNA molecules reach the size of the parental genes.
These genes can then be amplified with another PCR, this time with
the addition of primers that are designed to complement the ends of
the strands. The primers may have additional sequences added to
their 5' ends, such as sequences for restriction enzyme recognition
sites needed for ligation into a cloning vector. Further examples
of shuffling techniques are provided in US20050266541.
[0188] Homologous recombination mutagenesis involves recombination
between an exogenous DNA fragment and the targeted polynucleotide
sequence. After a double-stranded break occurs, sections of DNA
around the 5' ends of the break are cut away in a process called
resection. In the strand invasion step that follows, an overhanging
3' end of the broken DNA molecule then "invades" a similar or
identical DNA molecule that is not broken. The method can be used
to delete a gene, remove exons, add a gene, and introduce point
mutations. Homologous recombination mutagenesis can be permanent or
conditional. Typically, a recombination template is also provided.
A recombination template may be a component of another vector,
contained in a separate vector, or provided as a separate
polynucleotide. In some embodiments, a recombination template is
designed to serve as a template in homologous recombination, such
as within or near a target sequence nicked or cleaved by a
site-specific nuclease. A template polynucleotide may be of any
suitable length, such as about or more than about 10, 15, 20, 25,
50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In
some embodiments, the template polynucleotide is complementary to a
portion of a polynucleotide comprising the target sequence. When
optimally aligned, a template polynucleotide might overlap with one
or more nucleotides of a target sequences (e.g. about or more than
about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100
or more nucleotides). In some embodiments, when a template sequence
and a polynucleotide comprising a target sequence are optimally
aligned, the nearest nucleotide of the template polynucleotide is
within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500,
1000, 5000, 10000, or more nucleotides from the target sequence.
Non-limiting examples of site-directed nucleases useful in methods
of homologous recombination include zinc finger nucleases, CRISPR
nucleases, TALE nucleases, and meganuclease. For a further
description of the use of such nucleases, see e.g. U.S. Pat. No.
8,795,965 and US20140301990.
[0189] Mutagens that create primarily point mutations and short
deletions, insertions, transversions, and/or transitions, including
chemical mutagens or radiation, may be used to create genetic
variations. Mutagens include, but are not limited to, ethyl
methanesulfonate, methylmethane sulfonate, N-ethyl-N-nitrosurea,
triethylmelamine, N-methyl-N-nitrosourea, procarbazine,
chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide
monomer, melphalan, nitrogen mustard, vincristine,
dimethylnitrosamine, N-methyl-N'-nitro-Nitrosoguanidine,
nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene,
ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes
(diepoxyoctane, diepoxybutane, and the like),
2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine
dihydrochloride and formaldehyde.
[0190] Introducing genetic variation may be an incomplete process,
such that some bacteria in a treated population of bacteria carry a
desired mutation while others do not. In some cases, it is
desirable to apply a selection pressure so as to enrich for
bacteria carrying a desired genetic variation. Traditionally,
selection for successful genetic variants involved selection for or
against some functionality imparted or abolished by the genetic
variation, such as in the case of inserting antibiotic resistance
gene or abolishing a metabolic activity capable of converting a
non-lethal compound into a lethal metabolite. It is also possible
to apply a selection pressure based on a polynucleotide sequence
itself, such that only a desired genetic variation need be
introduced (e.g. without also requiting a selectable marker). In
this case, the selection pressure can comprise cleaving genomes
lacking the genetic variation introduced to a target site, such
that selection is effectively directed against the reference
sequence into which the genetic variation is sought to be
introduced. Typically, cleavage occurs within 100 nucleotides of
the target site (e.g. within 75, 50, 25, 10, or fewer nucleotides
from the target site, including cleavage at or within the target
site). Cleaving may be directed by a site-specific nuclease
selected from the group consisting of a Zinc Finger nuclease, a
CRISPR nuclease, a TALE nuclease (TALEN), or a meganuclease. Such a
process is similar to processes for enhancing homologous
recombination at a target site, except that no template for
homologous recombination is provided. As a result, bacteria lacking
the desired genetic variation are more likely to undergo cleavage
that, left unrepaired, results in cell death. Bacteria surviving
selection may then be isolated for use in exposing to plants for
assessing conferral of an improved trait.
[0191] A CRISPR nuclease may be used as the site-specific nuclease
to direct cleavage to a target site. An improved selection of
mutated microbes can be obtained by using Cas9 to kill non-mutated
cells. Plants are then inoculated with the mutated microbes to
re-confirm symbiosis and create evolutionary pressure to select for
efficient symbionts. Microbes can then be re-isolated from plant
tissues. CRISPR nuclease systems employed for selection against
non-variants can employ similar elements to those described above
with respect to introducing genetic variation, except that no
template for homologous recombination is provided. Cleavage
directed to the target site thus enhances death of affected
cells.
[0192] Other options for specifically inducing cleavage at a target
site are available, such as zinc finger nucleases, TALE nuclease
(TALEN) systems, and meganuclease. Zinc-finger nucleases (ZFNs) are
artificial DNA endonucleases generated by fusing a zinc finger DNA
binding domain to a DNA cleavage domain. ZFNs can be engineered to
target desired DNA sequences and this enables zinc-finger nucleases
to cleave unique target sequences. When introduced into a cell,
ZFNs can be used to edit target DNA in the cell (e.g., the cell's
genome) by inducing double stranded breaks. Transcription
activator-like effector nucleases (TALENs) are artificial DNA
endonucleases generated by fusing a TAL (Transcription
activator-like) effector DNA binding domain to a DNA cleavage
domain. TALENS can be quickly engineered to bind practically any
desired DNA sequence and when introduced into a cell, TALENs can be
used to edit target DNA in the cell (e.g., the cell's genome) by
inducing double strand breaks. Meganucleases (homing endonuclease)
are endodeoxyribonucleases characterized by a large recognition
site (double-stranded DNA sequences of 12 to 40 base pairs.
Meganucleases can be used to replace, eliminate or modify sequences
in a highly targeted way. By modifying their recognition sequence
through protein engineering, the targeted sequence can be changed.
Meganucleases can be used to modify all genome types, whether
bacterial, plant or animal and are commonly grouped into four
families: the LAGLIDADG family (SEQ ID NO: 1), the GIY-YIG family,
the His-Cyst box family and the HNH family. Exemplary homing
endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV,
I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII
and I-TevIII.
Genetic Variation--Methods of Identification
[0193] The microbes of the present disclosure may be identified by
one or more genetic modifications or alterations, which have been
introduced into said microbe. One method by which said genetic
modification or alteration can be identified is via reference to a
SEQ ID NO that contains a portion of the microbe's genomic sequence
that is sufficient to identify the genetic modification or
alteration.
[0194] Further, in the case of microbes that have not had a genetic
modification or alteration (e.g. a wild type, WT) introduced into
their genomes, the disclosure can utilize 16S nucleic acid
sequences to identify said microbes. A 16S nucleic acid sequence is
an example of a "molecular marker" or "genetic marker," which
refers to an indicator that is used in methods for visualizing
differences in characteristics of nucleic acid sequences. Examples
of other such indicators are restriction fragment length
polymorphism (RFLP) markers, amplified fragment length polymorphism
(AFLP) markers, single nucleotide polymorphisms (SNPs), insertion
mutations, microsatellite markers (SSRs), sequence-characterized
amplified regions (SCARs), cleaved amplified polymorphic sequence
(CAPS) markers or isozyme markers or combinations of the markers
described herein which defines a specific genetic and chromosomal
location. Markers further include polynucleotide sequences encoding
16S or 18S rRNA, and internal transcribed spacer (ITS) sequences,
which are sequences found between small-subunit and large-subunit
rRNA genes that have proven to be especially useful in elucidating
relationships or distinctions when compared against one another.
Furthermore, the disclosure utilizes unique sequences found in
genes of interest (e.g. nif H,D,K,L,A, glnE, amtB, etc.) to
identify microbes disclosed herein.
[0195] The primary structure of major rRNA subunit 16S comprise a
particular combination of conserved, variable, and hypervariable
regions that evolve at different rates and enable the resolution of
both very ancient lineages such as domains, and more modern
lineages such as genera. The secondary structure of the 16S subunit
include approximately 50 helices which result in base pairing of
about 67% of the residues. These highly conserved secondary
structural features are of great functional importance and can be
used to ensure positional homology in multiple sequence alignments
and phylogenetic analysis. Over the previous few decades, the 16S
rRNA gene has become the most sequenced taxonomic marker and is the
cornerstone for the current systematic classification of bacteria
and archaea (Yarza et al. 2014. Nature Rev. Micro. 12:635-45).
[0196] Thus, in certain aspects, the disclosure provides for a
sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to any sequence in Tables E, F, G, or H.
[0197] Thus, in certain aspects, the disclosure provides for a
microbe that comprises a sequence, which shares at least about 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 62-303.
These sequences and their associated descriptions can be found in
Tables F, G, and H.
[0198] In some aspects, the disclosure provides for a microbe that
comprises a 16S nucleic acid sequence, which shares at least about
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 85,
96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269,
277-283. These sequences and their associated descriptions can be
found in Tables G and H.
[0199] In some aspects, the disclosure provides for a microbe that
comprises a nucleic acid sequence, which shares at least about 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 86-95,
97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176,
263-268, 270-274, 275, 276, 284-295. These sequences and their
associated descriptions can be found in Tables G and H.
[0200] In some aspects, the disclosure provides for a microbe that
comprises a nucleic acid sequence, which shares at least about 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 177-260,
296-303. These sequences and their associated descriptions can be
found in Tables G and H.
[0201] In some aspects, the disclosure provides for a microbe that
comprises, or primer that comprises, or probe that comprises, or
non-native junction sequence that comprises, a nucleic acid
sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to SEQ ID NOs: 304-424. These sequences and their
associated descriptions can be found in Table E.
[0202] In some aspects, the disclosure provides for a microbe that
comprises a non-native junction sequence that shares at least about
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:
372-405. These sequences and their associated descriptions can be
found in Table E.
[0203] In some aspects, the disclosure provides for a microbe that
comprises an amino acid sequence, which shares at least about 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 77, 78, 81,
82, or 83. These sequences and their associated descriptions can be
found in Tables F and H.
Genetic Variation--Methods of Detection: Primers, Probes, and
Assays
[0204] The present disclosure teaches primers, probes, and assays
that are useful for detecting the microbes taught herein. In some
aspects, the disclosure provides for methods of detecting the WT
parental strains. In other aspects, the disclosure provides for
methods of detecting the non-intergeneric engineered microbes
derived from the WT strains. In aspects, the present disclosure
provides methods of identifying non-intergeneric genetic
alterations in a microbe.
[0205] In aspects, the genomic engineering methods of the present
disclosure lead to the creation of non-natural nucleotide
"junction" sequences in the derived non-intergeneric microbes.
These non-naturally occurring nucleotide junctions can be used as a
type of diagnostic that is indicative of the presence of a
particular genetic alteration in a microbe taught herein.
[0206] The present techniques are able to detect these
non-naturally occurring nucleotide junctions via the utilization of
specialized quantitative PCR methods, including uniquely designed
primers and probes. In some aspects, the probes of the disclosure
bind to the non-naturally occurring nucleotide junction sequences.
In some aspects, traditional PCR is utilized. In other aspects,
real-time PCR is utilized. In some aspects, quantitative PCR (qPCR)
is utilized.
[0207] Thus, the disclosure can cover the utilization of two common
methods for the detection of PCR products in real-time: (1)
non-specific fluorescent dyes that intercalate with any
double-stranded DNA, and (2) sequence-specific DNA probes
consisting of oligonucleotides that are labelled with a fluorescent
reporter which permits detection only after hybridization of the
probe with its complementary sequence. In some aspects, only the
non-naturally occurring nucleotide junction will be amplified via
the taught primers, and consequently can be detected via either a
non-specific dye, or via the utilization of a specific
hybridization probe. In other aspects, the primers of the
disclosure are chosen such that the primers flank either side of a
junction sequence, such that if an amplification reaction occurs,
then said junction sequence is present.
[0208] Aspects of the disclosure involve non-naturally occurring
nucleotide junction sequence molecules per se, along with other
nucleotide molecules that are capable of binding to said
non-naturally occurring nucleotide junction sequences under mild to
stringent hybridization conditions. In some aspects, the nucleotide
molecules that are capable of binding to said non-naturally
occurring nucleotide junction sequences under mild to stringent
hybridization conditions are termed "nucleotide probes."
[0209] In aspects, genomic DNA can be extracted from samples and
used to quantify the presence of microbes of the disclosure by
using qPCR. The primers utilized in the qPCR reaction can be
primers designed by Primer Blast
(https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify
unique regions of the wild-type genome or unique regions of the
engineered non-intergeneric mutant strains. The qPCR reaction can
be carried out using the SYBR GreenER qPCR SuperMix Universal
(Thermo Fisher P/N 11762100) kit, using only forward and reverse
amplification primers; alternatively, the Kapa Probe Force kit
(Kapa Biosystems P/N KK4301) can be used with amplification primers
and a TaqMan probe containing a RAM dye label at the 5' end, an
internal ZEN quencher, and a minor groove binder and fluorescent
quencher at the 3' end (Integrated DNA Technologies).
[0210] Certain primer, probe, and non-native junction sequences are
listed in Table E. qPCR reaction efficiency can be measured using a
standard curve generated from a known quantity of gDNA from the
target genome. Data can be normalized to genome copies per g fresh
weight using the tissue weight and extraction volume.
[0211] Quantitative polymerase chain reaction (qPCR) is a method of
quantifying, in real time, the amplification of one or more nucleic
acid sequences. The real time quantification of the PCR assay
permits determination of the quantity of nucleic acids being
generated by the PCR amplification steps by comparing the
amplifying nucleic acids of interest and an appropriate control
nucleic acid sequence, which may act as a calibration standard.
[0212] TaqMan probes are often utilized in qPCR assays that require
an increased specificity for quantifying target nucleic acid
sequences. TaqMan probes comprise a oligonucleotide probe with a
fluorophore attached to the 5' end and a quencher attached to the
3' end of the probe. When the TaqMan probes remain as is with the
5' and 3' ends of the probe in close contact with each other, the
quencher prevents fluorescent signal transmission from the
fluorophore. TaqMan probes are designed to anneal within a nucleic
acid region amplified by a specific set of primers. As the Taq
polymerase extends the primer and synthesizes the nascent strand,
the 5' to 3' exonuclease activity of the Taq polymerase degrades
the probe that annealed to the template. This probe degradation
releases the fluorophore, thus breaking the close proximity to the
quencher and allowing fluorescence of the fluorophore. Fluorescence
detected in the qPCR assay is directly proportional to the
fluorophore released and the amount of DNA template present in the
reaction.
[0213] The features of qPCR allow the practitioner to eliminate the
labor-intensive post-amplification step of gel electrophoresis
preparation, which is generally required for observation of the
amplified products of traditional PCR assays. The benefits of qPCR
over conventional PCR are considerable, and include increased
speed, ease of use, reproducibility, and quantitative ability
Improvement of Traits
[0214] Methods of the present disclosure may be employed to
introduce or improve one or more of a variety of desirable traits.
Examples of traits that may introduced or improved include: root
biomass, root length, height, shoot length, leaf number, water use
efficiency, overall biomass, yield, fruit size, grain size,
photosynthesis rate, tolerance to drought, heat tolerance, salt
tolerance, resistance to nematode stress, resistance to a fungal
pathogen, resistance to a bacterial pathogen, resistance to a viral
pathogen, level of a metabolite, and proteome expression. The
desirable traits, including height, overall biomass, root and/or
shoot biomass, seed germination, seedling survival, photosynthetic
efficiency, transpiration rate, seed/fruit number or mass, plant
grain or fruit yield, leaf chlorophyll content, photosynthetic
rate, root length, or any combination thereof, can be used to
measure growth, and compared with the growth rate of reference
agricultural plants (e.g., plants without the improved traits)
grown under identical conditions.
[0215] A preferred trait to be introduced or improved is nitrogen
fixation, as described herein. In some cases, a plant resulting
from the methods described herein exhibits a difference in the
trait that is at least about 5% greater, for example at least about
5%, at least about 8%, at least about 10%, at least about 15%, at
least about 20%, at least about 25%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
75%, at least about 80%, at least about 80%, at least about 90%, or
at least 100%, at least about 200%, at least about 300%, at least
about 400% or greater than a reference agricultural plant grown
under the same conditions in the soil. In additional examples, a
plant resulting from the methods described herein exhibits a
difference in the trait that is at least about 5% greater, for
example at least about 5%, at least about 8%, at least about 10%,
at least about 15%, at least about 20%, at least about 25%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 75%, at least about 80%, at least about
80%, at least about 90%, or at least 100%, at least about 200%, at
least about 300%, at least about 400% or greater than a reference
agricultural plant grown under similar conditions in the soil.
[0216] The trait to be improved may be assessed under conditions
including the application of one or more biotic or abiotic
stressors. Examples of stressors include abiotic stresses (such as
heat stress, salt stress, drought stress, cold stress, and low
nutrient stress) and biotic stresses (such as nematode stress,
insect herbivory stress, fungal pathogen stress, bacterial pathogen
stress, and viral pathogen stress).
[0217] The trait improved by methods and compositions of the
present disclosure may be nitrogen fixation, including in a plant
not previously capable of nitrogen fixation. In some cases,
bacteria isolated according to a method described herein produce 1%
or more (e.g. 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or
more) of a plant's nitrogen, which may represent an increase in
nitrogen fixation capability of at least 2-fold (e.g. 3-fold,
4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,
50-fold, 100-fold, 1000-fold, or more) as compared to bacteria
isolated from the first plant before introducing any genetic
variation. In some cases, the bacteria produce 5% or more of a
plant's nitrogen. The desired level of nitrogen fixation may be
achieved after repeating the steps of introducing genetic
variation, exposure to a plurality of plants, and isolating
bacteria from plants with an improved trait one or more times (e.g.
1, 2, 3, 4, 5, 10, 15, 25, or more times). In some cases, enhanced
levels of nitrogen fixation are achieved in the presence of
fertilizer supplemented with glutamine, ammonia, or other chemical
source of nitrogen. Methods for assessing degree of nitrogen
fixation are known, examples of which are described herein.
[0218] Microbe breeding is a method to systematically identify and
improve the role of species within the crop microbiome. The method
comprises three steps: 1) selection of candidate species by mapping
plant-microbe interactions and predicting regulatory networks
linked to a particular phenotype, 2) pragmatic and predictable
improvement of microbial phenotypes through intra-species crossing
of regulatory networks and gene clusters, and 3) screening and
selection of new microbial genotypes that produce desired crop
phenotypes. To systematically assess the improvement of strains, a
model is created that links colonization dynamics of the microbial
community to genetic activity by key species. The model is used to
predict genetic targets breeding and improve the frequency of
selecting improvements in microbiome-encoded traits of agronomic
relevance.
Measuring Nitrogen Delivered in an Agriculturally Relevant Field
Context
[0219] In the field, the amount of nitrogen delivered can be
determined by the function of colonization multiplied by the
activity.
Nitrogen delivered = .intg. Time & Space Colonization .times.
Activity ##EQU00001##
[0220] The above equation requires (1) the average colonization per
unit of plant tissue, and (2) the activity as either the amount of
nitrogen fixed or the amount of ammonia excreted by each microbial
cell. To convert to pounds of nitrogen per acre, corn growth
physiology is tracked over time, e.g., size of the plant and
associated root system throughout the maturity stages.
[0221] The pounds of nitrogen delivered to a crop per acre-season
can be calculated by the following equation:
Nitrogen delivered=.intg.Plant
Tissue(t).times.Colonization(t).times.Activity(t)dt
[0222] The Plant Tissue(t) is the fresh weight of corn plant tissue
over the growing time (t). Values for reasonably making the
calculation are described in detail in the publication entitled
Roots, Growth and Nutrient Uptake (Mengel. Dept. of Agronomy Pub.#
AGRY-95-08 (Rev. May-95. p. 1-8.).
[0223] The Colonization (t) is the amount of the microbes of
interest found within the plant tissue, per gram fresh weight of
plant tissue, at any particular time, t, during the growing season.
In the instance of only a single timepoint available, the single
timepoint is normalized as the peak colonization rate over the
season, and the colonization rate of the remaining timepoints are
adjusted accordingly.
[0224] Activity(t) is the rate of which N is fixed by the microbes
of interest per unit time, at any particular time, t, during the
growing season. In the embodiments disclosed herein, this activity
rate is approximated by in vitro acetylene reduction assay (ARA) in
ARA media in the presence of 5 mM glutamine or Ammonium excretion
assay in ARA media in the presence of 5 mM ammonium ions.
[0225] The Nitrogen delivered amount is then calculated by
numerically integrating the above function. In cases where the
values of the variables described above are discretely measured at
set timepoints, the values in between those timepoints are
approximated by performing linear interpolation.
Nitrogen Fixation
[0226] Described herein are methods of increasing nitrogen fixation
in a plant, comprising exposing the plant to bacteria comprising
one or more genetic variations introduced into one or more genes
regulating nitrogen fixation, wherein the bacteria produce 1% or
more of nitrogen in the plant (e.g. 2%, 5%, 10%, or more), which
may represent a nitrogen-fixation capability of at least 2-fold as
compared to the plant in the absence of the bacteria. The bacteria
may produce the nitrogen in the presence of fertilizer supplemented
with glutamine, urea, nitrates or ammonia. Genetic variations can
be any genetic variation described herein, including examples
provided above, in any number and any combination. The genetic
variation may be introduced into a gene selected from the group
consisting of nifA, nifL, ntrB, ntrC, glutamine synthetase, glnA,
glnB, glnK, draT, amtB, glutaminase, glnD, glnE, nifH, nifD, nifK,
nifY, nifE, nifN, nifU, nifS, nifW, nifZ, nifM, nifF, nifB, and
nifQ. The genetic variation may be a mutation that results in one
or more of: increased expression or activity of nifA or
glutaminase; decreased expression or activity of nifL, ntrB,
glutamine synthetase, glnB, glnK, draT, amtB; decreased
adenylyl-removing activity of GlnE; or decreased uridylyl-removing
activity of GlnD. The genetic variation introduced into one or more
bacteria of the methods disclosed herein may be a knock-out
mutation or it may abolish a regulatory sequence of a target gene,
or it may comprise insertion of a heterologous regulatory sequence,
for example, insertion of a regulatory sequence found within the
genome of the same bacterial species or genus. The regulatory
sequence can be chosen based on the expression level of a gene in a
bacterial culture or within plant tissue. The genetic variation may
be produced by chemical mutagenesis. The plants grown in step (c)
may be exposed to biotic or abiotic stressors.
[0227] The amount of nitrogen fixation that occurs in the plants
described herein may be measured in several ways, for example by an
acetylene-reduction (AR) assay. An acetylene-reduction assay can be
performed in vitro or in vivo. Evidence that a particular bacterium
is providing fixed nitrogen to a plant can include: I) total plant
N significantly increases upon inoculation, preferably with a
concomitant increase in N concentration in the plant; 2) nitrogen
deficiency symptoms are relieved under N-limiting conditions upon
inoculation (which should include an increase in dry matter); 3)
N.sub.2 fixation is documented through the use of an .sup.15N
approach (which can be isotope dilution experiments, .sup.15N.sub.2
reduction assays, or .sup.15N natural abundance assays); 4) fixed N
is incorporated into a plant protein or metabolite; and 5) all of
these effects are not be seen in non-inoculated plants or in plants
inoculated with a mutant of the inoculum strain.
[0228] The wild-type nitrogen fixation regulatory cascade can be
represented as a digital logic circuit where the inputs O.sub.2 and
NH.sub.4.sup.+ pass through a NOR gate, the output of which enters
an AND gate in addition to ATP. In some embodiments, the methods
disclosed herein disrupt the influence of NH.sub.4.sup.+ on this
circuit, at multiple points in the regulatory cascade, so that
microbes can produce nitrogen even in fertilized fields. However,
the methods disclosed herein also envision altering the impact of
ATP or O.sub.2 on the circuitry, or replacing the circuitry with
other regulatory cascades in the cell, or altering genetic circuits
other than nitrogen fixation. Gene clusters can be re-engineered to
generate functional products under the control of a heterologous
regulatory system. By eliminating native regulatory elements
outside of, and within, coding sequences of gene clusters, and
replacing them with alternative regulatory systems, the functional
products of complex genetic operons and other gene clusters can be
controlled and/or moved to heterologous cells, including cells of
different species other than the species from which the native
genes were derived. Once re-engineered, the synthetic gene clusters
can be controlled by genetic circuits or other inducible regulatory
systems, thereby controlling the products' expression as desired.
The expression cassettes can be designed to act as logic gates,
pulse generators, oscillators, switches, or memory devices. The
controlling expression cassette can be linked to a promoter such
that the expression cassette functions as an environmental sensor,
such as an oxygen, temperature, touch, osmotic stress, membrane
stress, or redox sensor.
[0229] As an example, the nifL, nifA, nifT, and nifX genes can be
eliminated from the nif gene cluster. Synthetic genes can be
designed by codon randomizing the DNA encoding each amino acid
sequence. Codon selection is performed, specifying that codon usage
be as divergent as possible from the codon usage in the native
gene. Proposed sequences are scanned for any undesired features,
such as restriction enzyme recognition sites, transposon
recognition sites, repetitive sequences, sigma 54 and sigma 70
promoters, cryptic ribosome binding sites, and rho independent
terminators. Synthetic ribosome binding sites are chosen to match
the strength of each corresponding native ribosome binding site,
such as by constructing a fluorescent reporter plasmid in which the
150 bp surrounding a gene's start codon (from -60 to +90) is fused
to a fluorescent gene. This chimera can be expressed under control
of the Ptac promoter, and fluorescence measured via flow cytometry.
To generate synthetic ribosome binding sites, a library of reporter
plasmids using 150 bp (-60 to +90) of a synthetic expression
cassette is generated. Briefly, a synthetic expression cassette can
consist of a random DNA spacer, a degenerate sequence encoding an
RBS library, and the coding sequence for each synthetic gene.
Multiple clones are screened to identify the synthetic ribosome
binding site that best matched the native ribosome binding site.
Synthetic operons that consist of the same genes as the native
operons are thus constructed and tested for functional
complementation. A further exemplary description of synthetic
operons is provided in US20140329326.
Bacterial Species
[0230] Microbes useful in the methods and compositions disclosed
herein may be obtained from any source. In some cases, microbes may
be bacteria, archaea, protozoa or fungi. The microbes of this
disclosure may be nitrogen fixing microbes, for example a nitrogen
fixing bacteria, nitrogen fixing archaea, nitrogen fixing fungi,
nitrogen fixing yeast, or nitrogen fixing protozoa. Microbes useful
in the methods and compositions disclosed herein may be spore
forming microbes, for example spore forming bacteria. In some
cases, bacteria useful in the methods and compositions disclosed
herein may be Gram positive bacteria or Gram negative bacteria. In
some cases, the bacteria may be an endospore forming bacteria of
the Firmicute phylum. In some cases, the bacteria may be a
diazatroph. In some cases, the bacteria may not be a
diazotroph.
[0231] The methods and compositions of this disclosure may be used
with an archaea, such as, for example, Methanothermobacter
thermoautotrophicus.
[0232] In some cases, bacteria which may be useful include, but are
not limited to, Agrobacterium radiobacter, Bacillus acidocaldarius,
Bacillus acidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus
albolactis, Bacillus alcalophilus, Bacillus alvei, Bacillus
aminoglucosidicus, Bacillus aminovorans; Bacillus amyloyticus (also
known as Paenibacillus amylolyticus) Bacillus amyloliquefaciens,
Bacillus aneurinolyticus, Bacillus atrophaeus, Bacillus
azotoformans, Bacillus badius, Bacillus cereus (synonyms: Bacillus
endorhythmos, Bacillus medusa), Bacillus chitinosporus, Bacillus
circulans, Bacillus coagulans, Bacillus endoparasiticus Bacillus
fastidiosus, Bacillus firmus, Bacillus kurstaki, Bacillus
lacticola, Bacillus lactimorbus, Bacillus lactis, Bacillus
laterosporus (also known as Brevibacillus laterosporus), Bacillus
lautus, Bacillus lentimorbus, Bacillus lentus, Bacillus
licheniformis, Bacillus maroccanus, Bacillus megaterium, Bacillus
metiens, Bacillus mycoides, Bacillus natio, Bacillus nematocida,
Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus,
Bacillus popillae, Bacillus psychrosaccharolyticus, Bacillus
pumilus, Bacillus sianensis, Bacillus smithii, Bacillus sphaericus,
Bacillus subtilis, Bacillus thuringiensis, Bacillus uniflagellatus,
Bradyrhizobium japonicum, Brevibacillus brevis Brevibacillus
laterosporus (formerly Bacillus laterosporus), Chromobacterium
subtsugae, Delftia acidovorans, Lactobacillus acidophilus,
Lysobacter antibioticus, Lysobacter enzymogenes, Paenibacillus
alvei, Paenibacillus polymyxa, Paenibacillus popilliae (formerly
Bacillus popilliae), Pantoea agglomerans, Pasteuria penetrans
(formerly Bacillus penetrates), Pasteuria usgae, Pectobacterium
carotovorum (formerly Erwinia carotovora), Pseudomonas aeruginosa,
Pseudomonas aureofaciens, Pseudomonas cepacia (formerly known as
Burkholderia cepacia), Pseudomonas chlororaphis, Pseudomonas
fluorescens, Pseudomonas proradix, Pseudomonas putida, Pseudomonas
syringae, Serratia entomophila, Serratia marcescens, Streptomyces
colombiensis, Streptomyces galbus, Streptomyces goshikiensis,
Streptomyces griseoviridis, Streptomyces lavendulae, Streptomyces
prasinus, Streptomyces saraceticus, Streptomyces venezuelae,
Xanthomonas campestris, Xenorhabdus luminescens, Xenorhabdus
nematophila, Rhodococcus globerulus AQ719 (NRRL Accession No.
B-21663), Bacillus sp. AQ175 (ATCC Accession No. 55608), Bacillus
sp. AQ 177 (ATCC Accession No. 55609), Bacillus sp. AQ178 (ATCC
Accession NO. 53522), and Streptomyces sp. strain NRRL Accession
No. 13-30145. In some cases the bacterium may be Azotobacter
chroococcum, Methanosarcina barker, Klesiella pneumoniae,
Azotobacter vinelandii, Rhodobacter spharoides, Rhodobacter
capsulatus, Rhodobcter palustris, Rhodosporillum rubrum, Rhizobium
leguminosarum or Rhizobium etli.
[0233] In some cases the bacterium may be a species of Clostridium,
for example Clostridium pasteurianum, Clostridium beijerinckii,
Clostridium perfringens, Clostridium tetani, Clostridium
acetobutylicum.
[0234] In some cases, bacteria used with the methods and
compositions of the present disclosure may be cyanobacteria.
Examples of cyanobacterial genuses include Anabaena (for example
Anagaena sp. PCC7120), Nostoc (for example Nostoc punctiforme), or
Synechocystis (for example Synechocystis sp. PCC6803).
[0235] In some cases, bacteria used with the methods and
compositions of the present disclosure may belong to the phylum
Chlorobi, for example Chlorobium tepidum.
[0236] In some cases, microbes used with the methods and
compositions of the present disclosure may comprise a gene
homologous to a known NifH gene. Sequences of known NifH genes may
be found in, for example, the Zehr lab NifH database,
(https://wwwzehr.pmc.aese.edu/nifH_Database_Public/, Apr. 4, 2014),
or the Buckley lab NifH database
(http://www.css.cornell.edu/faculty/buckley/nifh.htm, and Gaby,
John Christian, and Daniel H. Buckley. "A comprehensive aligned
nifH gene database: a multipurpose tool for studies of
nitrogen-fixing bacteria." Database 2014 (2014): bau001.). In some
cases, microbes used with the methods and compositions of the
present disclosure may comprise a sequence which encodes a
polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%,
98%, 99% or more than 99% sequence identity to a sequence from the
Zehr lab NifH database,
(https://wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, Apr. 4, 2014).
In some cases, microbes used with the methods and compositions of
the present disclosure may comprise a sequence which encodes a
polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%,
98%, 99% or more than 99% sequence identity to a sequence from the
Buckley lab NifH database, (Gaby, John Christian, and Daniel H.
Buckley. "A comprehensive aligned nifH gene database: a
multipurpose tool for studies of nitrogen-fixing bacteria."
Database 2014 (2014): bau001.).
[0237] Microbes useful in the methods and compositions disclosed
herein can be obtained by extracting microbes from surfaces or
tissues of native plants; grinding seeds to isolate microbes;
planting seeds in diverse soil samples and recovering microbes from
tissues; or inoculating plants with exogenous microbes and
determining which microbes appear in plant tissues. Non-limiting
examples of plant tissues include a seed, seedling, leaf, cutting,
plant, bulb or tuber. In some cases, bacteria are isolated from a
seed. The parameters for processing samples may be varied to
isolate different types of associative microbes, such as
rhizospheric, epiphytes, or endophytes. Bacteria may also be
sourced from a repository, such as environmental strain
collections, instead of initially isolating from a first plant. The
microbes can be genotyped and phenotyped, via sequencing the
genomes of isolated microbes; profiling the composition of
communities in planta; characterizing the transcriptomic
functionality of communities or isolated microbes; or screening
microbial features using selective or phenotypic media (e.g.,
nitrogen fixation or phosphate solubilization phenotypes). Selected
candidate strains or populations can be obtained via sequence data;
phenotype data; plant data (e.g., genome, phenotype, and/or yield
data); soil data (e.g., pH, N/P/K content, and/or bulk soil biotic
communities); or any combination of these.
[0238] The bacteria and methods of producing bacteria described
herein may apply to bacteria able to self-propagate efficiently on
the leaf surface, root surface, or inside plant tissues without
inducing a damaging plant defense reaction, or bacteria that are
resistant to plant defense responses. The bacteria described herein
may be isolated by culturing a plant tissue extract or leaf surface
wash in a medium with no added nitrogen. However, the bacteria may
be unculturable, that is, not known to be culturable or difficult
to culture using standard methods known in the art. The bacteria
described herein may be an endophyte or an epiphyte or a bacterium
inhabiting the plant rhizosphere (rhizospheric bacteria). The
bacteria obtained after repeating the steps of introducing genetic
variation, exposure to a plurality of plants, and isolating
bacteria from plants with an improved trait one or more times (e.g.
1, 2, 3, 4, 5, 10, 15, 25, or more times) may be endophytic,
epiphytic, or rhizospheric. Endophytes are organisms that enter the
interior of plants without causing disease symptoms or eliciting
the formation of symbiotic structures, and are of agronomic
interest because they can enhance plant growth and improve the
nutrition of plants (e.g., through nitrogen fixation). The bacteria
can be a seed-borne endophyte. Seed-borne endophytes include
bacteria associated with or derived from the seed of a grass or
plant, such as a seed-borne bacterial endophyte found in mature,
dry, undamaged (e.g., no cracks, visible fungal infection, or
prematurely germinated) seeds. The seed-borne bacterial endophyte
can be associated with or derived from the surface of the seed;
alternatively, or in addition, it can be associated with or derived
from the interior seed compartment (e.g., of a surface-sterilized
seed). In some cases, a seed-borne bacterial endophyte is capable
of replicating within the plant tissue, for example, the interior
of the seed. Also, in some cases, the seed-borne bacterial
endophyte is capable of surviving desiccation.
[0239] The bacterial isolated according to methods of the
disclosure, or used in methods or compositions of the disclosure,
can comprise a plurality of different bacterial taxa in
combination. By way of example, the bacteria may include
Proteobacteria (such as Pseudomonas, Enterobacter,
Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea,
Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter,
Duganella, Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas),
Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus,
Mycoplasma, and Acetabacterium), and Actinobacteria (such as
Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium). The
bacteria used in methods and compositions of this disclosure may
include nitrogen fixing bacterial consortia of two or more species.
In some cases, one or more bacterial species of the bacterial
consortia may be capable of fixing nitrogen. In some cases, one or
more species of the bacterial consortia may facilitate or enhance
the ability of other bacteria to fix nitrogen. The bacteria which
fix nitrogen and the bacteria which enhance the ability of other
bacteria to fix nitrogen may be the same or different. In some
examples, a bacterial strain may be able to fix nitrogen when in
combination with a different bacterial strain, or in a certain
bacterial consortia, but may be unable to fix nitrogen in a
monoculture. Examples of bacterial genuses which may be found in a
nitrogen fixing bacterial consortia include, but are not limited
to, Herbaspirillum, Azospirillum, Enterobacter, and Bacillus.
[0240] Bacteria that can be produced by the methods disclosed
herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp.,
and Sinorhizobium sp. In some cases, the bacteria may be selected
from the group consisting of: Azotobacter vinelandii,
Bradyrhizobium japonicum, Klebsiella pneumoniae, and Sinorhizobium
meliloti. In some cases, the bacteria may be of the genus
Enterobacter or Rahnella. In some cases, the bacteria may be of the
genus Frankia, or Clostridium. Examples of bacteria of the genus
Clostridium include, but are not limited to, Clostridium
acetobutilicum, Clostridium pasteurianum, Clostridium beijerinckii,
Clostridium perfringens, and Clostridium tetani. In some cases, the
bacteria may be of the genus Paenibacillus, for example
Paenibacillus azotofixans, Paenibacillus borealis, Paenibacillus
durus, Paenibacillus macerans, Paenibacillus polymyxa,
Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus
campinasensis, Paenibacillus chibensis, Paenibacillus
glucanolyticus, Paenibacillus illinoisensis, Paenibacillus larvae
subsp. Larvae, Paenibacillus larvae subsp. Pulvifaciens,
Paenibacillus lautus, Paenibacillus macerans, Paenibacillus
macquariensis, Paenibacillus macquariensis, Paenibacillus pabuli,
Paenibacillus peoriae, or Paenibacillus polymyxa.
[0241] In some examples, bacteria isolated according to methods of
the disclosure can be a member of one or more of the following
taxa: Achronobacter, Acidithiobacillus, Acidovorax, Acidovoraz,
Acinetobacter, Actinoplanes, Adlercreutzia, Aerococcus, Aeromonas,
Afipia, Agromyces, Ancylobacter, Arthrobacter, Atopostipes,
Azospirillum, Bacillus, Bdellovibrio, Beijerinckia, Bosea,
Bradyrhizobium, Brevibacillus, Brevundimonas, Burkholderia,
Candidatus Haloredivivus, Caulobacter, Cellulomonas, Cellvibrio,
Chryseobacterium, Citrobacter, Clostridium, Coraliomargarita,
Corynebacterium, Cupriavidus, Curtobacterium, Curvibacter,
Deinococcus, Delftia, Desemzia, Devosia, Dokdonella, Dyella,
Enhydrobacter, Enterobacter, Enterococcus, Erwinia, Escherichia,
Escherichia/Shigella, Exiguobacterium, Ferroglobus, Filimonas,
Finegoldia, Flavisolibacter, Flavobacterium, Frigoribacterium,
Gluconacetobacter, Hafnia, Halobaculum, Halomonas, Halosimplex,
Herbaspirillum, Hymenobacter, Klebsiella, Kocuria, Kosakonia,
Lactobacillus, Leclercia, Lentzea, Luteibacter, Luteimonas,
Massilia, Mesorhizobium, Methylobacterium, Microbacterium,
Micrococcus, Microvirga, Mycobacterium, Neisseria, Nocardia,
Oceanibaculum, Ochrobactrum, Okibacterium, Oligotropha, Oryzihumus,
Oxalophagus, Paenibacillus, Panteoa, Pantoea, Pelomonas,
Perlucidibaca, Plantibacter, Polynucleobacter, Propionibacterium,
Propioniciclava, Pseudoclavibacter, Pseudomonas, Pseudonocardia,
Pseudoxanthomonas, Psychrobacter, Rahnella, Ralstonia,
Rheinheimera, Rhizobium, Rhodococcus, Rhodopseudomonas, Roseateles,
Ruminococcus, Sebaldella, Sediminibacillus, Sediminibacterium,
Serratia, Shigella, Shigella, Sinorhizobium, Sinosporangium,
Sphingobacterium, Sphingomonas, Sphingopyxis, Sphingosinicella,
Staphylococcus, 25 Stenotrophomonas, Strenotrophomonas,
Streptococcus, Streptomyces, Stygiolobus, Sulfurisphaera,
Tatumella, Tepidimonas, Thermomonas, Thiobacillus, Variovorax,
WPS-2 genera incertae sedis, Xanthomonas, and Zimmermanella.
[0242] In some cases, a bacterial species selected from at least
one of the following genera are utilized: Enterobacter, Klebsiella,
Kosakonia, and Rahnella. In some cases, a combination of bacterial
species from the following genera are utilized: Enterobacter,
Klebsiella, Kosakonia, and Rahnella. In some cases, the species
utilized can be one or more of: Enterobacter sacchari, Klebsiella
variicola, Kosakonia sacchari, and Rahnella aquatilis.
[0243] In some cases, a Gram positive microbe may have a
Molybdenum-Iron nitrogenase system comprising: nifH, nifD, nifK,
nifB, nifE, nifN, nifX, hesA, nifV, nifU, nifW, nifU, nifS, nifl1,
and nifl2. In some cases, a Gram positive microbe may have a
vanadium nitrogenase system comprising: vnfDG, vnfK, vnfE, vnfN,
vupC, vupB, vupA, vnfV, vnfR1, vnfH, vnfR2, vnfA, (transcriptional
regulator). In some cases, a Gram positive microbe may have an
iron-only nitrogenase system comprising: anfK, anfG, anfD, anfH,
anfA (transcriptional regulator). In some cases a Gram positive
microbe may have a nitrogenase system comprising glnB, and glnK
(nitrogen signaling proteins). Some examples of enzymes involved in
nitrogen metabolism in Gram positive microbes include glnA
(glutamine synthetase), gdh (glutamate dehydrogenase), bdh
(3-hydroxybutyrate dehydrogenase), glutaminase, gltAB/gltB/gltS
(glutamate synthase), asnA/asnB (aspartate-ammonia
ligase/asparagine synthetase), and ansA/ansZ (asparaginase). Some
examples of proteins involved in nitrogen transport in Gram
positive microbes include amtB (ammonium transporter), glnK
(regulator of ammonium transport), glnPHQ/glnQHMP (ATP-dependent
glutamine/glutamate transporters), glnT/alsT/yrbD/yflA
(glutamine-like proton symport transporters), and
gltP/gltT/yhcl/nqt (glutamate-like proton symport
transporters).
[0244] Examples of Gram positive microbes which may be of
particular interest include Paenibacillus polymixa, Paenibacillus
riograndensis, Paenibacillus sp., Frankia sp., Heliobacterium sp.,
Heliobacterium chlorum, Heliobacillus sp., Heliophilum sp.,
Heliorestis sp., Clostridium acetobutylicum, Clostridium sp.,
Mycobacterium flaum, Mycobacterium sp., Arthrobacter sp., Agromyces
sp., Corynebacterium autitrophicum, Corynebacterium sp.,
Micromonspora sp., Propionibacteria sp., Streptomyces sp., and
Microbacterium sp.
[0245] Some examples of genetic alterations which may be make in
Gram positive microbes include: deleting glnR to remove negative
regulation of BNF in the presence of environmental nitrogen,
inserting different promoters directly upstream of the nif cluster
to eliminate regulation by GlnR in response to environmental
nitrogen, mutating glnA to reduce the rate of ammonium assimilation
by the GS-GOGAT pathway, deleting amtB to reduce uptake of ammonium
from the media, mutating glnA so it is constitutively in the
feedback-inhibited (FBI-GS) state, to reduce ammonium assimilation
by the GS-GOGAT pathway.
[0246] In some cases, glnR is the main regulator of N metabolism
and fixation in Paenibacillus species. In some cases, the genome of
a Paenibacillus species may not contain a gene to produce glnR. In
some cases, the genome of a Paenibacillus species may not contain a
gene to produce glnE or glnD. In some cases, the genome of a
Paenibacillus species may contain a gene to produce glnB or glnK.
For example Paenibacillus sp. WLY78 doesn't contain a gene for
glnB, or its homologs found in the archaeon Methanococcus
maripaludis, nifI1 and nifI2. In some cases, the genomes of
Paenibacillus species may be variable. For example, Paenibacillus
polymixa E681 lacks glnK and gdh, has several nitrogen compound
transporters, but only amtB appears to be controlled by GlnR. In
another example, Paenibacillus sp. JDR2 has glnK, gdh and most
other central nitrogen metabolism genes, has many fewer nitrogen
compound transporters, but does have glnPHQ controlled by GlnR.
Paenibacillus riograndensis SBR5 contains a standard glnRA operon,
an fdx gene, a main nil operon, a secondary nif operon, and an anf
operon (encoding iron-only nitrogenase). Putative glnR/tnrA sites
were found upstream of each of these operons. GlnR may regulate all
of the above operons, except the anf operon. GlnR may bind to each
of these regulatory sequences as a dimer.
[0247] Paenibacillus N-fixing strains may fall into two subgroups:
Subgroup I, which contains only a minimal nif gene cluster and
subgroup II, which contains a minimal cluster, plus an
uncharacterized gene between nifX and hesA, and often other
clusters duplicating some of the nif genes, such as nifH, nifHDK,
nifBEN, or clusters encoding vanadaium nitrogenase (vnf) or
iron-only nitrogenase (anf) genes.
[0248] In some cases, the genome of a Paenibacillus species may not
contain a gene to produce g/n/3 or &K. In some cases, the
genome of a Paenibacillus species may contain a minimal nif cluster
with 9 genes transcribed from a sigma-70 promoter. In some cases a
Paenibacillus nif cluster may be negatively regulated by nitrogen
or oxygen. In some cases, the genome of a Paenibacillus species may
not contain a gene to produce sigma-54. For example, Paenibacillus
sp. WLY78 does not contain a gene for sigma-54. In some cases, a
nif cluster may be regulated by glnR, and/or TnrA. In some cases,
activity of a nif cluster may be altered by altering activity of
glnR, and/or TnrA.
[0249] In Bacilli, glutamine synthetase (GS) is feedback-inhibited
by high concentrations of intracellular glutamine, causing a shift
in confirmation (referred to as FBI-GS). Nif clusters contain
distinct binding sites for the regulators GlnR and TnrA in several
Bacilli species. GlnR binds and represses gene expression in the
presence of excess intracellular glutamine and AMP. A role of GlnR
may be to prevent the influx and intracellular production of
glutamine and ammonium under conditions of high nitrogen
availability. TnrA may bind and/or activate (or repress) gene
expression in the presence of limiting intracellular glutamine,
and/or in the presence of FBI-GS. In some cases the activity of a
Bacilli nif cluster may be altered by altering the activity of
GlnR.
[0250] Feedback-inhibited glutamine synthetase (FBI-GS) may bind
GlnR and stabilize binding of GlnR to recognition sequences.
Several bacterial species have a GlnR/TnrA binding site upstream of
the nif cluster. Altering the binding of FBI-GS and GlnR may alter
the activity of the nif pathway.
Sources of Microbes
[0251] The bacteria (or any microbe according to the disclosure)
may be obtained from any general terrestrial environment, including
its soils, plants, fungi, animals (including invertebrates) and
other biota, including the sediments, water and biota of lakes and
rivers; from the marine environment, its biota and sediments (for
example, sea water, marine muds, marine plants, marine
invertebrates (for example, sponges), marine vertebrates (for
example, fish)); the terrestrial and marine geosphere (regolith and
rock, for example, crushed subterranean rocks, sand and clays); the
cryosphere and its meltwater; the atmosphere (for example, filtered
aerial dusts, cloud and rain droplets); urban, industrial and other
man-made environments (for example, accumulated organic and mineral
matter on concrete, roadside gutters, roof surfaces, and road
surfaces).
[0252] The plants from which the bacteria (or any microbe according
to the disclosure) are obtained may be a plant having one or more
desirable traits, for example a plant which naturally grows in a
particular environment or under certain conditions of interest. By
way of example, a certain plant may naturally grow in sandy soil or
sand of high salinity, or under extreme temperatures, or with
little water, or it may be resistant to certain pests or disease
present in the environment, and it may be desirable for a
commercial crop to be grown in such conditions, particularly if
they are, for example, the only conditions available in a
particular geographic location. By way of further example, the
bacteria may be collected from commercial crops gown in such
environments, or more specifically from individual crop plants best
displaying a trait of interest amongst a crop grown in any specific
environment: for example the fastest-growing plants amongst a crop
grown in saline-limiting soils, or the least damaged plants in
crops exposed to severe insect damage or disease epidemic, or
plants having desired quantities of certain metabolites and other
compounds, including fiber content, oil content, and the like, or
plants displaying desirable colors, taste or smell. The bacteria
may be collected from a plant of interest or any material occurring
in the environment of interest, including fungi and other animal
and plant biota, soil, water, sediments, and other elements of the
environment as referred to previously.
[0253] The bacteria (or any microbe according to the disclosure)
may be isolated from plant tissue. This isolation can occur from
any appropriate tissue in the plant, including for example root,
stem and leaves, and plant reproductive tissues. By way of example,
conventional methods for isolation from plants typically include
the sterile excision of the plant material of interest (e.g. root
or stem lengths, leaves), surface sterilization with an appropriate
solution (e.g. 2% sodium hypochlorite), after which the plant
material is placed on nutrient medium for microbial growth.
Alternatively, the surface-sterilized plant material can be crushed
in a sterile liquid (usually water) and the liquid suspension,
including small pieces of the crushed plant material spread over
the surface of a suitable solid agar medium, or media, which may or
may not be selective (e.g. contain only phytic acid as a source of
phosphorus). This approach is especially useful for bacteria which
form isolated colonies and can be picked off individually to
separate plates of nutrient medium, and further purified to a
single species by well-known methods. Alternatively, the plant root
or foliage samples may not be surface sterilized but only washed
gently thus including surface-dwelling epiphytic microorganisms in
the isolation process, or the epiphytic microbes can be isolated
separately, by imprinting and lifting off pieces of plant roots,
stem or leaves onto the surface of an agar medium and then
isolating individual colonies as above. This approach is especially
useful for bacteria, for example. Alternatively, the roots may be
processed without washing off small quantities of soil attached to
the roots, thus including microbes that colonize the plant
rhizosphere. Otherwise, soil adhering to the roots can be removed,
diluted and spread out onto agar of suitable selective and
non-selective media to isolate individual colonies of rhizospheric
bacteria.
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedures
[0254] The microbial deposits of the present disclosure were made
under the provisions of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purpose of
Patent Procedure (Budapest Treaty).
[0255] Applicants state that pursuant to 37 C.F.R. .sctn.
1.808(a)(2) "all restrictions imposed by the depositor on the
availability to the public of the deposited material will be
irrevocably removed upon the granting of the patent." This
statement is subject to paragraph (b) of this section (i.e. 37
C.F.R. .sctn. 1.808(b)).
[0256] Biologically pure cultures of Rahnella aquatilis and
Enterobacter sacchari were deposited on Jul. 14, 2015 with the
American Type Culture Collection (ATCC; an International Depositary
Authority), 10801 University Blvd., Manassas, Va. 20110, USA, and
assigned ATTC Patent Deposit Designation numbers PTA-122293 and
PTA-122294, respectively. The applicable deposit information is
found below in Table A.
[0257] The Enterobacter sacchari has now been reclassified as
Kosakonia sacchari, the name for the organism may be used
interchangeably throughout the manuscript.
[0258] Many microbes of the present disclosure are derived from two
wild-type strains, as depicted in FIG. 18 and FIG. 19. Strain CI006
is a bacterial species previously classified in the genus
Enterobacter (see aforementioned reclassification into Kosakonia),
and FIG. 19 identities the lineage of the mutants that have been
derived from CI006. Strain CI019 is a bacterial species classified
in the genus Rahnella, and FIG. 19 identifies the lineage of the
mutants that have been derived from CI019. With regard to FIG. 18
and FIG. 19, it is noted that strains comprising CM in the name are
mutants of the strains depicted immediately to the left of said CM
strain. The deposit information for the CI006 Kosakonia wild type
(WT) and CI019 Rahnella WT are found in the below Table A.
[0259] Some microorganisms described in this application were
deposited on Jan. 6, 2017 or Aug. 11, 2017 with the Bigelow
National Center for Marine Algae and Microbiota (NCMA), located at
60 Bigelow Drive, East Boothbay, Me. 04544, USA. As aforementioned,
all deposits were made under the terms of the Budapest Treaty on
the International Recognition of the Deposit of Microorganisms for
the Purposes of Patent Procedure. The Bigelow National Center for
Marine Algae and Microbiota accession numbers and dates of deposit
for the aforementioned Budapest Treaty deposits are provided in
Table A.
[0260] Biologically pure cultures of Kosakonia sacchari (WT),
Rahnella aquatilis (WT), and a variant Kosakonia sacchari strain
were deposited on Jan. 6, 2017 with the Bigelow National Center for
Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive,
East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit
Designation numbers 201701001, 201701003, and 201701002,
respectively. The applicable deposit information is found below in
Table A.
[0261] Biologically pure cultures of variant Kosakonia sacchari
strains were deposited on Aug. 11, 2017 with the Bigelow National
Center for Marine Algae and Microbiota (NCMA), located at 60
Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA
Patent Deposit Designation numbers 201708004, 201708003, and
201708002, respectively. The applicable deposit information is
found below in Table A.
[0262] A biologically pure culture of Klebsiella variicola (WV was
deposited on Aug. 11, 2017 with the Bigelow National Center for
Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive,
East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit
Designation number 201708001. Biologically pure cultures of two
Klebsiella variicola variants were deposited on Dec. 20, 2017 with
the Bigelow National Center for Marine Algae and Microbiota (NCMA),
located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA, and
assigned NCMA Patent Deposit Designation numbers 201712001 and
201712002, respectively. The applicable deposit information is
found below in Table A.
TABLE-US-00001 TABLE A Microorganisms Deposited under the Budapest
Treaty Pivot Strain Designation (some strains Depos- have multiple
Accession Date of itory designations) Taxonomy Number Deposit ATCC
Rahnella aquatilis PTA-122293 Jul. 14, 2015 ATCC Enterobacter
PTA-122294 Jul. 14, sacchari 2015 (taxonomically re- classified
after de- posit as Kosakonia sacchari) NCMA CI006, Kosakonia
sacchari 201701001 Jan. 6, PBC6.1, 6 (WT) 2017 NCMA CI019, 19
Rahnella aquatilis 201701003 Jan. 6, (WT) 2017 NCMA CM029,
Kosakonia sacchari 201701002 Jan. 6, 6-412 2017 NCMA 6-403
Kosakonia sacchari 201708004 Aug. 11, CM037 2017 NCMA 6-404,
Kosakonia sacchari 201708003 Aug. 11, CM38, 2017 PBC6.38 NCMA
CM094, Kosakonia sacchari 201708002 Aug. 11, 6-881, 2017 PBC6.94
NCMA CI137, 137, Klebsiella variicola 201708001 Aug. 11, PB137 (WT)
2017 NCMA 137-1034 Klebsiella variicola 201712001 Dec. 20, 2017
NCMA 137-1036 Klebsiella variicola 201712002 Dec. 20, 2017
Isolated and Biologically Pure Microorganisms
[0263] The present disclosure, in certain embodiments, provides
isolated and biologically pure microorganisms that have
applications, infer alia, in agriculture. The disclosed
microorganisms can be utilized in their isolated and biologically
pure states, as well as being formulated into compositions (see
below section for exemplary composition descriptions). Furthermore,
the disclosure provides microbial compositions containing at least
two members of the disclosed isolated and biologically pure
microorganisms, as well as methods of utilizing said microbial
compositions. Furthermore, the disclosure provides for methods of
modulating nitrogen fixation in plants via the utilization of the
disclosed isolated and biologically pure microbes.
[0264] In some aspects, the isolated and biologically pure
microorganisms of the disclosure are those from Table A. In other
aspects, the isolated and biologically pure microorganisms of the
disclosure are derived from a microorganism of Table A. For
example, a strain, child, mutant, or derivative, of a microorganism
from Table A are provided herein. The disclosure contemplates all
possible combinations of microbes listed in Table A, said
combinations sometimes forming a microbial consortia. The microbes
from Table A, either individually or in any combination, can be
combined with any plant, active (synthetic, organic, etc.),
adjuvant, carrier, supplement, or biological, mentioned in the
disclosure.
Compositions
[0265] Compositions comprising bacteria or bacterial populations
produced according to methods described herein and/or having
characteristics as described herein can be in the form of a liquid,
a foam, or a dry product. Compositions comprising bacteria or
bacterial populations produced according to methods described
herein and/or having characteristics as described herein may also
be used to improve plant traits. In some examples, a composition
comprising bacterial populations may be in the form of a dry
powder, a slurry of powder and water, or a flowable seed treatment.
The compositions comprising bacterial populations may be coated on
a surface of a seed, and may be in liquid form.
[0266] The composition can be fabricated in bioreactors such as
continuous stirred tank reactors, batch reactors, and on the farm.
In some examples, compositions can be stored in a container, such
as a jug or in mini bulk. In some examples, compositions may be
stored within an object selected from the group consisting of a
bottle, jar, ampule, package, vessel, bag, box, bin, envelope,
carton, container, silo, shipping container, truck bed, and/or
case.
[0267] Compositions may also be used to improve plant traits. In
some examples, one or more compositions may be coated onto a seed.
In some examples, one or more compositions may be coated onto a
seedling. In some examples, one or more compositions may be coated
onto a surface of a seed. In some examples, one or more
compositions may be coated as a layer above a surface of a seed. In
some examples, a composition that is coated onto a seed may be in
liquid form, in dry product form, in foam form, in a form of a
slurry of powder and water, or in a flowable seed treatment. In
some examples, one or more compositions may be applied to a seed
and/or seedling by spraying, immersing, coating, encapsulating,
and/or dusting the seed and/or seedling with the one or more
compositions. In some examples, multiple bacteria or bacterial
populations can be coated onto a seed and/or a seedling of the
plant. In some examples, at least two, at least three, at least
four, at least five, at least six, at least seven, at least eight,
at least nine, at least ten, or more than ten bacteria of a
bacterial combination can be selected from one of the following
genera: Acidovorax, Agrobacterium, Bacillus, Burkholderia,
Chryseobacterium, Curtobacterium, Enterobacter, Escherichia,
Methylobacterium, Paenibacillus, Pantoea, Pseudomonas, Ralstonia,
Saccharibacillus, Sphingomonas, and Stenotrophomonas.
[0268] In some examples, at least two, at least three, at least
four, at least five, at least six, at least seven, at least eight,
at least nine, at least ten, or more than ten bacteria and
bacterial populations of an endophytic combination are selected
from one of the following families: Bacillaceae, Burkholderiaceae,
Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae,
Methylobacteriaceae, Microbacteriaceae, Paenibacillileae,
Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae,
Cladosporiaceae, Gnomoniaceae, Incertae sedis, Lasiosphaeriaceae,
Netriaceae, and Pleosporaceae.
[0269] In some examples, at least two, at least three, at least
four, at least five, at least six, at least seven, at least eight,
at least night, at least ten, or more than ten bacteria and
bacterial populations of an endophytic combination are selected
from one of the following families: Bacillaceae, Burkholderiaceae,
Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae,
Methylobacteriaceae, Microbacteriaceae, Paenibacillileae,
Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae,
Cladosporiaceae, Gnomoniaceae, Incertae sedis, Lasiosphaeriaceae,
Netriaceae, Pleosporaceae.
[0270] Examples of compositions may include seed coatings for
commercially important agricultural crops, for example, sorghum,
canola, tomato, strawberry, barley, rice, maize, and wheat.
Examples of compositions can also include seed coatings for corn,
soybean, canola, sorghum, potato, rice, vegetables, cereals, and
oilseeds. Seeds as provided herein can be genetically modified
organisms (GMO), non-GMO, organic, or conventional. In some
examples, compositions may be sprayed on the plant aerial parts, or
applied to the roots by inserting into furrows in which the plant
seeds are planted, watering to the soil, or dipping the roots in a
suspension of the composition. In some examples, compositions may
be dehydrated in a suitable manner that maintains cell viability
and the ability to artificially inoculate and colonize host plants.
The bacterial species may be present in compositions at a
concentration of between 10.sup.8 to 10.sup.10 CFU/ml. In some
examples, compositions may be supplemented with trace metal ions,
such as molybdenum ions, iron ions, manganese ions, or combinations
of these ions. The concentration of ions in examples of
compositions as described herein may between about 0.1 mM and about
50 mM. Some examples of compositions may also be formulated with a
carrier, such as beta-glucan, carboxylmethyl cellulose (CMC),
bacterial extracellular polymeric substance (EPS), sugar, animal
milk, or other suitable carriers. In some examples, peat or
planting materials can be used as a carrier, or biopolymers in
which a composition is entrapped in the biopolymer can be used as a
carrier. The compositions comprising the bacterial populations
described herein can improve plant traits, such as promoting plant
growth, maintaining high chlorophyll content in leaves, increasing
fruit or seed numbers, and increasing fruit or seed unit
weight.
[0271] The compositions comprising the bacterial populations
described herein may be coated onto the surface of a seed. As such,
compositions comprising a seed coated with one or more bacteria
described herein are also contemplated. The seed coating can be
formed by mixing the bacterial population with a porous, chemically
inert granular carrier. Alternatively, the compositions may be
inserted directly into the furrows into which the seed is planted
or sprayed onto the plant leaves or applied by dipping the roots
into a suspension of the composition. An effective amount of the
composition can be used to populate the sub-soil region adjacent to
the roots of the plant with viable bacterial growth, or populate
the leaves of the plant with viable bacterial growth. In general,
an effective amount is an amount sufficient to result in plants
with improved traits (e.g. a desired level of nitrogen
fixation).
[0272] Bacterial compositions described herein can be formulated
using an agriculturally acceptable carrier. The formulation useful
for these embodiments may include at least one member selected from
the group consisting of a tackifier, a microbial stabilizer, a
fungicide, an antibacterial agent, a preservative, a stabilizer, a
surfactant, an anti-complex agent, an herbicide, a nematicide, an
insecticide, a plant growth regulator, a fertilizer, a rodenticide,
a dessicant, a bactericide, a nutrient, or any combination thereof.
In some examples, compositions may be shelf-stable. For example,
any of the compositions described herein can include an
agriculturally acceptable carrier (e.g., one or more of a
fertilizer such as a non-naturally occurring fertilizer, an
adhesion agent such as a non-naturally occurring adhesion agent,
and a pesticide such as a non-naturally occurring pesticide). A
non-naturally occurring adhesion agent can be, for example, a
polymer, copolymer, or synthetic wax. For example, any of the
coated seeds, seedlings, or plants described herein can contain
such an agriculturally acceptable carrier in the seed coating. In
any of the compositions or methods described herein, an
agriculturally acceptable carrier can be or can include a
non-naturally occurring compound (e.g., a non-naturally occurring
fertilizer, a non-naturally occurring adhesion agent such as a
polymer, copolymer, or synthetic wax, or a non-naturally occurring
pesticide). Non-limiting examples of agriculturally acceptable
carriers are described below. Additional examples of agriculturally
acceptable carriers are known in the art.
[0273] In some cases, bacteria are mixed with an agriculturally
acceptable carrier. The carrier can be a solid carrier or liquid
carrier, and in various forms including microspheres, powders,
emulsions and the like. The carrier may be any one or more of a
number of carriers that confer a variety of properties, such as
increased stability, wettability, or dispersability. Wetting agents
such as natural or synthetic surfactants, which can be nonionic or
ionic surfactants, or a combination thereof can be included in the
composition. Water-in-oil emulsions can also be used to formulate a
composition that includes the isolated bacteria (see, for example,
U.S. Pat. No. 7,485,451). Suitable formulations that may be
prepared include wettable powders, granules, gels, agar strips or
pellets, thickeners, and the like, microencapsulated particles, and
the like, liquids such as aqueous flowables, aqueous suspensions,
water-in-oil emulsions, etc. The formulation may include grain or
legume products, for example, ground grain or beans, broth or flour
derived from grain or beans, starch, sugar, or oil.
[0274] In some embodiments, the agricultural carrier may be soil or
a plant growth medium. Other agricultural carriers that may be used
include water, fertilizers, plant-based oils, humectants, or
combinations thereof. Alternatively, the agricultural carrier may
be a solid, such as diatomaceous earth, loam, silica, alginate,
clay, bentonite, vermiculite, seed cases, other plant and animal
products, or combinations, including granules, pellets, or
suspensions. Mixtures of any of the aforementioned ingredients are
also contemplated as carriers, such as but not limited to, pesta
(flour and kaolin clay), agar or flour-based pellets in loam, sand,
or clay, etc. Formulations may include food sources for the
bacteria, such as barley, rice, or other biological materials such
as seed, plant parts, sugar cane bagasse, hulls or stalks from
grain processing, ground plant material or wood from building site
refuse, sawdust or small fibers from recycling of paper, fabric, or
wood.
[0275] For example, a fertilizer can be used to help promote the
growth or provide nutrients to a seed, seedling, or plant.
Non-limiting examples of fertilizers include nitrogen, phosphorous,
potassium, calcium, sulfur, magnesium, boron, chloride, manganese,
iron, zinc, copper, molybdenum, and selenium (or a salt thereof).
Additional examples of fertilizers include one or more amino acids,
salts, carbohydrates, vitamins, glucose, NaCl, yeast extract,
NH.sub.4H.sub.2PO.sub.4, (NH.sub.4).sub.2SO.sub.4, glycerol,
valine, L-leucine, lactic acid, propionic acid, succinic acid,
malic acid, citric acid, KH tartrate, xylose, lyxose, and lecithin.
In one embodiment, the formulation can include a tackifier or
adherent (referred to as an adhesive agent) to help bind other
active agents to a substance (e.g., a surface of a seed). Such
agents are useful for combining bacteria with carriers that can
contain other compounds (e.g., control agents that are not
biologic), to yield a coating composition. Such compositions help
create coatings around the plant or seed to maintain contact
between the microbe and other agents with the plant or plant part.
In one embodiment, adhesives are selected from the group consisting
of: alginate, gums, starches, lecithins, formononetin, polyvinyl
alcohol, alkali formononetinate, hesperetin, polyvinyl acetate,
cephalins, Gum Arabic, Xanthan Gum, Mineral Oil, Polyethylene
Glycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, Methyl
Cellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate,
Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate,
Gellan Gum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum
Ghatti, and polyoxyethylene-polyoxybutylene block copolymers.
[0276] In some embodiments, the adhesives can be, e.g. a wax such
as carnauba wax, beeswax, Chinese wax, shellac wax, spermaceti wax,
candelilla wax, castor wax, ouricury wax, and rice bran wax, a
polysaccharide (e.g., starch, dextrins, maltodextrins, alginate,
and chitosans), a fat, oil, a protein (e.g., gelatin and zeins),
gum arables, and shellacs. Adhesive agents can be non-naturally
occurring compounds, e.g., polymers, copolymers, and waxes. For
example, non-limiting examples of polymers that can be used as an
adhesive agent include: polyvinyl acetates, polyvinyl acetate
copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl
alcohols, polyvinyl alcohol copolymers, celluloses (e.g.,
ethylcelluloses, methylcelluloses, hydroxymethylcelluloses,
hydroxypropylcelluloses, and carboxymethylcelluloses),
polyvinylpyrolidones, vinyl chloride, vinylidene chloride
copolymers, calcium lignosulfonates, acrylic copolymers,
polyvinylacrylates, polyethylene oxide, acylamide polymers and
copolymers, polyhydroxyethyl acrylate, methyl acrylamide monomers,
and polychloroprene.
[0277] In some examples, one or more of the adhesion agents,
anti-fungal agents, growth regulation agents, and pesticides (e.g.,
insecticide) are non-naturally occurring compounds (e.g., in any
combination). Additional examples of agriculturally acceptable
carriers include dispersants (e.g., polyvinylpyrrolidone/vinyl
acetate PVPIVA S-630), surfactants, binders, and filler agents.
[0278] The formulation can also contain a surfactant. Non-limiting
examples of surfactants include nitrogen-surfactant blends such as
Prefer 28 (Cenex), Surf-N(US), Inhance (Brandt), P-28 (Wilfann) and
Patrol (Helena); esterified seed oils include Sun-It II (AmCy), MSO
(UAP), Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); and
organo-silicone surfactants include Silwet L77 (UAP), Silikin
(Terra), Dyne-Amit (Helena), Kinetic (Helena), Sylgard 309
(Wilbur-Ellis) and Century (Precision). In one embodiment, the
surfactant is present at a concentration of between 0.01% v/v to
10% v/v. In another embodiment, the surfactant is present at a
concentration of between 0.1% v/v to 1% v/v.
[0279] In certain cases, the formulation includes a microbial
stabilizer. Such an agent can include a desiccant, which can
include any compound or mixture of compounds that can be classified
as a desiccant regardless of whether the compound or compounds are
used in such concentrations that they in fact have a desiccating
effect on a liquid inoculant. Such desiccants are ideally
compatible with the bacterial population used, and should promote
the ability of the microbial population to survive application on
the seeds and to survive desiccation. Examples of suitable
desiccants include one or more of trehalose, sucrose, glycerol, and
Methylene glycol. Other suitable desiccants include, but are not
limited to, non reducing sugars and sugar alcohols (e.g., mannitol
or sorbitol). The amount of desiccant introduced into the
formulation can range from about 5% to about 50% by weight/volume,
for example, between about 10% to about 40%, between about 15% to
about 35%, or between about 20% to about 30%. In some cases, it is
advantageous for the formulation to contain agents such as a
fungicide, an antibacterial agent, an herbicide, a nematicide, an
insecticide, a plant growth regulator, a rodenticide, bactericide,
or a nutrient. In some examples, agents may include protectants
that provide protection against seed surface-borne pathogens. In
some examples, protectants may provide some level of control of
soil-borne pathogens. In some examples, protectants may be
effective predominantly on a seed surface.
[0280] In some examples, a fungicide may include a compound or
agent, whether chemical or biological, that can inhibit the growth
of a fungus or kill a fungus. In some examples, a fungicide may
include compounds that may be fungistatic or fungicidal. In some
examples, fungicide can be a protectant, or agents that are
effective predominantly on the seed surface, providing protection
against seed surface-borne pathogens and providing some level of
control of soil-borne pathogens. Non-limiting examples of
protectant fungicides include captan, maneb, thiram, or
fludioxonil.
[0281] In some examples, fungicide can be a systemic fungicide,
which can be absorbed into the emerging seedling and inhibit or
kill the fungus inside host plant tissues. Systemic fungicides used
for seed treatment include, but are not limited to the following:
azoxystrobin, carboxin, mefenoxam, metalaxyl, thiabendazole,
trifloxystrobin, and various triazole fungicides, including
difenoconazole, ipconazole, tebuconazole, and triticonazole.
Mefenoxam and metalaxyl are primarily used to target the water mold
fungi Pythium and Phytophthora. Some fungicides are preferred over
others, depending on the plant species, either because of subtle
differences in sensitivity of the pathogenic fungal species, or
because of the differences in the fungicide distribution or
sensitivity of the plants. In some examples, fungicide can be a
biological control agent, such as a bacterium or fungus. Such
organisms may be parasitic to the pathogenic fungi, or secrete
toxins or other substances which can kill or otherwise prevent the
growth of fungi. Any type of fungicide, particularly ones that are
commonly used on plants, can be used as a control agent in a seed
composition.
[0282] In some examples, the seed coating composition comprises a
control agent which has antibacterial properties. In one
embodiment, the control agent with antibacterial properties is
selected from the compounds described herein elsewhere. In another
embodiment, the compound is Streptomycin, oxytetracycline, oxolinic
acid, or gentamicin. Other examples of antibacterial compounds
which can be used as part of a seed coating composition include
those based on dichlorophene and benzylalcohol hemi formal
(Proxel.RTM. from ICI or Acticide.RTM. RS from Thor Chemie and
Kathon.RTM. MK 25 from Rohm & Haas) and isothiazolinone
derivatives such as alkylisothiazolinones and benzisothiazolinones
(Acticide.RTM. NIBS from Thor Chemie).
[0283] In some examples, growth regulator is selected from the
group consisting of: Abscisic acid, amidochlor, ancymidol,
6-benzylaminopurine, brassinolide, butralin, chlormequat
(chlormequat chloride), choline chloride, cyclanilide, daminozide,
dikegulac, dimethipin, 2,6-dimethylpuridine, ethephon, flumetralin,
flurprimidol, fluthiacet, forchlorfenuron, gibberellic acid,
inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide,
mepiquat (mepiquat chloride), naphthaleneacetic acid,
N-6-benzyladenine, paclobutrazol, prohexadione phosphorotrithioate,
2,3,5-tri-iodobenzoic acid, trinexapac-ethyl and uniconazole.
Additional non-limiting examples of growth regulators include
brassinosteroids, cytokinines (e.g., kinetin and zeatin), auxins
(e.g., indolylacetic acid and indolylacetyl aspartate), flavonoids
and isoflavanoids (e.g., formononetin and diosmetin), phytoaixins
(e.g., glyceolline), and phytoalexin-inducing oligosaccharides
(e.g., pectin, chitin, chitosan, polygalacuronic acid, and
oligogalacturonic acid), and gibellerins. Such agents are ideally
compatible with the agricultural seed or seedling onto which the
formulation is applied (e.g., it should not be deleterious to the
growth or health of the plant). Furthermore, the agent is ideally
one which does not cause safety concerns for human, animal or
industrial use (e.g., no safety issues, or the compound is
sufficiently labile that the commodity plant product derived from
the plant contains negligible amounts of the compound).
[0284] Some examples of nematode-antagonistic biocontrol agents
include ARF18; 30 Arthrobotrys spp.; Chaetomium spp.;
Cylindrocarpon spp.; Exophilia spp.; Fusarium spp.; Gliocladium
spp.; Hirsutella spp.; Lecanicillium spp.; Monacrosporium spp.;
Myrothecium spp.; Neocosmospora spp.; Paecilomyces spp.; Pochonia
spp.; Stagonospora spp.; vesicular-arbuscular mycorrhizal fungi,
Burkholderia spp.; Pasteuria spp., Brevibacillus spp.; Pseudomonas
spp.; and Rhizobacteria. Particularly preferred
nematode-antagonistic biocontrol agents include ARF18, Arthrobotrys
oligospora, Arthrobotrys dactyloides. Chaetomium globosum,
Cylindrocarpon heteronema, Exophilia jeanselmei, Exophilia
pisciphila, Fusarium aspergilus, Fusarium solani, Gliocladium
catenulatum, Gliocladium roseum, Gliocladium vixens, Hirsutella
rhossiliensis, Hirsutella minnesotensis, Lecanicillium lecanii,
Monacrosporium drechsleri, Monacrosporium gephyropagum, Myrotehcium
verrucaria, Neocosmospora vasinfecta, Paecilomyces lilacinus,
Pochonia chlamydosporia, Stagonospora heteroderae, Stagonospora
phaseoli, vesicular-arbuscular mycorrhizal fungi, Burkholderia
cepacia, Pasteuria penetrans, Pasteuria thornei, Pasteuria
nishizawae, Pasteuria ramosa, Pastrueia usage, Brevibacillus
laterosporus strain G4, Pseudomonas fluorescens and
Rhizobacteria.
[0285] Some examples of nutrients can be selected from the group
consisting of a nitrogen fertilizer including, but not limited to
Urea, Ammonium nitrate, Ammonium sulfate, Non-pressure nitrogen
solutions, Aqua ammonia, Anhydrous ammonia, Ammonium thiosulfate,
Sulfur-coated urea, Urea-formaldehydes, IBDU, Polymer-coated urea,
Calcium nitrate, Ureaform, and Methylene urea, phosphorous
fertilizers such as Diammonium phosphate, Monoammonium phosphate,
Ammonium polyphosphate, Concentrated superphosphate and Triple
superphosphate, and potassium fertilizers such as Potassium
chloride, Potassium sulfate, Potassium-magnesium sulfate, Potassium
nitrate. Such compositions can exist as free salts or ions within
the seed coat composition. Alternatively, nutrients/fertilizers can
be complexed or chelated to provide sustained release over
time.
[0286] Some examples of rodenticides may include selected from the
group of substances consisting of 2-isovalerylindan-1,3-dione,
4-(quinoxalin-2-ylamino) benzenesulfonamide, alpha-chlorohydrin,
aluminum phosphide, antu, arsenous oxide, barium carbonate,
bisthiosemi, brodifacoum, bromadiolone, bromethalin, calcium
cyanide, chloralose, chlorophacinone, cholecalciferol, coumachlor,
coumafuryl, coumatetralyl, crimidine, difenacoum, difethialone,
diphacinone, ergocalciferol, flocoumafen, fluoroacetamide,
flupropadine, flupropadine hydrochloride, hydrogen cyanide,
iodomethane, lindane, magnesium phosphide, methyl bromide,
norbormide, phosacetim, phosphine, phosphorus, pindone, potassium
arsenite, pyrinuron, scilliroside, sodium arsenite, sodium cyanide,
sodium fluoroacetate, strychnine, thallium sulfate, warfarin and
zinc phosphide.
[0287] In the liquid form, for example, solutions or suspensions,
bacterial populations can be mixed or suspended in water or in
aqueous solutions. Suitable liquid diluents or carriers include
water, aqueous solutions, petroleum distillates, or other liquid
carriers.
[0288] Solid compositions can be prepared by dispersing the
bacterial populations in and on an appropriately divided solid
carrier, such as peat, wheat, bran, vermiculite, clay, talc,
bentonite, diatomaceous earth, fuller's earth, pasteurized soil,
and the like. When such formulations are used as wettable powders,
biologically compatible dispersing agents such as non-ionic,
anionic, amphoteric, or cationic dispersing and emulsifying agents
can be used.
[0289] The solid carriers used upon formulation include, for
example, mineral carriers such as kaolin clay, pyrophyllite,
bentonite, montmorillonite, diatomaceous earth, acid white soil,
vermiculite, and pearlite, and inorganic salts such as ammonium
sulfate, ammonium phosphate, ammonium nitrate, urea, ammonium
chloride, and calcium carbonate. Also, organic fine powders such as
wheat flour, wheat bran, and rice bran may be used. The liquid
carriers include vegetable oils such as soybean oil and cottonseed
oil, glycerol, ethylene glycol, polyethylene glycol, propylene
glycol, polypropylene glycol, etc.
Application of Bacterial Populations on Crops
[0290] The composition of the bacteria or bacterial population
described herein can be applied in furrow, in talc, or as seed
treatment. The composition can be applied to a seed package in
bulk, mini bulk, in a bag, or in talc.
[0291] The planter can plant the treated seed and grows the crop
according to conventional ways, twin row, or ways that do not
require tilling. The seeds can be distributed using a control
hopper or an individual hopper. Seeds can also be distributed using
pressurized air or manually. Seed placement can be performed using
variable rate technologies. Additionally, application of the
bacteria or bacterial population described herein may be applied
using variable rate technologies. In some examples, the bacteria
can be applied to seeds of corn, soybean, canola, sorghum, potato,
rice, vegetables, cereals, pseudocereals, and oilseeds. Examples of
cereals may include barley, Fonio, oats, palmer's grass, rye, pearl
millet, sorghum, spelt, teff, triticale, and wheat. Examples of
pseudocereals may include breadnut, buckwheat, cattail, chia, flax,
grain amaranth, hanza, quinoa, and sesame. In some examples, seeds
can be genetically modified organisms (GMO), non-GMO, organic or
conventional.
[0292] Additives such as micro-fertilizer, PGR, herbicide,
insecticide, and fungicide can be used additionally to treat the
crops. Examples of additives include crop protectants such as
insecticides, nematicides, fungicide, enhancement agents such as
colorants, polymers, pelleting, priming, and disinfectants, and
other agents such as inoculant, PGR, softener, and micronutrients.
PGRs can be natural or synthetic plant hormones that affect root
growth, flowering, or stem elongation. PGRs can include auxins,
gibberellins, cytokinins, ethylene, and abscisic acid (ABA).
[0293] The composition can be applied in furrow in combination with
liquid fertilizer. In some examples, the liquid fertilizer may be
held in tanks. NPK fertilizers contain macronutrients of sodium,
phosphorous, and potassium.
[0294] The composition may improve plant traits, such as promoting
plant growth, maintaining high chlorophyll content in leaves,
increasing fruit or seed numbers, and increasing fruit or seed unit
weight. Methods of the present disclosure may be employed to
introduce or improve one or more of a variety of desirable traits.
Examples of traits that may introduced or improved include: root
biomass, root length, height, shoot length, leaf number, water use
efficiency, overall biomass, yield, fruit size, grain size,
photosynthesis rate, tolerance to drought, heat tolerance, salt
tolerance, tolerance to low nitrogen stress, nitrogen use
efficiency, resistance to nematode stress, resistance to a fungal
pathogen, resistance to a bacterial pathogen, resistance to a viral
pathogen, level of a metabolite, modulation in level of a
metabolite, proteome expression. The desirable traits, including
height, overall biomass, root and/or shoot biomass, seed
germination, seedling survival, photosynthetic efficiency,
transpiration rate, seed/fruit number or mass, plant grain or fruit
yield, leaf chlorophyll content, photosynthetic rate, root length,
or any combination thereof, can be used to measure growth, and
compared with the growth rate of reference agricultural plants
(e.g., plants without the introduced and/or improved traits) grown
under identical conditions. In some examples, the desirable traits,
including height, overall biomass, root and/or shoot biomass, seed
germination, seedling survival, photosynthetic efficiency,
transpiration rate, seed/fruit number or mass, plant grain or fruit
yield, leaf chlorophyll content, photosynthetic rate, root length,
or any combination thereof, can be used to measure growth, and
compared with the growth rate of reference agricultural plants
(e.g., plants without the introduced and/or improved traits) grown
under similar conditions.
[0295] An agronomic trait to a host plant may include, but is not
limited to, the following: altered oil content, altered protein
content, altered seed carbohydrate composition, altered seed oil
composition, and altered seed protein composition, chemical
tolerance, cold tolerance, delayed senescence, disease resistance,
drought tolerance, ear weight, growth improvement, health
enhancement, heat tolerance, herbicide tolerance, herbivore
resistance improved nitrogen fixation, improved nitrogen
utilization, improved root architecture, improved water use
efficiency, increased biomass, increased root length, increased
seed weight, increased shoot length, increased yield, increased
yield under water-limited conditions, kernel mass, kernel moisture
content, metal tolerance, number of ears, number of kernels per
ear, number of pods, nutrition enhancement, pathogen resistance,
pest resistance, photosynthetic capability improvement, salinity
tolerance, stay-green, vigor improvement, increased dry weight of
mature seeds, increased fresh weight of mature seeds, increased
number of mature seeds per plant, increased chlorophyll content,
increased number of pods per plant, increased length of pods per
plant, reduced number of wilted leaves per plant, reduced number of
severely wilted leaves per plant, and increased number of
non-wilted leaves per plant, a detectable modulation in the level
of a metabolite, a detectable modulation in the level of a
transcript, and a detectable modulation in the proteome, compared
to an isoline plant grown from a seed without said seed treatment
formulation.
[0296] In some cases, plants are inoculated with bacteria or
bacterial populations that are isolated from the same species of
plant as the plant element of the inoculated plant. For example, an
bacteria or bacterial population that is normally found in one
variety of Zea mays (corn) is associated with a plant element of a
plant of another variety of Zea mays that in its natural state
lacks said bacteria and bacterial populations. In one embodiment,
the bacteria and bacterial populations is derived from a plant of a
related species of plant as the plant element of the inoculated
plant. For example, an bacteria and bacterial populations that is
normally found in Zea diploperennis Iltis et al., (diploperennial
teosinte) is applied to a Zea mays (corn), or vice versa. In some
cases, plants are inoculated with bacteria and bacterial
populations that are heterologous to the plant element of the
inoculated plant. In one embodiment, the bacteria and bacterial
populations is derived from a plant of another species. For
example, an bacteria and bacterial populations that is normally
found in dicots is applied to a monocot plant (e.g., inoculating
corn with a soybean-derived bacteria and bacterial populations), or
vice versa. In other cases, the bacteria and bacterial populations
to be inoculated onto a plant is derived from a related species of
the plant that is being inoculated. In one embodiment, the bacteria
and bacterial populations is derived from a related taxon, for
example, from a related species. The plant of another species can
be an agricultural plant. In another embodiment, the bacteria and
bacterial populations is part of a designed composition inoculated
into any host plant element.
[0297] In some examples, the bacteria or bacterial population is
exogenous wherein the bacteria and bacterial population is isolated
from a different plant than the inoculated plant. For example, in
one embodiment, the bacteria or bacterial population can be
isolated from a different plant of the same species as the
inoculated plant. In some cases, the bacteria or bacterial
population can be isolated from a species related to the inoculated
plant.
[0298] In some examples, the bacteria and bacterial populations
described herein are capable of moving from one tissue type to
another. For example, the present invention's detection and
isolation of bacteria and bacterial populations within the mature
tissues of plants after coating on the exterior of a seed
demonstrates their ability to move from seed exterior into the
vegetative tissues of a maturing plant. Therefore, in one
embodiment, the population of bacteria and bacterial populations is
capable of moving from the seed exterior into the vegetative
tissues of a plant. In one embodiment, the bacteria and bacterial
populations that is coated onto the seed of a plant is capable,
upon germination of the seed into a vegetative state, of localizing
to a different tissue of the plant. For example, bacteria and
bacterial populations can be capable of localizing to any one of
the tissues in the plant, including: the root, adventitious root,
seminal 5 root, root hair, shoot, leaf, flower, bud, tassel,
meristem, pollen, pistil, ovaries, stamen, fruit, stolon, rhizome,
nodule, tuber, trichome, guard cells, hydathode, petal, sepal,
glume, rachis, vascular cambium, phloem, and xylem. In one
embodiment, the bacteria and bacterial populations is capable of
localizing to the root and/or the root hair of the plant. In
another embodiment, the bacteria and bacterial populations is
capable of localizing to the photosynthetic tissues, for example,
leaves and shoots of the plant. In other cases, the bacteria and
bacterial populations is localized to the vascular tissues of the
plant, for example, in the xylem and phloem. In still another
embodiment, the bacteria and bacterial populations is capable of
localizing to the reproductive tissues (flower, pollen, pistil,
ovaries, stamen, fruit) of the plant. In another embodiment, the
bacteria and bacterial populations is capable of localizing to the
root, shoots, leaves and reproductive tissues of the plant. In
still another embodiment, the bacteria and bacterial populations
colonizes a fruit or seed tissue of the plant. In still another
embodiment, the bacteria and bacterial populations is able to
colonize the plant such that it is present in the surface of the
plant (i.e., its presence is detectably present on the plant
exterior, or the episphere of the plant). In still other
embodiments, the bacteria and bacterial populations is capable of
localizing to substantially all, or all, tissues of the plant. In
certain embodiments, the bacteria and bacterial populations is not
localized to the root of a plant. In other cases, the bacteria and
bacterial populations is not localized to the photosynthetic
tissues of the plant.
[0299] The effectiveness of the compositions can also be assessed
by measuring the relative maturity of the crop or the crop heating
unit (CHU). For example, the bacterial population can be applied to
corn, and corn growth can be assessed according to the relative
maturity of the corn kernel or the time at which the corn kernel is
at maximum weight. The crop heating unit (CHU) can also be used to
predict the maturation of the corn crop. The CHU determines the
amount of heat accumulation by measuring the daily maximum
temperatures on crop growth.
[0300] In examples, bacterial may localize to any one of the
tissues in the plant, including: the root, adventitious root,
seminal root, root hair, shoot, leaf, flower, bud tassel, meristem,
pollen, pistil, ovaries, stamen, fruit, stolon, rhizome, nodule,
tuber, trichome, guard cells, hydathode, petal, sepal, glume,
rachis, vascular cambium, phloem, and xylem. In another embodiment,
the bacteria or bacterial population is capable of localizing to
the photosynthetic tissues, for example, leaves and shoots of the
plant. In other cases, the bacteria and bacterial populations is
localized to the vascular tissues of the plant, for example, in the
xylem and phloem. In another embodiment, the bacteria or bacterial
population is capable of localizing to reproductive tissues
(flower, pollen, pistil, ovaries, stamen, or fruit) of the plant.
In another embodiment, the bacteria and bacterial populations is
capable of localizing to the root, shoots, leaves and reproductive
tissues of the plant. In another embodiment, the bacteria or
bacterial population colonizes a fruit or seed tissue of the plant.
In still another embodiment, the bacteria or bacterial population
is able to colonize the plant such that it is present in the
surface of the plant. In another embodiment, the bacteria or
bacterial population is capable of localizing to substantially all,
or all, tissues of the plant. In certain embodiments, the bacteria
or bacterial population is not localized to the root of a plant. In
other cases, the bacteria and bacterial populations is not
localized to the photosynthetic tissues of the plant.
[0301] The effectiveness of the bacterial compositions applied to
crops can be assessed by measuring various features of crop growth
including, but not limited to, planting rate, seeding vigor, root
strength, drought tolerance, plant height, dry down, and test
weight.
Plant Species
[0302] The methods and bacteria described herein are suitable for
any of a variety of plants, such as plants in the genera Hordeum,
Oryza, Zea, and Triticeae. Other non-limiting examples of suitable
plants include mosses, lichens, and algae. In some cases, the
plants have economic, social and/or environmental value, such as
food crops, fiber crops, oil crops, plants in the forestry or pulp
and paper industries, feedstock for biofuel production and/or
ornamental plants. In some examples, plants may be used to produce
economically valuable products such as a grain, a flour, a starch,
a syrup, a meal, an oil, a film, a packaging, a nutraceutical
product, a pulp, an animal feed, a fish fodder, a bulk material for
industrial chemicals, a cereal product, a processed human-food
product, a sugar, an alcohol, and/or a protein. Non-limiting
examples of crop plants include maize, rice, wheat, barley,
sorghum, millet, oats, rye triticale, buckwheat, sweet corn, sugar
cane, onions, tomatoes, strawberries, and asparagus.
[0303] In some examples, plants that may be obtained or improved
using the methods and composition disclosed herein may include
plants that are important or interesting for agriculture,
horticulture, biomass for the production of biofuel molecules and
other chemicals, and/or forestry. Some examples of these plants may
include pineapple, banana, coconut, lily, grasspeas and grass; and
dicotyledonous plants, such as, for example, peas, alfalfa,
tomatillo, melon, chickpea, chicory, clover, kale, lentil, soybean,
tobacco, potato, sweet potato, radish, cabbage, rape, apple trees,
grape, cotton, sunflower, Chale cress, canola, citrus (including
orange, mandarin, kumquat, lemon, lime, grapefruit, tangerine,
tangelo, citron, and pomelo), pepper, bean, lettuce, Panicum
virgatum (switch), Sorghum bicolor (sorghum, sudan), Miscanthus
giganteus (miscanthus), Saccharum sp. (energycane), Populus
balsamifera (poplar), Zea mays (corn), Glycine max (soybean),
Brassica napus (canola), Triticum aestivum (wheat), Gossypium
hirsutum (cotton), Oryza sativa (rice), Helianthus annuus
(sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet),
Pennisetum glaucum (pearl millet), Panicum spp. Sorghum spp.,
Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp.,
Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.
(eucalyptus), Triticosecale spp. (triticum-25 wheat X rye), Bamboo,
Carthamus tinctorius (safflower), Jatropha curcas (Jatropha),
Ricinus communis (castor), Elaeis guineensis (oil palm), Phoenix
dactylifera (date palm), Archontophoenix cunninghamiana (king
palm), Syagrus romanzoffiana (queen palm), Linum usitatissimum
(flax), Brassica juncea, Manihot esculenta (cassaya), Lycopersicon
esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca
(banana), Solanum tuberosum (potato), Brassica oleracea (broccoli,
cauliflower, brussel sprouts), Camellia sinensis (tea), Fragaria
ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica
(coffee), Vitis vinifera (grape), Ananas comosus (pineapple),
Capsicum annum (hot & sweet pepper), Allium cepa (onion),
Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima
(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),
Citrullus lanatus (watermelon), Abelmoschus esculentus (okra),
Solanum melongena (eggplant), Papaver somniferum (opium poppy),
Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia
annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus,
Vinca rosea, Cinchona officinalis, Coichicum autumnale, Veratrum
californica, Digitalis lanata, Digitalis purpurea, Dioscorea 5
spp., Andrographis paniculata, Atropa belladonna, Datura stomonium,
Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp.,
Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium
serratum (Huperzia serrata), Lycopodium spp., Rauwolfia serpentina,
Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula
officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum
parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber),
Mentha spicata (mint), Mentha piperita (mint), Bixa orellana,
Alstroemeria spp., Rosa spp. (rose), Dianthus caryophyllus
(carnation), Petunia spp. (petunia), Poinsettia pulcherrima
(poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin),
Uniola paniculata (oats), Hordeum vulgare (barley), and Lolium spp.
(rye).
[0304] In some examples, a monocotyledonous plant may be used.
Monocotyledonous plants belong to the orders of the Alismatales,
Arales, Arecales, Bromeliales, Commelinales, Cyclanthales,
Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales,
Najadales, Orchidales, Pandanales, Poales, Restionales,
Triuridales, Typhales, and Zingiberales. Plants belonging to the
class of the Gymnospermae are Cycadales, Ginkgoales, Gnetales, and
Pinales. In some examples, the monocotyledonous plant can be
selected from the group consisting of a maize, rice, wheat, barley,
and sugarcane.
[0305] In some examples, a dicotyledonous plant may be used,
including those belonging to the orders of the Aristochiales,
Asterales, Batales, Campanulales, Capparales, Caryophyllales,
Casuarinales, Celastrales, Cornales, Diapensales, Dilleniales,
Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales,
Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales,
Middles, Juglandales, Lamiales, Laurales, Lecythidales,
Leitneriales, Magniolales, Malvales, Myricales, Myrtales,
Nymphaeales, Papeverales, Piperales, Plantaginales, Plumbaginales,
Podostemales, Polemoniales, Polygalales, Polygonales, Primulales,
Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales,
Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae,
Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales,
and Violates. In some examples, the dicotyledonous plant can be
selected from the group consisting of cotton, soybean, pepper, and
tomato.
[0306] In some cases, the plant to be improved is not readily
amenable to experimental conditions. For example, a crop plant may
take too long to grow enough to practically assess an improved
trait serially over multiple iterations. Accordingly, a first plant
from which bacteria are initially isolated, and/or the plurality of
plants to which genetically manipulated bacteria are applied may be
a model plant, such as a plant more amenable to evaluation under
desired conditions. Non-limiting examples of model plants include
Setaria, Brachypodium, and Arabidopsis. Ability of bacteria
isolated according to a method of the disclosure using a model
plant may then be applied to a plant of another type (e.g. a crop
plant) to confirm conferral of the improved trait.
[0307] Traits that may be improved by the methods disclosed herein
include any observable characteristic of the plant, including, for
example, growth rate, height, weight, color, taste, smell, changes
in the production of one or more compounds by the plant (including
for example, metabolites, proteins, drugs, carbohydrates, oils, and
any other compounds). Selecting plants based on genotypic
information is also envisaged (for example, including the pattern
of plant gene expression in response to the bacteria, or
identifying the presence of genetic markers, such as those
associated with increased nitrogen fixation). Plants may also be
selected based on the absence, suppression or inhibition of a
certain feature or trait (such as an undesirable feature or trait)
as opposed to the presence of a certain feature or trait (such as a
desirable feature or trait).
Concentrations and Rates of Application of Agricultural
Compositions
[0308] As aforementioned, the agricultural compositions of the
present disclosure, which comprise a taught microbe, can be applied
to plants in a multitude of ways. In two particular aspects, the
disclosure contemplates an in-furrow treatment or a seed
treatment
[0309] For seed treatment embodiments, the microbes of the
disclosure can be present on the seed in a variety of
concentrations. For example, the microbes can be found in a seed
treatment at a cfu concentration, per seed of: 1.times.10.sup.1,
1.times.10.sup.2, 1.times.10.sup.3, 1.times.10.sup.4,
1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.17,
1.times.10.sup.8, 1.times.10.sup.9, 1.times.10.sup.10, or more. In
particular aspects, the seed treatment compositions comprise about
1.times.10.sup.4 to about 1.times.10.sup.8 cfu per seed. In other
particular aspects, the seed treatment compositions comprise about
1.times.10.sup.5 to about 1.times.10.sup.7 cfu per seed. In other
aspects, the seed treatment compositions comprise about
1.times.10.sup.6 cfu per seed.
[0310] In the United States, about 10% of corn acreage is planted
at a seed density of above about 36,000 seeds per acre; 1/3 of the
corn acreage is planted at a seed density of between about 33,000
to 36,000 seeds per acre; 1/3 of the corn acreage is planted at a
seed density of between about 30,000 to 33,000 seeds per acre, and
the remainder of the acreage is variable. See, "Corn Seeding Rate
Considerations," written by Steve Butzen, available at:
https://www.pioneer.com/home/site/us/agronomy/librarykorn-seeding-rate-co-
nsiderations/
[0311] Table B below utilizes various cfu concentrations per seed
in a contemplated seed treatment embodiment (rows across) and
various seed acreage planting densities (1.sup.st column: 15K-41K)
to calculate the total amount of cfu per acre, which would be
utilized in various agricultural scenarios (i.e. seed treatment
concentration per seed.times.seed density planted per acre). Thus,
if one were to utilize a seed treatment with 1.times.10.sup.6 cfu
per seed and plant 30,000 seeds per acre, then the total cfu
content per acre would be 3.times.10.sup.10 (i.e.
30K*1.times.10.sup.6).
TABLE-US-00002 TABLE B Total CFU Per Acre Calculation for Seed
Treatment Embodiments Corn Population (i.e, seeds per acre)
1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08
1.00E+09 15,000 1.50E+06 1.50E+07 1.50E+08 1.50E+09 1.50E+10
1.50E+11 1.50E+12 1.50E+13 16,000 1.60E+06 1.60E+07 1.60E+08
1.60E+09 1.60E+10 1.60E+11 1.60E+12 1.60E+13 17,000 1.70E+06
1.70E+07 1.70E+08 1.70E+09 1.70E+10 1.70E+11 1.70E+12 1.70E+13
18,000 1.80E+06 1.80E+07 1.80E+08 1.80E+09 1.80E+10 1.80E+11
1.80E+12 1.80E+13 19,000 1.90E+06 1.90E+07 1.90E+08 1.90E+09
1.90E+10 1.90E+11 1.90E+12 1.90E+13 20,000 2.00E+06 2.00E+07
2.00E+08 2.00E+09 2.00E+10 2.00E+11 2.00E+12 2.00E+13 21,000
2.10E+06 2.10E+07 2.10E+08 2.10E+09 2.10E+10 2.10E+11 2.10E+12
2.10E+13 22,000 2.20E+06 2.20E+07 2.20E+08 2.20E+09 2.20E+10
2.20E+11 2.20E+12 2.20E+13 23,000 2.30E+06 2.30E+07 2.30E+08
2.30E+09 2.30E+10 2.30E+11 2.30E+12 2.30E+13 24,000 2.40E+06
2.40E+07 2.40E+08 2.40E+09 2.40E+10 2.40E+11 2.40E+12 2.40E+13
25,000 2.50E+06 2.50E+07 2.50E+08 2.50E+09 2.50E+10 2.50E+11
2.50E+12 2.50E+13 26,000 2.60E+06 2.60E+07 2.60E+08 2.60E+09
2.60E+10 2.60E+11 2.60E+12 2.60E+13 27,000 2.70E+06 2.70E+07
2.70E+08 2.70E+09 2.70E+10 2.70E+11 2.70E+12 2.70E+13 28,000
2.80E+06 2.80E+07 2.80E+08 2.80E+09 2.80E+10 2.80E+11 2.80E+12
2.80E+13 29,000 2.90E+06 2.90E+07 2.90E+08 2.90E+09 1.90E+10
2.90E+11 2.90E+12 2.90E+13 30,000 3.00E+06 3.00E+07 3.00E+08
3.00E+09 3.00E+10 3.00E+11 3.00E+12 3.00E+13 31,000 3.10E+06
3.10E+07 3.10E+08 3.10E+09 3.10E+10 3.10E+11 3.10E+12 3.10E+13
32,000 3.20E+06 3.20E+07 3.20E+08 3.20E+09 3.20E+10 3.20E+11
3.20E+12 3.20E+13 33,000 3.30E+06 3.30E+07 3.30E+08 3.30E+09
3.30E+10 3.30E+11 3.30E+12 3.30E+13 34,000 3.40E+06 3.40E+07
3.40E+08 3.40E+09 3.40E+10 3.40E+11 3.40E+12 3.40E+13 35,000
3.50E+06 3.50E+07 3.50E+08 3.50E+09 3.50E+10 3.50E+11 3.50E+12
3.50E+13 36,000 3.60E+06 3.60E+07 3.60E+08 3.60E+09 3.60E+10
3.60E+11 3.60E+12 3.60E+13 37,000 3.70E+06 3.70E+07 3.70E+08
3.70E+09 3.70E+10 3.70E+11 3.70E+12 3.70E+13 38,000 3.80E+06
3.80E+07 3.80E+08 3.80E409 3.80E+10 3.80E+11 3.80E+12 3.80E+13
39,000 3.90E+06 3.90E+07 3.90E+08 3.90E+09 3.90E+10 3.90E+11
3.90E+12 3.90E+13 40,000 4.00E+06 4.00E+07 4.00E+08 4.00E+09
4.00E+10 4.00E+11 4.00E+12 4.00E+13 41,000 4.10E+06 4.10E+07
4.10E+08 4.10E+09 4.10E+10 4.10E+11 4.10E+12 4.10E+13
[0312] For in-furrow embodiments, the microbes of the disclosure
can be applied at a cfu concentration per acre of:
1.times.10.sup.6, 3.20.times.10.sup.10, 1.60.times.10.sup.11,
3.20.times.10.sup.11, 8.0.times.10.sup.11, 1.6.times.10.sup.12,
3.20.times.10.sup.12, or more. Therefore, in aspects, the liquid
in-furrow compositions can be applied at a concentration of between
about 1.times.10.sup.6 to about 3.times.10.sup.12 cfu per acre.
[0313] In some aspects, the in-furrow compositions are contained in
a liquid formulation. In the liquid in-furrow embodiments, the
microbes can be present at a cfu concentration per milliliter of:
1.times.10.sup.1, 1.times.10.sup.2, 1.times.10.sup.3,
1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9,
1.times.10.sup.10, 1.times.10.sup.11, 1.times.10.sup.12,
1.times.10.sup.13, or more. In certain aspects, the liquid
in-furrow compositions comprise microbes at a concentration of
about 1.times.10.sup.6 to about 1.times.10.sup.11 cfu per
milliliter. In other aspects, the liquid in-furrow compositions
comprise microbes at a concentration of about 1.times.10.sup.7 to
about 1.times.10.sup.10 cfu per milliliter. In other aspects, the
liquid in-furrow compositions comprise microbes at a concentration
of about 1.times.10.sup.8 to about 1.times.10.sup.9 cfu per
milliliter. In other aspects, the liquid in-furrow compositions
comprise microbes at a concentration of up to about
1.times.10.sup.13 cfu per milliliter.
Examples
[0314] The examples provided herein describe methods of bacterial
isolation, bacterial and plant analysis, and plant trait
improvement. The examples are for illustrative purposes only and
are not to be construed as limiting in any way.
Example 1: Isolation of Microbes from Plant Tissue
[0315] Topsoil was obtained from various agricultural areas in
central California. Twenty soils with diverse texture
characteristics were collected, including heavy clay, peaty clay
loam, silty clay, and sandy loam. Seeds of various field corn,
sweet corn, heritage corn and tomato were planted into each soil,
as shown in Table 1.
TABLE-US-00003 TABLE 1 Crop Type and Varieties planted into soil
with diverse characteristics Crop Type Field Corn Sweet Corn
Heritage Corn Tomato Varieties Mo17 Ferry-Morse Victory Seeds
Ferry-Morse Roma `Golden Cross `Moseby Prolific` VF Bantam T-51`
B73 Ferry-Morse `Silver Victory Seeds `Reid's Stover Roma Queen
Hybrid` Yellow Dent` DKC 66- Ferry-Morse `Sugar Victory Seeds
Totally Tomatoes 40 Dots` `Hickory King` `Micro Tom Hybrid` DKC 67-
Heinz 1015 07 DKC 70- Heinz 2401 01 Heinz 3402 Heinz 5508 Heinz
5608 Heinz 8504
[0316] Plants were uprooted after 2-4 weeks of growth and excess
soil on root surfaces was removed with deionized water. Following
soil removal, plants were surface sterilized with bleach and rinsed
vigorously in sterile water. A cleaned, 1 cm section of root was
excised from the plant and placed in a phosphate buffered saline
solution containing 3 mm steel beads. A slurry was generated by
vigorous shaking of the solution with a Qiagen TissueLyser II.
[0317] The root and saline slurry was diluted and inoculated onto
various types of growth media to isolate rhizospheric, endophytic,
epiphytic, and other plant-associated microbes. R2A and Nfb agar
media were used to obtain single colonies, and semisolid Nfb media
slants were used to obtain populations of nitrogen fixing bacteria.
After 2-4 weeks incubation in semi-solid Nfb media slants,
microbial populations were collected and streaked to obtain single
colonies on R2A agar, as shown in FIG. 1A-B. Single colonies were
resuspended in a mixture of R2A and glycerol, subjected to PCR
analysis, and frozen at -80.degree. C. for later analysis.
Approximately 1,000 single colonies were obtained and designated
"isolated microbes."
[0318] Isolates were then subjected to a colony PCR screen to
detect the presence of the nifH gene in order to identify
diazotrophs. The previously-described primer set Ueda 19F/388R,
which has been shown to detect over 90% of diazotrophs in screens,
was used to probe the presence of the nif cluster in each isolate
(Ueda et al. 1995; J. Bacteriol. 177: 1414-1417). Single colonies
of purified isolates were picked, resuspended in PBS, and used as a
template for colony PCR, as shown in FIG. 2. Colonies of isolates
that gave positive PCR bands were re-streaked, and the colony PCR
and re-streaking process was repeated twice to prevent false
positive identification of diazotrophs. Purified isolates were then
designated "candidate microbes."
Example 2: Characterization of Isolated Microbes
Sequencing, Analysis and Phylogenetic Characterization
[0319] Sequencing of 16S rDNA with the 515f-806r printer set was
used to generate preliminary phylogenetic identities for isolated
and candidate microbes (see e.g. Vernon et al.; BMC Microbiol. 2002
Dec. 23; 2:39.). The microbes comprise diverse genera including:
Enterobacter, Burkholderia, Klebsiella, Bradyrhizobium, Rahnella,
Xanthomonas, Raoultella, Pantoea, Pseudomonas, Brevundimonas,
Agrobacterium, and Paenibacillus, as shown in Table 2.
TABLE-US-00004 TABLE 2 Diversity of microbes isolated from tomato
plants as determined by deep 16S rDNA sequencing. Genus Isolates
Achromobacter 7 Agrobacterium 117 Agromyces 1 Alicyclobacillus 1
Asticcacaulis 6 Bacillus 131 Bradyrhizobium 2 Brevibacillus 2
Burkholderia 2 Coulobacter 17 Chryseobacterium 42 Comamonas 1
Dyadobacter 2 Flavobacterium 46 Halomonas 3 Leptothrix 3 Lysobacter
2 Neisseria 13 Paenibacillus 1 Paenisporosarcina 3 Pantoea 14
Pedobacter 16 Pimelobacter 2 Pseudomonas 212 Rhizobium 4 Rhodoferax
1 Sphingobacterium 13 Sphingobium 23 Sphingomonas 3 Sphingopyxis 1
Stenotrophomonas 59 Streptococcus 3 Variovorax 37 Xylanimicrobium 1
unidentified 75
[0320] Subsequently, the genomes of 39 candidate microbes were
sequenced using Illumina Miseq platform. Genomic DNA from pure
cultures was extracted using the QIAmp DNA mini kit (QIAGEN), and
total DNA libraries for sequencing were prepared through a third
party vendor (SeqMatic, Hayward). Genome assembly was then carried
out via the AS pipeline (Tritt et al. 2012; PLoS One 7(9):e42304).
Genes were identified and annotated, and those related to
regulation and expression of nitrogen fixation were noted as
targets for mutagenesis.
Transcriptomic Profiling of Candidate Microbes
[0321] Transcriptomic profiling of strain 0010 was performed to
identify promoters that are active in the presence of environmental
nitrogen. Strain 0010 was cultured in a defined, nitrogen-free
media supplemented with 10 mM glutamine. Total RNA was extracted
from these cultures (QIAGEN RNeasy kit) and subjected to RNAseq
sequencing via Illumina HiSeq (SeqMatic, Fremont Calif.).
Sequencing reads were mapped to CI010 genome data using Geneious,
and highly expressed genes under control of proximal
transcriptional promoters were identified.
[0322] Tables 3A-C lists genes and their relative expression level
as measured through RNASeq sequencing of total RNA. Sequences of
the proximal promoters were recorded for use in mutagenesis of nif
pathways, nitrogen utilization related pathways, or other genes
with a desired expression level.
TABLE-US-00005 TABLE 3A Name Minimum Maximum Length Direction
murein lipoprotein CDS 2,929,898 2,930,134 237 forward membrane
protein CDS 5,217,517 5,217,843 327 forward zinc/cadmium-binding
3,479,979 3,480,626 648 forward protein CDS acyl carrier protein
CDS 4,563,344 4,563,580 237 reverse ompX CDS 4,251,002 4,251,514
513 forward DNA-binding protein HU- 375,156 375,428 273 forward
beta CDS sspA CDS 629,998 630,636 639 reverse tatE CDS 3,199,435
3,199,638 204 reverse LexA repressor CDS 1,850,457 1,851,065 609
forward hisS CDS <3999979 4,001,223 >1245 forward
TABLE-US-00006 TABLE 3B RNASeq_ RNASeq_ RNASeq_ RNASeq_
Differential nifL- nifL- WT- WT- Expression Differential Raw Raw
Raw Raw Absolute Expression Read Transcript Read Transcript Name
Confidence Ratio Count Count Count Count murein 1000 -1.8 12950.5
10078.9 5151.5 4106.8 lipoprotein CDS membrane 1000 -1.3 9522.5
5371.3 5400 3120 protein CDS zinc/cadmium- 3.3 1.1 6461 1839.1 5318
1550.6 binding protein CDS acyl carrier 25.6 1.6 1230.5 957.6
1473.5 1174.7 protein CDS ompX CDS 1.7 1.1 2042 734.2 1687.5 621.5
DNA-binding 6.9 -1.3 1305 881.7 725 501.8 protein HU- beta CDS sspA
CDS 0.2 1 654 188.8 504.5 149.2 tatE CDS 1.4 1.3 131 118.4 125
115.8 LexA 0.1 -1.1 248 75.1 164 50.9 repressor CDS hisS CDS 0 -1.1
467 69.2 325 49.3
TABLE-US-00007 TABLE 3C Prm (In Forward SEQ SEQ SEQ direction, -250
ID Expressed ID Neighbor ID Name to +10 region) NO: Sequence NO:
Sequence NO: murein GCCTCTCGGGGC 3 ATGAATCGTACT 13 ATGAAAAAGACC 23
lipoprotein GCTTTTTTTTATT AAACTGGTACTG AAAATTGTTTGC CDS
CCGGCACTAGCC GGCGCGGTAATC ACCATCGGTCCG GCTATTAATAAA CTGGGTTCTACTC
AAAACCGAATCC AATGCAAATCGG TGCTGGCTGGTT GAAGAGATGTTG AATTTACTATTTA
GCTCCAGCAATG ACCAAAATGCTG ACGCGAGATTAT CTAAAATCGATC GACGCGGGCATG
CTAAGATGAATC AGCTGTCTTCTGA AACGTTATGCGT CGATGGAAGCGC CGTTCAGACTCT
CTGAACTTCTCTC GCTGTTTTCACTC GAACGCTAAAGT ACGGTGACTATG GCCTTTTTAAAGT
TGACCAGCTGAG CGGAACACGGTC TACGTGATGATTT CAACGACGTGAA AGCGCATCCAGA
CGATGCTTCTTTG CGCAATGCGTTC ATCTGCGCAATG AGCGAACGATCA CGACGTTCAGGC
TGATGAGTAAAA AAAATAAGCGTA TGCTAAAGATGA CCGGTAAGAAAG TTCAGGTAAAAA
CGCAGCTCGCGC CGGCAATCCTGC AATATTCTCATCA TAACCAGCGTCT TGGACACCAAAG
CAAAAAAGTTTG GGACAACGCAGC GTCCGGAAATCC TGTAATACTTGTA TACTAAATACCG
GTACCATTAAGC ACGCT--- TAAGTAA TGGAAGGCGGCA ACATGGAGATTA
ACGACGTCTCCC ACTC TGAAAGCGGGCC AGACCTTCACCTT CACCACCGATAA
ATCCGTTGTCGGT AATAACGAAATC GTTGCGGTGACC TATGAAGGCTTC ACCAGCGACCTG
AGCGTTGGCAAC ACGGTACTGGTT GACGATGGTCTG ATCGGTATGGAA GTGACCGCTATC
GAAGGCAACAAA GTTGTTTGTAAA GTGCTGAACAAC GGCGACCTCGGC GAGAACAAAGGC
GTTAACCTGCCG GGCGTATCTATC GCGCTGCCGGCG CTGGCTGAAAAA GACAAACAGGAT
CTGATCTTCGGTT GCGAACAGGGCG TTGACTTTGTTGC GGCATCCTTTATC CGTAAGCGTTCT
GACGTTGTTGAA ATCCGTGAGCAC CTGAAAGCCCAC GGCGGCGAGAAG ATCCAGATCATC
TCCAAAATCGAA AACCAGGAAGGC CTGAACAACTTC GACGAAATCCTC GAAGCCTCTGAC
GGCATCATGGTA GCCCGTGGCGAC CTGGGCGTTGAA ATCCCGGTTGAA GAAGTTATCTTC
GCGCAGAAGATG ATGATCGAGAAA TGTATCCGCGCG CGTAAAGTCGTT ATCACCGCGACC
CAGATGCTGGAT TCCATGATCAAA AACCCGCGTCCG ACCCGTGCGGAA GCAGGCGACGTG
GCCAACGCCATC CTCGACGGCACC GACGCAGTTATG CTGTCCGGCGAA TCCGCGAAAGGT
AAATACCCGCTG GAAGCGGTCACC ATCATGGCGACC ATCTGCGAACGT ACCGACCGCGTC
ATGACCAGCCGT CTTGAGTACAAC AACGACAACCGT AAGCTGCGCATC ACCGAAGCGGTG
TGCCGCGGTGCG GTAGAAACGGCT GAAAAACTGGAA GCGCCGCTGATC GTTGTGGCAACC
CAGGGCGGTAAA TCCGCGCGCGCC GTACGTAAATAC TTCCCGGATGCC ACTATCCTGGCG
CTGACCACCAAC GAAACCACCGCG CGTCAGCTGGTG CTGAGCAAAGGC GTTGTGGCACAG
CTGGTTGAAGAT ATCTCCTCTACCG ATGCGTTCTACAT CCAGGGTAAAGA ACTGGCGCTGCA
GAGCGGTCTGGC GCGTAAAGGCGA CGTGGTTGTTATG GTTTCCGGCGCG TTAGTCCCGAGC
GGAACCACCAAT ACCGCTTCCGTG CACGTGCTGTAA membrane GGTTCACATAAA 4
ATGGCCAACCGA 14 ATGTATTTAAGA 24 protein CATAATTATCGC GCAAACCGCAAC
CCCGATGAGGTG CDS CACGGCGATAGC AACGTAGAAGAG GCGCGTGTTCTTG
CGTACGCTTTTTG AGCGCTGAAGAT AAAAAGCCGGCT CGTCACAACATC ATCCATAACGAT
TCACCATGGATG CATGGTGAAGCC GTCAGCCAATTA TTGTGACGCAAA GGCTTTTTCAAG
GCGGATACGCTG AAGCGTACGGCT AACACGCGCCAC GAAGAGGTGCTG ATCGCCGTGGCG
CTCATCGGGTCTT AAATCGTGGGGC ATAATTATGTTTA AATACATACTC AGCGACGCCAAA
TGTGAACCGTGA ATTCCTCATTATC GACGAAGCGGAG AGCTCGTATGGG TTTTACCGCACGT
GCCGCGCGCAAA GCGTACCGCGTT TAACCTTACCTTA AAAGCGCAGGCG AATTATTCATCCG
TTCATTAAAGGC CTGCTGAAAGAG GCTTTAAAAGAG AACGCTTTCGGA ACCCGCGCCCGG
CGCAGCACAACG ATATTCCATAAA CTTAACGGCAAC CTTGCGGAGCCC GGGCTATTTACA
AACCGCGTCCAG GCGTCGGATATC GCATAATTCAAA CAGGCGGCGTGC AAAACCTGCGAT
ATCTTGTCCTACA GACGCCATGGGC CATTATGAGCAG CTTATAGACTCA TGCGCTGACAGC
TTCCCGCTCTATT ATGGAATTAAGG TACGTGCGCGAC TAGCGCTGGGATG GA
AAACCGTGGCAA CTCAACACTCATT AGCGTCGGCGCC ATGGTATTCCAC GCAGCAGCCGTT
ACGGGTTCAGTT GGGGTATTTATT CGCGAATGGCGC GGCGTATTACTG TTGAGCGTTTTCT
AATTTACGTCGA GAGTGGCCTGTT TAA TGGCGAAACGCA GTATAGCTGA zinc/
GCGCGGAAAATC 5 ATGACCAAAAAG 15 ATGGATAGCGAC 25 cadmium-
GACGCATAGCGC ATTTCCGCCCTAG ATTAATCAGGTC binding ATTCTCAGAAGC
CGTTTGGCATTG ATTGATTCTTTTG protein CGGCCTGGTCTC GCATGGTAATGG
TTAAAGGCCCGG CDS GGTGGAAAAGCG CGAGCAGCCAGG CGGTCGTGGGAA
AATCTTTCCCACG CTTTTGCCCACGG AGATTCGCTTTTC ACCGCCGGGCCT TCACCATAGTCA
CACCGAGACCAG TTAACAAAAGAA TGGCCCGGCGCT GCCGGCTTCTGA TCAATGACCTGA
GACCGAAGCGGA GAATGCGCTATG TTAATGTCGCTAT ACAAAAGGCGAG CGTCGATTTTCCG
CCATTCTCTCTCC TGAAGGCATTTTT CGCCTCGAAATC GCGTAATGCGAT GCTGACCAGGAC
ATGCTTGCGGGT CTTTTTTCATCAT CTTAAAGGACAGG CAGCTTCACGAT ACCTAACAAACT
GCGCTGAGCGAC CCGGCGATTAAA GGCAGAGGGAAA TGGGAGGGGATC GCCGATCGCGCC
AGCCGCGCGGTT TGGCAGTCGGTT CAGCTCATGCCG TTTCTGCGAAGT AACCCCTATCTG
CACGATGTGCTG GTATTGTAAGAT CTGAACGGGGAT TATATTCCCGCTG TTGTTTGATATGT
TTAGATCCGGTTC GCGGATGGAATG TATATCGTAACA TGGAGCAGAAGG ACCCGCAATGGC
TATTATTGCAAA CCAAAAAGGCCG TGGCGCCCTCCA CAT GTAAAAGCGTGG
CTCTGCTCACTAT CGGAATATCGGG CTTATTTGGTAAA AATATTATAAGA CAGCAGCTGGAA
AGGGCTACGCTA TTCGTCCTGCGCC CCGATGTCGACC ACTGGGACGGCA AGATTGGTATCG
GCGCGCTTAACG AGGATAACGTCA TGCTGGATAAAC TGGAGTTTCACG AGCAGGTTCCGC
TCGGGAAAACCG GCCGCGGTCCCC TCAACGCCTGTA GGGTCGGCTCTTT AGTACAGCTATT
TCTGCTGCAGGC CCGGTTACAAAA GCTGAATGAAAT TTCTGACCTACGC GCAGATGCAGCC
ATCCGGTAAAAA GCGGGAGCAGCA AGGCGTGCGCTA CACGGCCCGCTT CCTGTTCGAATG
TATTGTCACCAG CCAGCAGGCGGA CCTGCTCAGCCA TTCAAAAGCGCC CTGTGCCGATCT
GAAGTTTGTTCA GCTGGGCAGCCA GTTTAGCGATCA GGTACAAACCTC CACCATCGCGCC
ATCGCGCAGCCA ACGCAAGTCCCA GGCGCTTTTTGA GCATTTCCACATC AGCGATTCGTAA
TTTATGGGCAAT GCATATTGACGC GAGTCCCAGGAA CCACTTTGCCGA GCGCTGCTGAAA
CCCGTTAACCCG GAGATGGATAAC GGAGTCGGTGGC TGGCCAACCTAC GCAGGCGTTTTA
TATCCTTATGCGC CCTCTCGCCAAA TGCATAAAGAGC CTATCTATCCCAC AGATTGTCGACG
CTGTTCCAGAAA AAATGCTGCACC TGCGGGCCAATG ACTAA GGCTTTAACGAG
TATCTGAATCAC ATCCGCCTGGAG CAGGCCAGAATG CTGTTAAAAGGC CACGATATGAAA
GTGAAAGATATC GCCCACGCCTGC GGTTTCGCCGAC AGCAACTACTTC TGCCGCCTGTTTC
GCAAAAACACCG AACGCTCGCCGT CGGAGTATCGCC GTCAATATCACA GCCAGCTGACGG
AAAAAACAGCCC CGGCAAAAAACT AG acyl CTGACGAAGCGA 6 ATGAGCACTATC 16
ATGAGTTTTGAA 26 carrier GTFACATCACCG GAAGAACGCGTT GGAAAAATCGCG
protein GTGAAACTCTGC AAGAAAATTATC CTGGTTACCGGT CDS ACGTCAACGGCG
GGCGAACAGCTG GCAAGTCGCGGG GAATGTATATGG GGCGTTAAGCAG ATTGGCCGCGCA
TCTGACCGAGAT GAAGAAGTTACC ATCGCTGAAACG TTGCGCAAAACG AACAATGCTTCC
CTCGTTGCCCGTG CTCAGGAACCGC TTCGTTGAAGAC GCGCGAAAGTTA GCAGTCTGTGCG
CTGGGCGCTGAT TCGGGACTGCGA GTTCACTGTAAT TCTCTTGACACCG CCAGCGAAAGCG
GTTTTGTACAAA TTGAGCTGGTAA GCGCGCAGGCGA ATGATTTGCGTTA TGGCTCTGGAAG
TCAGCGATTATTT TGAGGGCAAACA AAGAGTTTGATA AGGTGCTAACGG GCCGCAAAATAG
CTGAGATTCCGG TAAAGGTCTGCT CGTAAAATCGTG ACGAAGAAGCTG GCTGAATGTGAC
GTAAGACCTGCC AGAAAATCACTA CGATCCTGCATCT GGGATTTAGTTG CTGTTCAGGCTG
ATTGAATCTGTTC CAAATTTTTCAAC CCATTGATTACAT TGGGAAATATTC
ATTTTATACACTA CAACGGCCACCA GCGCAGAATTTG CGAAAACCATCG GGCGTAA
GTGAAGTTGATA CGAAAGCGAGTT TCCTGGTGAACA TTGA ATGCCGGGATCA
CTCGTGATAACC
TGTTAATGCGCA TGAAAGATGATG AGTGGAACGATA TTATCGAAACCA ACCTGTCATCTGT
TTTCCGTCTGTCA AAAGCGGTAATG CGCGCTATGATG AAAAAGCGTCAT GGACGTATTATC
ACTATCGGTTCTG TGGTTGGTACCA TGGGAAATGCGG GTCAGGCCAACT ACGCTGCGGCGA
AAGCGGGTCTGA TTGGCTTCAGTA AATCACTGGCTC GCGAAGTTGCGT CCCGCGGTATTA
CTGTAAACGTTG TTGCTCCGGGCTT TATTGAAACGGA CATGACGCGTGC GCTGACCGATGA
GCAGCGTGCGGG TACGCTGGCGGC AGTTCCTGCGGG GCGCCTCGGCTC TCCAAATGAAAT
CGCCAGTGCGGT GGCATTTTTAGCC TCTGACGAAGCG AGTTACATCACC GGTGAAACTCTG
CACGTCAACGGC GGAATGTATATG GTCTGA ompX ACGCCTGGGGCG 7 ATGAATAAAATT
17 ATGCCCGGCTCG 27 CDS CCGACCAGCGGG GCACGTTTTTCAG TCTCGTAAGGTA
AAGAGTGATTTG CACTGGCCGTTG CCGGCATGGTTG GCCAACGAGGCG TTCTGGCTGCATC
CCGATACTGGTT CCGCTCTGAATG CGTAGGTACCAC ATTTTAATCGCCA GAAATCATGGCG
TGCTTTCGCTGCG TGATTTCCAT ATTAAAATAACC ACTTCTACCGTTA AGTATCGGCAAC
CCGGTGGCTACG CATGCCGGTACC CGCAGAGCGACA TTACGAGACGAG TGCAGGGTGAAG
CCGGGCATCCTTT CGAACAAAGCTG CTCCTGTCAATTT GCGGTTTCAACC TGTCAAATGCGG
TGAAGTACCGCT TAAAGGTTCCAG ACGAGCAAGACA TGTAATTGAATT ACAACCCGCTGG
ACCCCGCGCCGG GTGTTATCGGTTC TTGAGCTAATGTT TTTCACCTACACC GAAAAAAAGGGT
GAAAAAGATCGT CTTAAAAGCAGT TCTGAATCTGGC ACAATAGGGCGG GTTTACAAAAAA
GTCTGAAGATAA GGCCAGTACTAC TTTCA GGCATCACCGCA GGTCCGGCTTAC
CGTCTGAACGAC TGGGCTAGCATC TACGGCGTAGTG GGTGTTGGTTAC GGTAAATTCCAG
GACAACAGCTAC CCGAACAAATCT GATATGAGCGAC TACGGTTTCTCTT ACGGCGCTGGTC
TGCAGTTCAACC CGATCGAAAACG TTGCCCTGGACTT CTCCTACGAGCA GTCTCGCATTCGT
AACGTTGACGTT GGCACCTGGATT GCTGGCGTAGGT TACCGCTTCTAA DNA-
TCTGATTCCTGAT 8 GTGAATAAATCT 18 ATGAATCCTGAG 28 binding
GAAAATAAACGC CAACTGATTGAC CGTTCTGAACGC protein GACCTTGAAGAA
AAAATTGCTGCC ATTGAAATCCCC HU-beta ATTCCGGATAAC GGTGCGGACATT
GTATTGCCGTTGC CDS GTTATCGCCGATT TCTAAAGCCGCA GCGATGTGGTGG
TAGATATCCATC GCTGGACGTGCG TTTATCCGCACAT CGGTGAAACGAA TTAGATGCTTTAA
GGTCATACCCCT TCGAGGAAGTTC TCGCTTCTGTTAC GTTTGTAGGGCG TGGCACTTGCGC
TGAATCTCTGCA GGAAAAATCTAT TACAGAACGAAC GGCTGGAGATGA CCGTTGTCTCGA
CGTTTGGAATGG CGTTGCGCTGGT AGCAGCCATGGA AAGTCGTCACGG AGGGTTTGGTAC
CCATGATAAAAA CAAAATAGTGAT TTTTGCTGTTAAA AATCATGCTGGT TTCGCGCAAATA
GAGCGCGCTGCC TGCGCAGAAAGA GCGCTAAGAAAA CGTACTGGTCGC AGCCTCGACGGA
ATAGGGCTGGTA AATCCGCAAACA TGAGCCGGGTGT AGTAAATTCGTA GGCAAAGAAATC
AAACGATCTTTTC CTTGCCAGCCTTT ACCATTGCTGCT ACCGTCGGGACC TTTTGTGTAGCTA
GCTAAAGTTCCG GTGGCGTCTATTT ACTTAGATCGCT GGTTTCCGCGCA TGCAAATGCTGA
GGCAGGGGGGTC GGTAAAGCGCTG AGCTACCGGACG AATT AAAGACGCGGTA
GTACTGTTAAAG AACTGA TGCTGGTCGAAG GTTTGCAGCGCG CGCGCATCTCTG
CGCTGTCTGATA ATGGCGAACATT TTTCGGCGAAGG CGGAATACCTTG AATCGCCGGCGA
TTGACGAACGCG AGCAGGAAGTGC TGGTTCGTACCG CTATCAGCCAGT TTGAAGGCTACA
TCAAGCTGAACA AAAAAATCCCTC CGGAAGTGCTGA CGTCGCTGAATA GCATCGACGATC
CGGCGCGTCTGG CGGATACCATCG CTGCGCATATGC CGCTGAAGCTGG CGGACAAACAGT
CCGTGCTGGAGA TGTCCGACGTTA ACGAGCGTCTGG AATATCTGATGG CGATGATGGAGT
CGGAAATCGATC TGCTGCAGGTGG AGAAGCGTATTC GCAACCGCGTGA AAAAGCAGATGG
AGAAATCTCAGC GCGAGTACTATC TGAATGAGCAAA TGAAAGCCATTC AAAAAGAGCTCG
GCGAGATGGACG ACCTCCCCGGACG AGAACGAAGCGC TGAAGCGTAAGA TCGACGCGGCGA
AAATGCCGAAAG AGGCAAAAGAGA AAACCGAAGCGG AACTGCAAAAAC TGAAAATGATGT
CCCCGATGTCGG CGGAAGCGACCG TCGTTCGCGGCT ACATCGACTGGA TGGTGCAGGTAC
CGTGGAACGCTC GCAGCAAGGTTA AAAAAGACCTGC GTCAGGCTCAGG AGATCCTCGATA
CCGATCACTACG GCCTTGAGCGCG TGAAGGATCGCA TTCTTGAGTACCT CGCGGTGCAGAG
CCGTGTTAACAA GCTCAAAGGGCC GATCCTGTGCCT GGTTGGCTCCTCC GGGGGTAGGTAA
AACCTCTCTCGG CCAATCCATCGC CAAAGCAACTGG ACGCAAATATGT GCGTATGGCGCT
GGGCGGCGTGCG TGATGAAGCGGA AATCCGCGGTCA CCGCCGTACCTA TATTGGCTCAAT
GCCGGGCAAACT GATCCAGAAAAT GGCTAAAGTGGG CGTTAAAAACCC GCTGTTCTTGCTG
GATGAGATCGAC AAGATGTCTTCT GACATGCGCGGC GATCCGGCCTCG GCGCTGCTGGAG
GTGTTGGATCCG GAACAGAACGTG GCCTTTAACGAC CACTATCTGGAA GTGGATTACGAT
CTCAGCGACGTG ATGTTCGTTGCG ACCTCTAACTCC ATGAACATCCCG GCGCCGCTGCTG
GATCGTATGGAA GTGATCCGCCTCT CCGGCTATACCG AAGATGAGAAGC TAAACATCGCCA
AACGCCATCTGC TGTCAAAACAGA TTGAGCGTAACG CGCTCAAGAAAG GCGAGCTGACGG
TGGATGACAGCG CGATTATCGGCA TCATTCGCTACTA CACCCGTGAAGC AGGCGTGCGTGG
TCTGGAGCGTGA AATCTCGAAACT GTGCCGCAAAGC GGTGAAACAGCT GCTGCTGGATAA
GTCGCTGAAACA CATCGAGATTAA CGGCGACAACCT GCACGATTTCCTT GGCGTGCAGCGC
TACGACTATGGT CGTGCGGATAGC GAAAACCGCGTA GGTCAGGTGACC GGACTGGCGTGG
ACGGAAGTGGGC GGCGATCTGCTG ACCATTGAAACC GCCTGCGTTCCG GGTAAAGGCAAA
CTGACCTACACC GGTTCACTGGGT GAAGTCATGCAG GAATCCATCCAG GCGGCGCTGACG
GTGGTTCGTTCAC GTGCGGATAAGC TGGGTATTAACT CAGACTTTTACG AAAAACGTGATA
TTCACGTTCACGT GCCGGAAGGCGC GACGCCGAAGGA TGGTCCAAGCGC
CGGTATCGCGAT GTGCACCGCGCT GGTTTCCTGTCTG ACGGGTAATCCG GTACGCGCCGAC
GTGGCGATGACC GGTGAGATTACC CTCCGTGGCCAG GTATTGCCGATT GGTGGTCTGAAG
GAAAAACTGTTG GCCGCGCATCGC GGCGGCATTAAG ACTGTTCTGATTC CTGATGAAAATA
AACGCGACCTTG AAGAAATTCCGG ATAACGTTATCG CCGATTTAGATA TCCATCCGGTGA
AACGAATCGAGG AAGTTCTGGCAC TTGCGCTACAGA ACGAACCGTTTG GAATGGAAGTCG
TCACGGCAAAAT AG sspA CDS GTAAGAAAGTCG 9 ATGGCTGTCGCT 19
ATGGCTGAAAAT 29 GCCTGCGTAAAG GCCAACAAACGT CAATACTACGGC CACGTCGTCGTC
TCGGTAATGACG ACCGGTCGCCGC CTCAGTTCTCCAA CTGTTTTCTGGTC AAAAGTTCCGCA
ACGTTAATTGTTT CTACTGACATCT GCTCGCGTTTTCA TCTGCTCACGCA ATAGCCATCAGG
TCAAACCGGGCA GAACAATTTGCG TCCGCATCGTGCT ACGGTAAAATCG AAAAAACCCGCT
GGCCGAAAAAGG TTATCAACCAGC TCGGCGGGTTTTT TGTTAGTTTTGAG GTTCTCTGGAAC
TTATGGATAAAT ATAGAGCACGTG AGTACTTCGGTC TTGCCATTTTCCC GAGAAGGACAAC
GTGAAACTGCCC TCTACAAACGCC CCGCCTCAGGAT GCATGGTAGTTC CCATTGTTACCAC
CTGATTGACCTC GTCAGCCGCTGG TTTTTCAGCATTT AACCCGAATCAA AACTGGTCGACA
CCAGAATCCCCT AGCGTACCGACG TGGTTGAGAAAT CACCACAACGTC CTTGTGGATCGT
TAGATCTGTACA TTCAAAATCTGG GAGCTCACTCTG TCACCGTTAAAG TAAACTATCATC
TGGGAATCTCGC GTGGTGGTATCT CAATTTTCTGCCC ATCATTATGGAA CTGGTCAGGCTG
AAATGCAGGTGA TATCTGGATGAG GTGCGATCCGTC TTGTTCATTTTT CGTTTCCCGCATC
ACGGTATCACCC CGCCGCTCATGC GCGCTCTGATGG CGGTTTACCCGG AGTACGACGAGT
TGGCGCGTGGGG CCCTGCGTGGCG AAAGCCGTCTGT AACTGCGTAAAG ATATGCAGCGTA
CTGGTTTCGTTAC TCGAAAAGGACT TCGTGATGCTCGT GGTATTCGTTGAT CAGGTTGAACGT
GAATACCATTCA AAGAAAGTCGGC GACCGGTACCGC CTGCGTAAAGCA TGCGCAGGCTGA
CGTCGTCGTCCTC TACTGCGCGTAA AGTTCTCCAAAC GCAGCTGCGTGA GTTAA
AGAACTACAGGC GATTGCGCCAGT TTTCACCCAGAA GCCCTACTTCCTG AGCGATGAGTTC
AGCCTGGTGGAC TGCTACCTGGCA CCACTGCTGTGG CGTCTGCCGGTTC TCGGCGTAGAGC
TGGTCGGCGCTG GCGCGAAAGAGC TTAAAGGCTATA TGACTCGCGTATT TGAGCGCGACTC
TTTCCTCGCTTCT TTAACTGAAGCC GAACGTGAAATG CGTCTCGGTCGG GGCTAA tatE
CDS GTCAAAGCCGTA 10 ATGGGTGAGATT 20 ATGTTTGTTGCTG 30 TTATCGACCCCTT
AGTATTACCAAA CCGGACAATTTG AGGGACAACGCT CTGCTGGTAGTC CCGTAACGCCGG
TGCCGGGGCGGG GCAGCGCTGATT ACTGGACGGGAA AGAGCGGCCGCA ATCCTGGTGTTTG
ACGCGCAGACCT GTTGATTTTTGCC GTACCAAAAAGT GCGTCAGCATGA GAACTTTCAGCT
TACGCACGCTGG TGCGCCAGGCCG GATTATATTCAG GTGGAGACCTGG CGGAGCGGGGGG
CAGGTACGCGAG GCTCGGCTATCA CGTCGCTTCTGGT CGCCTGCCGGTG AAGGCTTTAAAA
TCTGCCTGAGGC TTGCGCAATCGC AAGCCATGAGCG GTTGCTGGCGCG CGCTTTGCGCCA
ATGACGATGACA AGACGATAACGA CCGCAATTATTAT GTGCGAAGAAGA TGCGGATTTATC
GACGTTTTTTTAA CCAGTGCTGAAG GGTTAAATCCGC ACAAGGCTTGAT AAGCGCCGGCAC
CCAGCAGCTGGA TCACCTTGTTACA AGAAGCTCTCTC TGGCGGCTTCTTA GATTGCTATTGTG
ATAAAGAGTAA CAGCTCTTGCTG TCCGCGCGTCAA GCGGAGAGCGAA ATAGCCGTTAAT
AACAGCGCTTTG TGTATGCGTGTAT ACGACGGTGCTG GATGGCGTATTC ACCCTGCATATC G
CCTTCCGGCGAA GGTCGAGCGACG AATACGCTGGTG GCCCTGCGTCAG GGGAAGATTGTG
GCGCAATATCAG AAACTGCATCTC TATGATGCGTTC AATATCCAGGAA TCCAGGCTGGTC
GATGCCGGGCGG CAAATTCCGCCG CTGATCGAAGTC GACGGGATGCGC GTCGGGCTGATG
ACCTGCTACGAT TTACGTTTCCCTG AGCTGGCGCTGT CGTTAGCGCTCA GCGGCGCGCAGC
TCATAGTGTTGCC TGCCGCGTGGGT AAAAGGGCCGCT GAAGGAACATCA CTGGGCGACGCT
GCTGGCGGCGCG GGCGCTGGATAC AACCTGCTATATT GTCGCCGCAGGA GAGTGCGGGACG
CGTAATATCGGT CAAAGCCGTATT ATCGACCCCTTA GGGACAACGCTT GCCGGGGCGGGA
GAGCGGCCGCAG TTGATTTTTGCCG AACTTTCAGCTG ATTATATTCAGC AGGTACGCGAGC
GCCTGCCGGTGT TGCGCAATCGCC GCTTTGCGCCAC CGCAATTATTAT GA LexA
GAGGCGGTGGTT 11 ATGAAAGCGTTA 21 ATGGCCAATAAT 31 repressor
GACCGTATCGGT ACGACCAGGCAG ACCACTGGGTTA CDS CCCGAGCATCAT
CAAGAGGTGTTT ACCCGAATTATT GAGCTTTCGGGG GATCTCATTCGG AAAGCGGCCGGG
CGAGCGAAAGAT GATCATATCAGC TATTCCTGGAAA ATGGGATCGGCG CAGACGGGCATG
GGATTCCGTGCG GCGGTACTGCTG CCGCCGACGCGT GCGTGGGTCAAT GCGATTATCATC
GCGGAGATTGCT GAGGCCGCATTT GCGCTGATCGCG CAGCGCTTGGGG CGTCAGGAAGGC
TGGGGAACGCTG TTTCGCTCCCCAA ATCGCGGCCGTT CTGTGGGCGAAC ACGCGGCGGAAG
ATTGCCGTGGCG TACCGCTAAGTC AGCATCTGAAAG ATCGCCTGCTGG TTGTCGTAGCTGC
CGCTGGCGCGTA TTGGACGTCGAT TCGCAAAACGGA AAGGCGCAATCG GCCATCACGCGG
AAGAAACTCCTG AGATCGTTTCCG GTGCTGCTCATTA ATTTTTGTGTGAA GCGCCTCCCGCG
GCTCGGTCCTGTT ATGTGGTTCCAA GTATTCGTCTGCT AGTGATGATAGT AATCACCGTTAG
GACGGAAGAAGA TGAAATTATCAA CTGTATATACTCA AACCGGTCTGCC TAGCGCGATTGA
CAGCATAACTGT GCTTATTGGCCG GGCGGTGGTTGA ATATACACCCAG CGTCGCGGCAGG
CCGTATCGCTCC GGGGC TGAGCCGCTGCT CGAGCATCATGA AGCGCAGCAGCA
GCTTTCGGGGCG CATTGAAGGCCA AGCGAAAGATAT CTACCAGGTGGA GGGATCGGCGGC
CCCGGCCATGTTT GGTACTGCTGGC AAGCCGAACGCC GATTATCATCGC GATTTTCTGCTGC
GCTGATCGCGTG GTGTTAGCGGTA GGGAACGCTGCT TGTCGATGAAGG GTGGGCGAACTA
ATATCGGTATTCT CCGCTAA CGATGGCGACCT GCTGGCTGTCCA TAAAACGCAGGA
TGTGCGCAATGG TCAGGTGGTTGT GGCGCGTATCGA CGAAGAAGTGAC CGTGAAGCGTCT
GAAAAAACAGGG TAACGTCGTGGA ATTGCTGCCGGA AAACAGCGAATT CTCGCCGATCGT
GGTCGACCTTCG CGAACAAAGCTT TACTATTGAAGG CCTGGCCGTCGG CGTTATCCGCAA
CGGCAACTGGCA ATAA hisS CDS TAAGAAAAGCGG 12 ...ATGAACGATTA 22
ATGCATAACCAG 32 CCTGTACGAAGA TCTGCCGGGCGA GCTCCGATTCAA CGGCGTACGTAA
AACCGCTCTCTG CGTAGAAAATCA AGACACTGCTGGA GCAGCGCATTGA AAACGAATTTAC
TAACGACGATAT AGGCTCACTGAA GTTGGGAATGTG GATCGATCAGCT GCAGGTGCTTGG
CCGATTGGCGAT GGAAGCGCGTAT TAGCTACGGTTA GGCGCCCCCATC TCGCGCTAAAGC
CAGCGAAATCCG GCCGTACAGTCG ATCGATGCTGGA TTTGCCGATTGTA ATGACAAACACG
TGAGGCGCGTCG GAGCAGACCCCG CGCACCACCGAT TATCGATATCCA TTATTCAAACGC
GTGGCGGCGACG GCAGGTTGAAGC GCTATCGGCGAA GTAAATCAAATT GAAATAACGTGT
GTGACCGACGTG AAAGCCCTCGAG TGCTGAAGCGATA GTTGAAAAAGAG CGCGTTGGCGCG
CGCTTCCCGTGTA ATGTACACCTTTG GATATCGTGCGC TGATTGAACCTG AGGACCGTAACG
GTTTCGGTGCCG CGGGCGCGAGGC GCGATAGCCTGA ACGATGGATGCG GCCGGGGTTCAT
CTCTACGTCCGG GCGGAAGCGTTC TTTTGTATATATA AAGGCACGGCTG AAACTTATCAAA
AAGAGAATAAAC GCTGCGTACGCG CAGCAGGTTAAC GTGGCAAAGAAC CCGGTATCGAAC
GTCCCGCTGGTT ATTCAA ATGGTCTCCTGTA GCCGATATCCAC CAATCAAGAACA
TTCGATTACCGC GCGCCTGTGGTA ATTGCGCTGAAG CATTGGGCCGAT GTAGCGGAATAC
GTTCCGCCACGA GGCGTTGATTGC ACGTCCGCAAAA CTGCGTATTAAC AGGCCGCTACCG
CCGGGCAATATC TCAGTTCCACCA GGCAACGAAGAG GATTGGCGCCGA CGTATCCGCATG
AGCGTTTGGCCT GTGGTGGACTGC GCAGGGGCCGGA GCTCGCGATAAA TATCGATGCCGA
AATATTCCTATCC GCTGATTATGCT GTATCGGGGTAA GACCGCCCGCTG ACGCCGGTTCTCT
GTGGCGCGAGCT GGAAAAAGATCT GGGCATCTCCGG CCAGGAAAAATA CCACGTTGCGCT
CGGCGAACCGAC GGAGCTGAACTC TCCGCAGGCGCT TATCGGTTCGCTG GCTGGAATCGGC
GAGGCTCGCGCT AATGCGCCATGT AACTATCGCGAC TGATCATCTCGAT GCGCTGGTGGCC
CGTCTCAACTTCG TATCTTGAGCAG ATCAGTTTAAAG TTTAAAGATAAG TCAGCGTAAAAG
CTGGACGAAGAC CCTCCGATGTGTT TGCAAACGCCGC CCTCGCGGTTGA ATGTACACCAAC
ATCCTATCGCCTG CCGCTGCGCGTG TTGGCGAAACAG CTGGATTCTAAA
ATCGATCAGCCT
AACCCGGACGTC CTGCACCTCGGG CAGGCGCTGCTG ATCACCGAAGCG AACGACGCCCCG
GGCGGCGCGCGC ACGCTGGGCGAC AGCGGCGCGGTG TATCTTGATGAA AAGTCCGCGATC
GAGTCCAAAACG GGCCTCGGCCTG CATTTTGCCGGG CTGCTGTCTGAA CTGTGCGCGCTG
GGGATTGGCGAT CTGGATGATGCC ACGCTGCGCGTC GGTATTCGCTAT TCTCTGGCGGCG
ACCGTGAATCAG GATCCCGTTGAA CGTCTGGTACGC GAGATCAAAGTG GGTCTCGACTAC
GGCTTCGATATTC TACAACCGCACC TCAAGTCGCTGC GTGTTTGAGTGG GTATTCGCTCTCG
GTCACCACCAGC CGGGATCAACTT CTCGGTTCCCAG TATTGCCTGCCCG GGCACCGTCTGC
ACCTGTTCACGTC GCCGGAGGCCGT AGGAGTTTGACG TACGATGGTCTG TTATCGGTACCGT
GTTGAGCAGCTT TAACGCGCTGGA GGCGGTCGCGCT GCAGCGCCTGGA ACCCCTGGCGTC
AGATATCATTAC GGCTTTGCGATG GCCGATGGATAT GGGCTGGAACGT TTCGATCATTGGC
CTTGTTTTACTGG TGCGTGGTAAAC TTCAGGCAGTGA GGTCCCGGCGAG ATCCGGAATTTA
GCGCTGGTTTCC AAGCCGATCCTG ACCCTCGGCGTA TTGTCGATATATA ACCGGCGGCAAT
CCTGGTAGCCTC AAGAAAAGCGGC CGGAACTGACAC CTGTACGAAGAC CCAGTCCGCAGC
GGCGTACGTAAA AATGCGTCTGGC GACAGGCTGGAT TGAACAGGTACG AACGACGATATG
CGATGCGTTACC ATCGATCAGCTG CGGCGTTAAGCT GAAGCGCGTATT GATGACCAACCA
CGCGCTAAAGCA TGGCGGCGGCAA TCGATGCTGGAT CTTTAAGAAGCA GAGGCGCGTCGT
GTTTGCGCGCGC ATCGATATCCAG TGATAAATGGGG CAGGTTGAAGCG CGCTCGCGTTGC
AAATAA GCTGGTGCTGGG CGAATCAGAAAT CGCCGACGGAAA CGTGGTAGTGAA
AGATTTACGCTC AGGTGAGCAAAC TACCGTAACGCA GGATAGCGTTGC TGCGCATTTGCG
CACACTTCTGGG TTAA
TABLE-US-00008 Table of Strains First Current Universal Mutagenic
DNA Gene 1 Gene 2 Sort Reference Name Name Lineage Description
Genotype mutation mutation 1 Application CI006 CI006 Isolated None
WT text strain from Enterobacter genera 2 Application CI008 CI008
Isolated None WT text strain from Burkholderia genera 3 Application
CI010 CI010 Isolated None WT text strain from Klebsiella genera 4
Application CI019 CI019 Isolated None WT text strain from Rahnella
genera 5 Application CI028 CI028 Isolated None WT text strain from
Enterobacter genera 6 Application CI050 CI050 Isolated None WT text
strain from Klebsiella genera 7 Application CM002 CM002 Mutant of
Disruption of nifL .DELTA.nifL::KanR SEQ ID text CI050 gene with a
NO: 33 kanamycin resistance expression cassette (KanR) encoding the
aminoglycoside O- phosphotransferase gene aph1 inserted. 8
Application CM011 CM011 Mutant of Disruption of nifL
.DELTA.nifL::SpecR SEQ ID text CI019 gene with a NO: 34
spectinomycin resistance expression cassette (SpecR) encoding the
streptomycin 3''-O- adenylyltransferase gene aadA inserted. 9
Application CM013 CM013 Mutant of Disruption of nifL
.DELTA.nifL::KanR SEQ ID text CI006 gene with a NO: 35 kanamycin
resistance expression cassette (KanR) encoding the aminoglycoside
O- phosphotransferase gene aph1 inserted. 10 FIG. 4A CM004 CM004
Mutant of Disruption of amtB .DELTA.amtB::KanR SEQ ID CI010 gene
with a NO: 36 kanamycin resistance expression cassette (KanR)
encoding the aminoglycoside O- phosphotransferase gene aph1
inserted. 11 FIG. 4A CM005 CM005 Mutant of Disruption of nifL
.DELTA.nifL::KanR SEQ ID CI010 gene with a NO: 37 kanamycin
resistance expression cassette (KanR) encoding the aminoglycoside
O- phosphotransferase gene aph1 inserted. 12 FIG. 4B CM015 CM015
Mutant of Disruption of nifL .DELTA.nifL::Prm5 SEQ ID CI006 gene
with a fragment NO: 38 of the region upstream of the ompX gene
inserted (Prm5). 13 FIG. 4B CM021 CM021 Mutant of Disruption of
nifL .DELTA.nifL::Prm2 SEQ ID CI006 gene with a fragment NO: 39 of
the region upstream of an unanotated gene and the first 73 bp of
that gene inserted (Prm2). 14 FIG. 4B CM023 CM023 Mutant of
Disruption of nifL .DELTA.nifL::Prm4 SEQ ID CI006 gene with a
fragment NO: 40 of the region upstream of the acpP gene and the
first 121 bp of the acpP gene inserted (Prm4). 15 FIG. 10A CM014
CM014 Mutant of Disruption of nifL .DELTA.nifL::Prm1 SEQ ID CI006
gene with a fragment NO: 41 of the region upstream of the lpp gene
and the first 29 bp of the lpp gene inserted (Prm1). 16 FIG. 10A
CM016 CM016 Mutant of Disruption of nifL .DELTA.nifL::Prm9 SEQ ID
CI006 gene with a fragment NO: 42 of the region upstream of the
lexA 3 gene and the first 21 bp of the lexA 3 gene inserted (Prm9).
17 FIG. 10A CM022 CM022 Mutant of Disruption of nifL
.DELTA.nifL::Prm3 SEQ ID CI006 gene with a fragment NO: 43 of the
region upstream of the mntP 1 gene and the first 53 bp of the mntP
1 gene inserted (Prm3). 18 FIG. 10A CM024 CM024 Mutant of
Disruption of nifL .DELTA.nifL::Prm7 SEQ ID CI006 gene with a
fragment NO: 44 of the region upstream of the sspA gene inserted
(Prm7). 19 FIG. 10A CM025 CM025 Mutant of Disruption of nifL
.DELTA.nifL::Prm10 SEQ ID CI006 gene with a fragment NO: 45 of the
region upstream of the hisS gene and the first 52 bp of the hisS
gene inserted (Prm10). 20 FIG. 10B CM006 CM006 Mutant of Disruption
of glnB .DELTA.glnB::KanR SEQ ID CI010 gene with a NO: 46 kanamycin
resistance expression cassette (KanR) encoding the aminoglycoside
O- phosphotransferase gene aph1 inserted. 21 FIG. 10C CI028 CM017
Mutant of Disruption of nifL .DELTA.nifL::KanR SEQ ID nifL:KanR
CI028 gene with a NO: 47 kanamycin resistance expression cassette
(KanR) encoding the aminoglycoside O- phosphotransferase gene aph1
inserted. 22 FIG. 10C CI019 CM011 Mutant of Disruption of nifL
.DELTA.nifL::SpecR SEQ ID nifL:SpecR CI019 gene with a NO: 48
spectinomycin resistance expression cassette (SpecR) encoding the
streptomycin 3''-O- adenylyltransferase gene aadA inserted. 23 FIG.
10C CI016 CM013 Mutant of Disruption of nifL .DELTA.nifL::KanR SEQ
ID nifL:KanR CI006 gene with a NO: 49 kanamycin resistance
expression cassette (KanR) encoding the aminoglycoside O-
phosphotransferase gene aph1 inserted. 24 FIG. 10C CI010 CM005
Mutant of Disruption of nifL .DELTA.nifL::KanR SEQ ID nifL:KanR
CI010 gene with a NO: 50 kanamycin resistance expression cassette
(KanR) encoding the aminoglycoside O- phosphotransferase gene aph1
inserted. 25 FIG. 4C Strain 2 CI006 Isolated None WT strain from
Enterobaeter genera 26 FIG. 4C Strain 4 CI010 Isolated None WT
strain from Klebsiella genera 27 FIG. 4C Strain 1 CI019 Isolated
None WT strain from Rahnella genera 28 FIG. 4C Strain 3 CI028
Isolated None WT strain from Enterobacter genera 29 FIG. 4B Strain
2 CI006 Isolated None WT strain from Enterobacter genera 30 FIG. 4B
High CM014 Mutant of Disruption of nifL .DELTA.nifL::Prm1 SEQ ID
CI006 gene with a fragment NO: 51 of the region upstream of the lpp
gene and the first 29 bp of the lpp gene inserted (Prm1). 31 FIG.
4B Med CM015 Mutant of Disruption of nifL .DELTA.nifL::Prm5 SEQ ID
CI006 gene with a fragment NO: 52 of the region upstream of the
ompX gene inserted (Prm5). 32 FIG. 4B Low CM023 Mutant of
Disruption of nifL .DELTA.nifL::Prm4 SEQ ID CI006 gene with a
fragment NO: 53 of the region upstream of the acpP gene and the
first 121 bp of the acpP gene inserted (Prm4). 33 FIG. 4D Strain 2
CI006 Isolated None WT strain from Enterobacter genera 34 FIG. 4D
Evolved CM029 Mutant of Disruption of nifL .DELTA.nifL::Prm5 SEQ ID
SEQ ID CI006 gene with a fragment .DELTA.glnE- NO: 54 NO: 61 of the
region AR_KO1 upstream of the ompX gene inserted (Prm5) and
deletion of the 1287 bp after the start codon of the glnE gene
containing the adenylyl- removing domain of glutamate-ammonia-
ligase adenylyltransferase (.DELTA.glnE-AR_KO1). 35 FIG. 14C Wild
CI006 Isolated None WT strain from Enterobacter genera 36 FIG. 14C
Evolved CM014 Mutant of Disruption of nifL .DELTA.nifL::Prm1
SEQ ID CI006 gene with a fragment NO: 55 of the region upstream of
the lpp gene and the first 29 bp of the lpp gene inserted (Prm1).
37 FIG. 14B Wild CI019 Isolated None WT strain from Rahnella genera
38 FIG. 14B Evolved CM011 Mutant of Disruption of nifL
.DELTA.nifL::SpecR SEQ ID CI019 gene with a NO: 56 spectinomycin
resistance expression cassette (SpecR) encoding the streptomycin
3''-O- adenylyltransferase gene aadA inserted. 39 FIG. 14A Evolved
CM011 Mutant of Disruption of nifL .DELTA.nifL::SpecR SEQ ID CI019
gene with a NO: 57 spectinomycin resistance expression cassette
(SpecR) encoding the streptomycin 3''-O- adenylyltransferase gene
aadA inserted. 40 FIG. 15A Wild CI006 Isolated None WT strain from
Enterobacter genera 41 FIG. 15A Evolved CM013 Mutant of Disruption
of nifL .DELTA.nifL::KanR SEQ ID CI006 gene with a NO: 58 kanamycin
resistance expression cassette (KanR) encoding the aminoglycoside
O- phosphotransferase gene aph1 inserted. 42 FIG. 15B No CM011
Mutant of Disruption of nifL .DELTA.nifL::SpecR SEQ ID name CI019
gene with a NO: 59 spectinomycin resistance expression cassette
(SpecR) encoding the streptomycin 3''-O- adenylyltransferase gene
aadA inserted. 43 FIG. 16B Strain 5 CI008 Isolated None WT strain
from Burkholderia genera 44 FIG. 16B Strain 1 CM011 Mutant of
Disruption of nifL .DELTA.nifL::SpecR SEQ ID CI019 gene with a NO:
60 spectinomycin resistance expression cassette (SpecR) encoding
the streptomycin 3''-O- adenylyltransferase gene aadA inserted.
TABLE-US-00009 Table of Strains sequences SEQ ID NO: Sequence 33
ATGAGCCATATTCAACGGGAAACGTCTTGCTCCAGGCCGCGATTAAATT
CCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGT
CGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCG
CCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTCTCCAATGATGTTA
CAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCC
GACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCA
CTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGA
TTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGC
ATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTC
TCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGA
TTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAA
ATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGA
TTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTA
TTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCAT
CCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTT
TTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCAT
TTGATGCTCGATGAGTTTTTCTAATAAGCCTGCCTGGTTCTGCGTTTCCC
GCTCTTTAATACCCTGACCGGAGGTGAGCAATGA 34
ATGAGCATCACGGCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTCGCCGCCCGACTGCTGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCGGAGGCTTAAAATGA 35
CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTT
GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCAGGCCGCGATTAAAT
TCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACC
ACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTT
GCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTC
GTCTCCTCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAG
TGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAA
GAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGG
TGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTT
GTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGC
CATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGC
TTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTT
CATTTGATGCTCGATGAGTTTTTCTAATAAGCCTTGACCCTACGATTCCC
GCTATTTCATTCACTGACCGGAGGTTCAAAATGA 36
ATGAAGATAGCAACAATGAAAACAGGTCTGGGAGCGTTGGCTCTTCTTC
CCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTT
GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCCGTCCGCGCTTAAAC
TCCAACATGGACGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCCGTCTCAACTGGCTGACGGAGTTTATGCCTCTCC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCGTGGTTACTCACC
ACCGCGATTCCTGGGAAAACAGCCTTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTTTTGATGCGCTGGCCGTGTTCCTGCGCCGGTTA
CATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGTGTATTTCGT
CTTGCTCAGGCGCAATCACGCATGAATAACGGTTTGGTTGATGCGAGTG
ATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA
AATGCACAAGCTCTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTG
ATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGT
ATTGATGTTGGACGGGTCGGAATCGCAGACCGTTACCAGGACCTTGCCA
TTCTTTGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTT
TTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCA
TTTGATGCTCGATGAGTTTTTCTAATAAGCCTGTGAAGGGCTGGACGTA
AACAGCCACGGCGAAAACGCCTACAACGCCTGA 37
ATGACCCTGAATATGATGCTCGATAACGCCGTACCCGAGGCGATTGCCG
GCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGT
TGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCCGTCCGCGCTTAAAC
TCCAACATGGACGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCCGTCTCAACTGGCTGACGGAGTTTATGCCTCTCC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCGTGGTTACTCACC
ACCGCGATTCCTGGGAAAACAGCCTTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCCGTGTTCCTGCGCCGGTTA
CATTCGATTCCTCTTTTGTAATTGTCCTTTTAACAGCGATCGTGTATTTCGT
CTTGCTCAGGCGCAATCACGCATGAATAACGGTTTGGTTGATGCGAGTG
ATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA
AATGCACAAGCTCTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTG
ATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGT
ATTGATGTTGGACGGGTCGGAATCGCAGACCGTTACCAGGACCTTGCCA
TTCTTTGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTT
TTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCA
TTTGATGCTCGATGAGTTTTTCTAATAAGCCTTGGTTCTGCGTTTCCCGCT
CTTTAATACCCTGACCGGAGGTGAGCAATGA 38
ATGACCCTGAATATGATGATGGATGCCGGCGGACATCATCGCGACAAAC
AATATTAATACCGGCAACCACACCGGCAATTTACGAGACTGCGCAGGCA
TCCTTTCTCCCGTCAATTTCTGTCAAATAAAGTAAAAGAGGCAGTCTACT
TGAATTACCCCCGGCTGGTTGAGCGTTTGTTGAAAAAAAGTAACTGAAA
AATCCGTAGAATAGCGCCACTCTGATGGTTAATTAACCTATTCAATTAA
GAATTATCTGGATGAATGTGCCATTAAATGCGCAGCATAATGGTGCGTT
GTGCGGGAAAACTGCTTTTTTTTGAAAGGGTTGGTCAGTAGCGGAAACA
ACTCACTTCACACCCCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCG
CTATTTCATTCACTGACCGGAGGTTCAAAATGA 39
ATGACCCTGAATATGATGATGGATGCCGGCTCACCACGGCGATAACCAT
AGGTTTTCGGCGTGGCCACATCCATGGTGAATCCCACTTTTTCCAGCACG
CGCGCCACTTCATCGGGTCTTAAATACATAGATTTTCCTCGTCATCTTTC
CAAAGCCTCGCCACCTTACATGACTGAGCATGGACCGTGACTCAGAAAA
TTCCACAAACGAACCTGAAAGGCGTGATTGCCGTCTGGCCTTAAAAATT
ATGGTCTAAACTAAAATTTACATCGAAAACGAGGGAGGATCCTATGTTT
AACAAACCGAATCGCCGTGACGTAGATGAAGGTGTTGAGGATATTAACC
ACGATGTTAACCAGCTCGAACTCACTTCACACCCCGAAGGGGGAAGTTG
CCTGACCCTACGATTCCCGCTATTTCATTCACTGACCGGAGGTTCAAAAT GA 40
ATGACCCTGAATATGATGATGGATGCCGGCTGACGAGGCAGGTTACATC
ACTGGTGAAACCCTGCACGTCAATGGCGGAATGTATATGGTTTAACCAC
GATGAAAATTATTTGCGTTATTAGGGCGAAAGGCCTCAAAATAGCGTAA
AATCGTGGTAAGAACTGCCGGGATTTAGTTGCAAATTTTTCAACATTTTA
TACACTACGAAAACCATCGCGAAAGCGAGTTTTGATAGGAAATTTAAGA
GTATGAGCACTATCGAAGAACGCGTTAAGAAAATTATCGGCGAACAGCT
GGGCGTTAAGCAGGAAGAAGTTACCAACAATGCTTCCTTCGTTGAAGAC
CTGGGCGCTGATTCTCTTGACACCGAACTCACTTCACACCCCGAAGGGG
GAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGACCGGAGGT TCAAAATGA 41
ATGACCCTGAATATGATGATGGATGCCGGCCGTCCTGTAATAATAACCG
GACAATTCGGACTGATTAAAAAAGCGCCCTTGTGGCGCTTTTTTTATATT
CCCGCCTCCATTTAAAATAAAAAATCCAATCGGATTTCACTATTTAAACT
GGCCATTATCTAAGATGAATCCGATGGAAGCTCGCTGTTTTAACACGCG
TTTTTTAACCTTTTATTGAAAGTCGGTGCTTCTTTGAGCGAACGATCAAA
TTTAAGTGGATTCCCATCAAAAAAATATTCTCAACCTAAAAAAGTTTGT
GTAATACTTGTAACGCTACATGGAGATTAACTCAATCTAGAGGGTATTA
ATAATGAATCGTACTAAACTGGTACTGGGCGCAACTCACTTCACACCCC
GAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGA CCGGAGGTTCAAAATGA
42 ATGACCCTGAATATGATGATGGATGCCGGCATATTGACACCATGACCYCG
CGTAATGCTGATTGGTTCTGTGACGCTGGTAATGATTGTCGAAATTCTGA
ACAGTGCCATCGAAGCCGTAGTAGACCGTATTGGTGCAGAATTCCATGA
ACTTTCCGGGCGGGCGAAGGATATGGGGTCCTGCGGCGGTGCTGATGTCC
ATCCTGCTGGCGATGTTTACCTGGATCGCATTACTCTGGTCACATTTTCG
ATAACGCTTCCAGAATTCGATAACGCCCTGGTTTTTTGCTTAAATTTGGT
TCCAAAATCGCCTTTAGCTGTATATACTCACAGCATAACTGTATATACAC
CCAGGGGGCGGGATGAAAGCATTAACGGCCAGGAACTCACTTCACACC
CCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACT
GACCGGAGGTTCAAAATGA 43
ATGACCCTGAATATGATGATGGATGCCGGCATCATATTGCGCTCCCTGG
TTATCATTTGTTACTAAATGAAATGTTATAATATAACAATTATAAATACC
ACATCGCTTTCAATTCACCAGCCAAATGAGAGGAGCGCCGTCTGACATA
GCCAGCGCTATAAAACATAGCATTATCTATATGTTTATGATTAATAACTG
ATTTTTGCGTTTTGGATTTGGCTGTGGCATCCTTGCCGCTCTTTTCGCAGC
GTCTGCGTTTTTGCCCTCCGGTCAGGGCATTTAAGGGTCAGCAATGAGTT
TTTACGCAATTACGATTCTTGCCTTCGGCATGTCGATGGATGCTTTAACT
CACTTCACACCCCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTA
TTTCATTCACTGACCGGAGGTTCAAAATGA 44
ATGACCCTGAATATGATGATGGATGCCGGCCGCGTCAGGTTGAACGTAA
AAAAGTCGGTCTGCGCAAAGCACGTCGTCGTCCGCAGTTCTCCAAACGT
TAATTGGTTTCTGCTTCGGCAGAACGATTGGCGAAAAAACCCGGTGCGA
ACCGGGTTTTTTTATGGATAAAGATCGTGTTATCCACAGCAATCCATTGA
TTATCTCTTCTTTTTCAGCATTTCCAGAATCCCCTCACCACAAAGCCCGC
AAAATCTGGTAAACTATCATCCAATTTTCTGCCCAAATGGCTGGGATTGT
TCATTTTTTGTTTGCCTTACAACGAGAGTGACAGTACGCGCGGGTAGTTA
ACTCAACATCTGACCGGTCGATAACTCACTTCACACCCCGAAGGGGGAA
GTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGACCGGAGGTTCA AAATGA 45
ATGACCCTGAATATGATGATGGATGCCGGCCCTGTATGAAGATGGCGTG
CGCAAAGATCGCCTGGATAACAGCGATATGATTAGCCAGCTTGAAGCCC
GCATTCGCGCGAAAGCGTCAATGCTGGACGAAGCGCGTCGTATTCGATGT
GCAACAGGTAGAAAAATAAGGTTGCTGGGAAGCGGCAGGCTTCCCGTG
TATGATGAACCCGCCCGGCGCGACCCGTTGTTCGTCGCGGCCCCGAGGG
TTCATTTTTTGTATTAATAAAGAGAATAAACGTGGCAAAAAATATTCAA
GCCATTCGCGGCATGAACGATTATCTGCCTGGCGAACTCACTTCACACC
CCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACT
GACCGGAGGTTCAAAATGA 46
ATGAAAAAGATTGATGCGATTATTAAACCTTTCAAACTGGATGACGTGC
GCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGT
TGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCCGTCCGCGCTTAAAC
TCCAACATGGACGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCCGTCTCAACTGGCTGACGGAGTTTATGCCTCTCC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCGTGGTTACTCACC
ACCGCGATTCCTGGGAAAACAGCCTTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTCTGCCGTGTTCCTGCGCCGGTTA
CATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGTGTATTTCGT
CTTGCTCAGGCGCAATCACGCATGAATAACGGTTTGGTTGATGCGAGTG
ATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA
AATGCACAAGCTCTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTG
ATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGT
ATTGATGTTGGACGGGTCGGAATCGCAGACCGTTACCAGGACCTTGCCA
TTCTTTGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTT
TTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCA
TTTGATGCTCGATGAGTTTTTCTAATAAGCCTCGCGCGTGATTCGTATCC
GCACCGGCGAAGAAGACGACGCGGCGATTTAA 47
ATGACCATGAACCTGATGACGGATGTCGTCTCAGCCACCGGGATCGCCG
GGTTGCTTTCACGACAACACCCGACGCTGTTTTTTACACTAATTGAACAG
GCCCCCGTGGCGATCACGCTGACGGATACCGCTGCCCGCATTGTCTATG
CCAACCCGGGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGCTCGC
CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTT
GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCAGGCCGCGATTAAAT
TCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACC
ACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTT
GCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTC
GTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAG
TGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAA
GAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGG
TGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTT
GTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGC
CATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGC
TTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTT
CATTTGATGCTCGATGAGTTTTTCTAATAAGCCTGACCGGTGGTGAATTT
AATCTCGCTGACGTGTAGACATTCATCGATCTGCATCCACGGTCCGGCG
GCGGTACCTGCCTGACGCTACGTTTACCGCTCTTTTATGAACTGACCGGA GGCCCAAGATGA 48
ATGAGCATCACGCCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGTGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCGGAGGCTTAAAATGA 49
CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTT
GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCAGGCCGCGATTAAAT
TCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACC
ACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTT
GCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTC
GTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAG
TGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAA
GAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGG
TGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTT
GTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGC
CATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGC
TTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTT
CATTTGATGCTCGATGAGTTTTTCTAATAAGCCTTGACCCTACGATTCCC
GCTATTTCATTCACTGACCGGAGGTTCAAAATGA 50
ATGACCCTGAATATGATGCTCGATAACGCCGTACCCGAGGCGATTGCCG
GCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGT
TGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCCGTCCGCGCTTAAAC
TCCAACATGGACGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCCGTCTCAACTGGCTGACGGAGTTTATGCCTCTCC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCGTGGTTACTCACC
ACCGCGATTCCTGGGAAAACAGCCTTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCCGTGTTCCTGCGCCGGTTA
CATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGTGTATTTCGT
CTTGCTCAGGCGCAATCACGCATGAATAACGGTTTGGTTGATGCGAGTG
ATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA
AATGCACAAGCTCTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTG
ATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGT
ATTGATGTTGGACGGGTCGGAATCGCAGACCGTTACCAGGACCTTGCCA
TTCTTTGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTT
TTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCA
TTTGATGCTCGATGAGTTTTTCTAATAAGCCTTGGTTCTGCGTTTCCCGCT
CTTTAATACCCTGACCGGAGGTGAGCAATGA 51
ATGACCCTGAATATGATGATGGATGCCGGCCGTCCTGTAATAATAACCG
GACAATTCGGACTGATTAAAAAAGCGCCCTTGTGGCGCTTTTTTTATATT
CCCGCCTCCATTTAAAATAAAAAATCCAATCGGATTTCACTATTTAAACT
GGCCATTATCTAAGATGAATCCGATGGAAGCTCGCTGTTTTAACACGCG
TTTTTTAACCTTTTATTGAAAGTCGGTGCTTCTTTGAGCGAACGATCAAA
TTTAAGTGGATTCCCATCAAAAAAATATTCTCAACCTAAAAAAGTTTGT
GTAATACTTGTAACGCTACATGGAGATTAACTCAATCTAGAGGGTATTA
ATAATGAATCGTACTAAACTGGTACTGGGCGCAACTCACTTCACACCCC
GAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGA CCGGAGGTTCAAAATGA
52 ATGACCCTGAATATGATGATGGATGCCGGCGGACATCATCGCGACAAAC
AATATTAATACCGGCAACCACACCGGCAATTTACGAGACTGCGCAGGCA
TCCTTTCTCCCGTCAATTTCTGTCAAATAAAGTAAAAGAGGCAGTCTACT
TGAATTACCCCCGGCTGGTTGAGCGTTTGTTGAAAAAAAGTAACTGAAA
AATCCGTAGAATAGCGCCACTCTGATGGTTAATTAACCTATTCAATTAA
GAATTATCTGGATGAATGTGCCATTAAATGCGCAGCATAATGGTGCGTT
GTGCGGGAAAACTGCTTTTTTTTGAAAGGGTTGGTCAGTAGCGGAAACA
ACTCACTTCACACCCCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCG
CTATTTCATTCACTGACCGGAGGTTCAAAATGA 53
ATGACCCTGAATATGATGATGGATGCCGGCTGACGAGGCAGGTTACATC
ACTGGTGAAACCCTGCACGTCAATGGCGGAATGTATATGGTTTAACCAC
GATGAAAATTATTTGCGTTATTAGGGCGAAAGGCCTCAAAATAGCGTAA
AATCGTGGTAAGAACTGCCGGGATTTAGTTGCAAATTTTTCAACATTTTA
TACACTACGAAAACCATCGCGAAAGCGAGTTTTGATAGGAAATTTAAGA
GTATGAGCACTATCGAAGAACGCGTTAAGAAAATTATCGGCGAACAGCT
GGGCGTTAAGCAGGAAGAAGTTACCAACAATGCTTCCTTCGTTGAAGAC
CTGGGCGCTGATTCTCTTGACACCGAACTCACTTCACACCCCGAAGGGG
GAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGACCGGAGGT TCAAAATGA 54
ATGACCCTGAATATGATGATGGATGCCGGCGGACATCATCGCGACAAAC
AATATTAATACCGGCAACCACACCGGCAATTTACGAGACTGCGCAGGCA
TCCTTTCTCCCGTCAATTTCTGTCAAATAAAGTAAAAGAGGCAGTCTACT
TGAATTACCCCCGGCTGGTTGAGCGTTTGTTGAAAAAAAGTAACTGAAA
AATCCGTAGAATAGCGCCACTCTGATGGTTAATTAACCTATTCAATTAA
GAATTATCTGGATGAATGTGCCATTAAATGCGCAGCATAATGGTGCGTT
GTGCGCTGAAAACTGCTTTTTTTTGAAAGGGTTGGTCAGTAGCGGAAACA
ACTCACTTCACACCCCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCG
CTATTTCATTCACTGACCGGAGGTTCAAAATGA 55
ATGACCCTGAATATGATGATGGATGCCGGCCGTCCTGTAATAATAACCG
GACAATTCGGACTGATTAAAAAAGCGCCCTTGTGGCGCTTTTTTTATATT
CCCGCCTCCATTTAAAATAAAAAATCCAATCGGATTTCACTATTTAAACT
GGCCATTATCTAAGATGAATCCGATGGAAGCTCGCTGTTTTAACACGCG
TTTTTTAACCTTTTATTGAAAGTCGGTGCTTCTTTGAGCGAACGATCAAA
TTTAAGTGGATTCCCATCAAAAAAATATTCTCAACCTAAAAAAGTTTGT
GTAATACTTGTAACGCTACATGGAGATTAACTCAATCTAGAGGGTATTA
ATAATGAATCGTACTAAACTGGTACTGGGCGCAACTCACTTCACACCCC
GAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGA CCGGAGGTTCAAAATGA
56 ATGAGCATCACGGCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGCTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGCCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTTCGCCGCCCGACTGGGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCGGAGGCTTAAAATGA 57
ATGAGCATCACGGCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TEGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCCTGCGCGGCTTAACTCAACTCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCGGAGGCTTAAAATGA 58
CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTT
GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCAGGCCGCGATTAAAT
TCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATCTGTTACTCACC
ACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTT
GCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTC
GTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAG
TGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAA
GAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGG
TGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTT
GTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGC
CATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGC
TTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTT
CATTTGATGCTCGATGAGTTTTTCTAATAAGCCTTGACCCTACGATTCCC
GCTATTTCATTCACTGACCGGAGGTTCAAAATGA 59
ATGAGCATCACGGCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCCTGAGGCTTAAAATGA 60
ATGAGCATCACGGCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCACCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGTATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCGGAGGCTTAAAATGA 61
ATGTTTAACGATCTGATTGGCGATGATGAAACGGATTCGCCGGAAGATG
CGCTTTCTGAGAGCTGGCGCGAATTGTGGCAGGATGCGTTGCAGGAGGA
GGATTCCACGCCCGTGCTGGCGCATCTCTCAGAGGACGATCGCCGCCGC
GTGGTGGCGCTGATTGCCGATTTTCGCAAAGAGTTGGATAAACGCACCA
TTGGCCCGCGAGGGCGGCAGGTACTCGATCACTTAATGCCGCATCTGCT
CAGCGATGTATGCTCGCGCGACGATGCGCCAGTACCGCTGTCACGCCTG
ACGCCGCTGCTCACCGGAATTATTACCCGCACCACTTACCTTGAGCTGCT
AAGTGAATTTCCCGGCGCACTGAAACACCTCATTTCCCTGTGTGCCGCGT
CGCCGATGGTTGCCAGTCAGCTGGCGCGCTACCCGATCCTGCTTGATGA
ATTGCTCGACCCGAATACGCTCTATCAACCGACGGCGATGAATGCCTAT
CGCGATGAGCTGCGCCAATACCTGCTGCGCGTGCCGGAAGATGATGAAG
AGCAACAGCTTGAGGCGCTGCGGCAGTTTAAGCAGGCGCAGTTGCTGCG
CGTGGCGGCGGCGGATATTGCCGGTACGTTGCCAGTAATGAAAGTGAGC
GATCACTTAACCTGGCTGGCGGAAGCGATTATTGATGCGGTGGTGCAGC
AAGCCTGGGGGCAGATGGTGGCGCGTTATGGCCAGCCAACGCATCTGCA
CGATCGCGAAGGGCGCGCTTTTTGCGGTGGTCGGTTATGGCAAGCTGGGC
GGCTGGGAGCTGGGTTACAGCTCCGATCTGGATCTGGTATTCCTGCACG
ACTGCCCGATGGATGTGATGACCGATGGCGAGCGTGAAATCGATGGTCG
CCAGTTCTATTTGCGTCTCGCGCAGCGCGTGATGCACCTGTTTAGCACGC
GCACGTCGTCCGGCATCCTTTATGAAGTTGATGCGCGTCTGCGTCCATCT
GGCGCTGCGGGGATGCTGGTCACTACTACGGAATCGTTCGCCGATTACC
AGCAAAACGAAGCCTGGACGTGGGAACATCAGGCGCTGGCCCGTGCGC
GCGTGGTGTACGGCGATCCGCAACTGACCGCCGAATTTGACGCCATTCG
CCGCGATATTCTGATGACGCCTCGCGACGGCGCAACGCTGCAAACCGAC
GTGCGAGAAATGCGCGAGAAAATGCGTGCCCATCTTGGCAACAAGCAT
AAAGACCGCTTCGATCTGAAAGCCGATGAAGGCGGTATCACCGACATCG
AGTTTATCGCCCAATATCTGGTGCTGCGCTTTGCCCATGACAACTCCGAA
ACTGACGCGCTGGTCGGATAATGTGCGCATTCTCGAAGGGCTGGCGCAA
AACCTGCATCATGGAGGAGCAGGAAGCGCAGGCATTGACGCTGGCGTAC
ACCACATTGCGTGATGAGCTGCACCACCTGGCGCTGCAAGAGTTGCCGG
GACATGTGGCGCTCTCCTGTTTTGTCGCCGAGCGTGCGCTTATTAAAACC
AGCTGGGACAAGTGGCTGGTGGAACCGTGCGCCCCGGCGTAA
Assessment of Genetic Tractability
[0323] Candidate microbes were characterized based on
transformability and genetic tractability. First, optimal carbon
source utilization was determined by growth on a small panel of
relevant media as well as a growth curve in both nitrogen-free and
rich media. Second, the natural antibiotic resistance of each
strain was determined through spot-plating and growth in liquid
culture containing a panel of antibiotics used as selective markers
for mutagenesis. Third, each strain was tested for its
transformability through electroporation of a collection of
plasmids. The plasmid collection comprises the combinatorial
expansion of seven origins of replication, i.e., p15a, pSC101,
CloDF, colA, RK2, pBBR1, and pRO1600 and four antibiotic resistance
markers, i.e., CmR, KmR, SpecR, and TetR. This systematic
evaluation of origin and resistance marker compatibility was used
to identify vectors for plasmid-based mutagenesis in candidate
microbes.
Example 3: Mutagenesis of Candidate Microbes
Lambda-Red Mediated Knockouts
[0324] Several mutants of candidate microbes were generated using
the plasmid pKD46 or a derivative containing a kanamycin resistance
marker (Datsenko et al. 2000; PNAS 97(12): 6640-6645). Knockout
cassettes were designed with 250 bp homology flanking the target
gene and generated via overlap extension PCR. Candidate microbes
were transformed with pKD46, cultured in the presence of arabinose
to induce Lambda-Red machinery expression, prepped for
electroporation, and transformed with the knockout cassettes to
produce candidate mutant strains. Four candidate microbes and one
laboratory strain, Klebsiella oxytoca M5A1, were used to generate
thirteen candidate mutants of the nitrogen fixation regulatory
genes nifL, glnB, and amtB, as shown in Table 4.
TABLE-US-00010 TABLE 4 List of single knockout mutants created
through Lambda-red mutagenesis Strain nifL glnB amtB M5A1 X X X
CI006 X X X CI010 X X X CI019 X X CI028 X X
[0325] Oligo-Directed Mutagenesis with Cas9 Selection
[0326] Oligo-directed mutagenesis was used to target genomic
changes to the rpoB gene in E. coli DH10B, and mutants were
selected with a CRISPR-Cas system. A mutagenic oligo (ss1283:
"G*T*T*G*ATCAGACCGATGTTCGGACCTTCcaagGTTTCGATCGGACATACGCGAC
CGTAGTGGGTCGGGTGTACGTCTCGAACTTCAAAGCC" (SEQ ID NO: 2), where *
denotes phosphorothioate bond) was designed to confer rifampicin
resistance through a 4-bp mutation to the rpoB gene. Cells
containing a plasmid encoding Cas9 were induced for Cas9
expression, prepped for electroporation, and then electroporated
with both the mutagenic oligo and a plasmid encoding constitutive
expression of a guide RNA (gRNA) that targets Cas9 cleavage of the
WT rpoB sequence. Electroporated cells were recovered in
nonselective media overnight to allow sufficient segregation of the
resulting mutant chromosomes. After plating on selection for the
gRNA-encoding plasmid, two out of ten colonies screened were shown
to contain the desired mutation, while the rest were shown to be
escape mutants generated through protospacer mutation in the gRNA
plasmid or Cas9 plasmid loss.
Lambda-Red Mutagenesis with Cas9 Selection
[0327] Mutants of candidate microbes CI006 and CI010 were generated
via lambda-red mutagenesis with selection by CRISPR-Cas. Knockout
cassettes contained an endogenous promoter identified through
transcriptional profiling (as described in Example 2 and depicted
in Tables 3A-C) and .about.250 bp homology regions flanking the
deletion target. CI006 and CI010 were transformed with plasmids
encoding the Lambda-red recombination system (exo, beta, gam genes)
under control of an arabinose inducible promoter and Cas9 under
control of an IPTG inducible promoter. The Red recombination and
Cas9 systems were induced in resulting transformants, and strains
were prepared for electroporation. Knockout cassettes and a
plasmid-encoded selection gRNA were subsequently transformed into
the competent cells. After plating on antibiotics selective for
both the Cas9 plasmid and the gRNA plasmid, 7 of the 10 colonies
screened showed the intended knockout mutation, as shown in FIG.
3.
Example 4: In Vitro Phenotyping of Candidate Molecules
[0328] The impact of exogenous nitrogen on nitrogenase biosynthesis
and activity in various mutants was assessed. The Acetylene
Reduction Assay (ARA) (Temme et. al. 2012; 10908): 7085-7090) was
used to measure nitrogenase activity in pure culture conditions.
Strains were grown in air-tight test tubes, and reduction of
acetylene to ethylene was quantified with an Agilent 6890 gas
chromatograph. ARA activities of candidate microbes and counterpart
candidate mutants grown in nitrogen fixation media supplemented
with 0 to 10 mM glutamine are shown in FIGS. 4A-B and FIGS.
10A-C.
[0329] Under anaerobic culture conditions, a range of glutamine and
ammonia concentrations was tested to quantify impact on nitrogen
fixation activity. In wild-type cells, activity quickly diminished
as glutamine concentrations increased. However, in a series of
initial knock-out mutations, a class of mutation was validated
enabling expression of nitrogen fixation genes under concentrations
of glutamine that would otherwise shut off activity in wild type.
This profile was generated in four different species of
diazotrophs, as seen in FIG. 4C. In addition, by rewiring the
regulatory network using genetic parts that have been identified,
the nitrogen fixation activity level was tuned predictably. This is
seen in FIG. 4B, which illustrates strains CM023, CM021, CM015, and
CI006. Strain CM023 is an evolved strain low; strain CM021 is an
evolved strain high; strain CM015 is an evolved strain mid; strain
CI006 is a wild-type (strain 2). Ammonia excreted into culture
supernatants was tested using a enzymatic-based assay (MEGAZYME).
The assay measures the amount of NADPH consumed in the absorbance
of 340 nm. The assay was conducted on bacterial cultures grown in
nitrogen-free, anaerobic environment with a starting density of 1E9
CFU/ml. Across a panel of six evolved strains, one strain excreted
up to 100 .mu.M of ammonia over a course of a 48 hour period, as
seen in FIG. 4D. Further, a double mutant exhibited higher ammonia
excretion than the single mutant from which it was derived, as seen
in FIG. 11. This demonstrates a microbial capacity to produce
ammonia in excess of its physiological needs.
Transcription Profiling of Pure Cultures
[0330] Transcriptional activity of CI006 was measured using the
Nanostring Elements platform. Cells were grown in nitrogen-free
media and 10E8 cells were collected after 4 hours incubation. Total
RNA was extracted using the Qiagen RNeasy kit. Purified RNA was
submitted to Core Diagnostics in Palo Alto, Calif., for probe
hybridization and Digital Analyzer analysis, as shown in FIG.
5.
Example 5: In Planta Phenotyping of Candidate Microbes
Colonization of Plants by Candidate Microbes
[0331] Colonization of desired host plants by a candidate microbe
was quantified through short-term plant growth experiments. Corn
plants were inoculated with strains expressing RFP either from a
plasmid or from a Tn5-integrated RFP expression cassette. Plants
were grown in both sterilized sand and nonsterile peat medium, and
inoculation was performed by pipetting 1 mL of cell culture
directly over the emerging plant coleoptile three days
post-germination. Plasmids were maintained by watering plants with
a solution containing the appropriate antibiotic. After three
weeks, plant roots were collected, rinsed three times in sterile
water to remove visible soil, and split into two samples. One root
sample was analyzed via fluorescence microscopy to identify
localization patterns of candidate microbes. Microscopy was
performed on 10 mm lengths of the finest intact plant roots, as
shown in FIG. 6.
[0332] A second quantitative method for assessing colonization was
developed. A quantitative PCR assay was performed on whole DNA
preparations from the roots of plants inoculated with the
endophytes. Seeds of corn (Dekalb DKC-66-40) were germinated in
previously autoclaved sand in a 2.5 inch by 2.5 inch by 10 inch
pot. One day after planting, 1 ml of endophyte overnight culture
(SOB media) was drenched right at the spot of where the seed was
located. 1 mL of this overnight culture is roughly equivalent to
about 10 9 cfu, varying within 3-fold of each other, depending on
which strain is being used. Each seedling was fertilized 3.times.
weekly with 50 mL modified Hoagland's solution supplemented with
either 2.5 mM or 0.25 mM ammonium nitrate. At four weeks after
planting, root samples were collected for DNA extraction. Soil
debris were washed away using pressurized water spray. These tissue
samples were then homogenized using QIAGEN Tissuelyzer and the DNA
was then extracted using QIAmp DNA Mini Kit (QIAGEN) according to
the recommended protocol. qPCR assay was performed using Stratagene
Mx3005P RT-PCR on these DNA extracts using primers that were
designed (using NCBI's Primer BLAST) to be specific to a loci in
each of the endophyte's genome. The presence of the genome copies
of the endophytes was quantified. To further confirm the identity
of the endophytes, the PCR amplification products were sequenced
and are confirmed to have the correct sequence. The summary of the
colonization profile of strain CI006 and CI008 from candidate
microbes are presented in Table 5. Colonization rate as high as 10
7.times.cfu/g fw of root was demonstrated in strain CI008.
TABLE-US-00011 TABLE 5 Colonization of corn as measured by qPCR
Strain Colonization Rate (CFU/g fw) CI006 1.45 .times.
10{circumflex over ( )}5 CI008 1.24 .times. 10{circumflex over (
)}7
In Planta RNA Profiling
[0333] Biosynthesis of nif pathway components in planta was
estimated by measuring the transcription of nif genes. Total RNA
was obtained from root plant tissue of CI006 inoculated plants
(planting methods as described previously). RNA extraction was
performed using RNEasy Mini Kit according to the recommended
protocol (QIAGEN). Total RNA from these plant tissues was then
assayed using Nanostring Elements kits (NanoString Technologies,
Inc.) using probes that were specific to the nif genes in the
genome of strain CI006. The data of nif gene expression in planta
is summarized in Table 6. Expression of nifH genes was detected in
plants inoculated by CM013 strains whereas nifH expression was not
detectable in CI006 inoculated plants. Strain CM013 is a derivative
of strain CI006 in which the nifL gene has been knocked out.
[0334] Highly expressed genes of CM011, ranked by transcripts per
kilobase million (TPM), were measured in planta under fertilized
condition. The promoters controlling expression of some of these
highly expressed genes were used as templates for homologous
recombination into targeted nitrogen fixation and assimilation
loci. RNA samples from greenhouse grown CM011 inoculated plant were
extracted, rRNA removed using Ribo-Zero kit, sequenced using
Illumina's Truseq platform and mapped back to the genome of CM011.
Highly expressed genes from CM011 are listed in Table 7.
TABLE-US-00012 TABLE 6 Expression of nifH in planta Strains
Relative Transcript Expression CI006 9.4 CM013 103.25
TABLE-US-00013 TABLE 7 TPM Raw (Transcripts Gene Read Per Kilobase
Gene Name Location Direction Count Million) rpsH CDS 18196-18588
reverse 4841.5 27206.4 rplQ CDS 11650-12039 reverse 4333 24536.2
rpsJ CDS 25013-25324 reverse 3423 24229 rplV CDS 21946-22278
reverse 3367.5 22333 rpsN CDS 18622-18927 reverse 2792 20150.1 rplN
CDS 19820-20191 reverse 3317 19691.8 rplF CDS 17649-18182 reverse
4504.5 18628.9 rpsD CDS 13095-13715 reverse 5091.5 18106.6 rpmF CDS
8326-8493 forward 1363.5 17923.8 rplW CDS 23429-23731 reverse 2252
16413.8 rpsM CDS 14153-14509 reverse 2269 14036.2 rplR CDS
17286-17639 reverse 2243.5 13996.1 rplC CDS 24350-24979 reverse
3985 13969.2 rplK CDS 25526-25954 reverse 2648.5 13634.1 rplP CDS
20807-21217 reverse 2423 13019.5 rplX CDS 19495-19809 reverse 1824
12787.8 rpsQ CDS 20362-20616 reverse 1460.5 12648.7 bhsA 3 CDS
79720-79977 reverse 1464 12531.5 rpmC CDS 20616-20807 reverse 998.5
11485 rpoA CDS 12080-13069 reverse 4855 10830.2 rplD CDS
23728-24333 reverse 2916.5 10628.5 bhsA 1 CDS 78883-79140 reverse
1068 9141.9 rpsS CDS 22293-22571 reverse 1138.5 9011.8 rpmA CDS
2210-2467 forward 1028.5 8803.7 rpmD CDS 16585-16764 reverse 694.5
8520.8 rplB CDS 22586-23410 reverse 3132 8384 rpsC CDS 21230-21928
reverse 2574.5 8133.9 rplE CDS 18941-19480 reverse 1972.5 8066.9
rplO CDS 16147-16581 reverse 1551 7874.2 preprotein translocase
14808-16139 reverse 4657 7721.2 subunit SecY CDS rpsE CDS
16771-17271 reverse 1671.5 7368 rpsK CDS 13746-14135 reverse 1223.5
6928.2 tufA CDS 27318-28229 reverse 2850 6901.3 rpmI CDS
38574-38771 forward 615 6859.5 rplU CDS 1880-2191 forward 935.5
6621.7 rplT CDS 38814-39170 forward 1045 6464.4 bhsA 2 CDS
79293-79550 reverse 754 6454.1 rpmB CDS 8391-8627 reverse 682
6355.1 rplJ CDS 23983-24480 reverse 1408 6243.9 fusA 2 CDS 481-2595
reverse 5832 6089.6 rpsA CDS 25062-26771 reverse 4613 5957.6 rpmJ
CDS 14658-14774 reverse 314 5926.9 rpsR CDS 52990-53217 forward 603
5840.7 rpsG CDS 2692-3162 reverse 1243 5828.2 rpsI CDS 11354-11746
reverse 980.5 5509.8 cspC 1 CDS 8091-8300 reverse 509 5352.8 rpsF
CDS 52270-52662 forward 916 5147.4 rpsT CDS 55208-55471 reverse 602
5035.9 infC CDS 38128-38478 forward 755 4750.3 cspG CDS 30148-30360
forward 446 4624.2
.sup.15N Assay
[0335] The primary method for demonstrating fixation uses the
nitrogen isotope 15N, which is found in the atmosphere at a set
rate relative to 14N. By supplementing either fertilizer or
atmosphere with enriched levels of 15N, one can observe fixation
either directly, in heightened amounts of 15N fixed from an
atmosphere supplemented with 15N2 gas (Yoshida 1980), or inversely,
through dilution of enriched fertilizer by atmospheric N2 gas in
plant tissues (Iniguez 2004). The dilution method allows for the
observation of cumulative fixed nitrogen over the course of plant
growth, while the 15N.sub.2 gas method is restricted to measuring
the fixation that occurs over the short interval that a plant can
be grown in a contained atmosphere (rate measurement). Therefore,
the gas method is superior in specificity (as any elevated
15N.sub.2 levels in the plant above the atmospheric rate can be
attributed unambiguously to fixation) but cannot show cumulative
activity.
[0336] Both types of assay has been performed to measure fixation
activity of improved strains relative to wild-type and uninoculated
corn plants, and elevated fixation rates were observed in planta
for several of the improved strains (FIG. 12, FIG. 14A, and FIG.
14B). These assays are instrumental in demonstrating that the
activity of the strains observed in vitro translates to in vivo
results. Furthermore, these assays allow measurement of the impact
of fertilizer on strain activity, suggesting suitable functionality
in an agricultural setting. Similar results were observed when
setaria plants were inoculated with wild-type and improved strains
(FIG. 13). In planta fixation activity shown in FIGS. 14A-14C is
further backed up by transcriptomic data. Evolved strains exhibit
increased nifH transcript level relative to wild-type counterparts.
Furthermore, the microbe derived nitrogen level in planta is also
correlated with the colonization level on a plant by plant basis.
These results (FIG. 12, FIG. 13, FIGS. 14A-14C, FIG. 15A, and FIG.
15B) support the hypothesis that the microbe, through the improved
regulation of the nif gene cluster, is the likely reason for the
increase in atmospheric derived nitrogen seen in the plant tissue.
In addition to measuring fixation directly, the impact of
inoculating plants with the improved strains in a nitrogen-stressed
plant biomass assay was measured. While plant biomass may be
related to many possible microbe interactions with the plant, one
would expect that the addition of fixed nitrogen would impact the
plant phenotype when nitrogen is limited. Inoculated plants were
grown in the complete absence of nitrogen, and significant
increases in leaf area, shoot fresh and dry weight, and root fresh
and dry weight in inoculated plants relative to untreated controls
was observed (FIG. 14C). Although these differences cannot be
attributed to nitrogen fixation exclusively, they support the
conclusion that the improved strains are actively providing
nitrogen to the plant. Corn and setaria plants were grown and
inoculated as described above. Fertilizer comprising 1.2% .sup.15N
was regularly supplied to plants via watering. Nitrogen fixation by
microbes was quantified by measuring the .sup.15N level in the
plant tissue. Fourth leaf tissue was collected and dried at 4 weeks
after planting. Dried leaf samples were homogenized using beads
(QIAGEN Tissuelyzer) and aliquoted out into tin capsules for IRMS
(MBL Stable Isotope Laboratory at The Ecosystems Center, Woods
Hole, Mass.). Nitrogen derived from the atmosphere (NDFA) was
calculated, and nitrogen production by CI050 and CM002 are shown in
FIG. 7.
Phytohormone Production Assay
[0337] The dwarf tomato (Solanum lycopersicum) cultivar `Micro-Tom`
has previously been used to study the influence of indole-3-acetic
acid on fruit ripening through an in vitro assay (Cohen 1996; J Am
Soc Hortic Sci 121: 520-524). To evaluate phytohormone production
and secretion by candidate microbes, a plate-based screening assay
using immature Micro-Tom fruit was developed. Twelve-well tissue
culture test plates were prepared by filling wells with agar
medium, allowing it to solidify, and spotting 10 uL of overnight
microbial cultures onto the agar surface, as shown in FIG. 8. Wells
with agar containing increasing amounts of gibberellic acid (GA)
but no bacterial culture were used as a positive control and
standards. Flowers one day post-anthesis abscised from growing
Micro-Tom plants were inserted, stem-first, into the agar at the
point of the bacterial spot culture. These flowers were monitored
for 2-3 weeks, after which the fruits were harvested and weighed.
An increase in plant fruit mass across several replicates indicates
production of plant hormone by the inoculant microbe, as shown in
FIG. 9.
Example 6: Cyclical Host-Microbe Evolution
[0338] Corn plants were inoculated with CM013 and grown 4 weeks to
approximately the V5 growth stage. Those demonstrating improved
nitrogen accumulation from microbial sources via .sup.15N analysis
were uprooted, and roots were washed using pressurized water to
remove bulk soil. A 0.25 g section of root was cut and rinsed in
PBS solution to remove fine soil particles and non-adherent
microbes. Tissue samples were homogenized using 3 mm steel beads in
QIAGEN TissueLyser II. The homogenate was diluted and plated on SOB
agar media. Single colonies were resuspended in liquid media and
subjected to PCR analysis of 16s rDNA and mutations unique to the
inoculating strain. The process of microbe isolation, mutagenesis,
inoculation, and re-isolation can be repeated iteratively to
improve microbial traits, plant traits, and the colonization
capability of the microbe.
Example 7: Compatibility Across Geography
[0339] The ability of the improved microbes to colonize an
inoculated plant is critical to the success of the plant under
field conditions. While the described isolation methods are
designed to select from soil microbes that may have a close
relationship with crop plants such as corn, many strains may not
colonize effectively across a range of plant genotypes,
environments, soil types, or inoculation conditions. Since
colonization is a complex process requiring a range of interactions
between a microbial strain and host plant, screening for
colonization competence has become a central method for selecting
priority strains for further development. Early efforts to assess
colonization used fluorescent tagging of strains, which was
effective but time-consuming and not scalable on a per-strain
basis. As colonization activity is not amenable to straightforward
improvement, it is imperative that potential product candidates are
selected from strains that are natural colonizers.
[0340] An assay was designed to test for robust colonization of the
wild-type strains in any given host plant using qPCR and primers
designed to be strain-specific in a community sample. This assay is
intended to rapidly measure the colonization rate of the microbes
from corn tissue samples. Initial tests using strains assessed as
probable colonizers using fluorescence microscopy and plate-based
techniques indicated that a qPCR approach would be both
quantitative and scalable.
[0341] A typical assay is performed as follows: Plants, mostly
varieties of maize and wheat, are grown in a peat potting mix in
the greenhouse in replicates of six per strain. At four or five
days after planting, a 1 mL drench of early stationary phase
cultures of bacteria diluted to an OD590 of 0.6-1.0 (approximately
5E+08 CFU/mL) is pipetted over the emerging coleoptile. The plants
are watered with tap water only and allowed to grow for four weeks
before sampling, at which time, the plants are uprooted and the
roots washed thoroughly to remove most peat residues. Samples of
clean root are excised and homogenized to create a slurry of plant
cell debris and associated bacterial cells. We developed a
high-throughput DNA extraction protocol that effectively produced a
mixture of plant and bacterial DNA to use as template for qPCR.
Based on bacterial cell spike-in experiments, this DNA extraction
process provides a quantitative bacterial DNA sample relative to
the fresh weight of the roots. Each strain is assessed using
strain-specific primers designed using Primer BLAST (Ye 2012) and
compared to background amplification from uninoculated plants.
Since some primers exhibit off-target amplification in uninoculated
plants, colonization is determined either by presence of
amplification or elevated amplification of the correct product
compared to the background level.
[0342] This assay was used to measure the compatibility of the
microbial product across different soil geography. Field soil
qualities and field conditions can have a huge influence on the
effect of a microbial product. Soil pH, water retention capacity,
and competitive microbes are only a few examples of factors in soil
that can affect inoculum survival and colonization ability. A
colonization assay was performed using three diverse soil types
sampled from agricultural fields in California as the plant growth
medium (FIG. 16A). An intermediate inoculation density was used to
approximate realistic agricultural conditions. Within 3 weeks,
Strain 5 colonized all plants at 1E+06 to 1E+07 CFU/g FW. After 7
weeks of plant growth, an evolved version of Strain 1 exhibited
high colonization rates (1E+06 CFU/g FW) in all soil types. (FIG.
16B).
[0343] Additionally, to assess colonization in the complexity of
field conditions, a 1-acre field trial in San Luis Obispo in June
of 2015 was initiated to assess the impacts and colonization of
seven of the wild-type strains in two varieties of field corn.
Agronomic design and execution of the trial was performed by a
contract field research organization, Pacific Ag Research. For
inoculation, the same peat culture seed coating technique tested in
the inoculation methods experiment was employed. During the course
of the growing season, plant samples were collected to assess for
colonization in the root and stem interior. Samples were collected
from three replicate plots of each treatment at four and eight
weeks after planting, and from all six reps of each treatment
shortly before harvest at 16 weeks. Additional samples were
collected from all six replicate plots of treatments inoculated
with Strain 1 and Strain 2, as well as untreated controls, at 12
weeks. Numbers of cells per gram fresh weight of washed roots were
assessed as with other colonization assays with qPCR and
strain-specific primers. Two strains, Strain 1 and Strain 2, showed
consistent and widespread root colonization that peaked at 12 weeks
and then declined precipitously (FIG. 16C). While Strain 2 appeared
to be present in numbers an order of magnitude lower than Strain 1,
it was found in more consistent numbers from plant to plant. No
strains appeared to effectively colonize the stem interior. In
support of the qPCR colonization data, both strains were
successfully re-isolated from the root samples using plating and
16S sequencing to identify isolates of matching sequence.
Example 8: Microbe Breeding
[0344] Examples of microbe breeding can be summarized in the
schematic of FIG. 17A. FIG. 17A depicts microbe breeding wherein
the composition of the microbiome can be first measured and a
species of interest is identified. The metabolism of the microbiome
can be mapped and linked to genetics. Afterwards, a targeted
genetic variation can be introduced using methods including, but
not limited to, conjugation and recombination, chemical
mutagenesis, adaptive evolution, and gene editing. Derivative
microbes are used to inoculate crops. In some examples, the crops
with the best phenotypes are selected.
[0345] As provided in FIG. 17A, the composition of the microbiome
can be first measured and a species of interest is identified. FIG.
17B depicts an expanded view of the measurement of the microbiome
step. The metabolism of the microbiome can be mapped and linked to
genetics. The metabolism of nitrogen can involve the entrance of
ammonia (NW) from the rhizosphere into the cytosol of the bacteria
via the AmtB transporter. Ammonia and L-glutamate (L-Glu) are
catalyzed by glutamine synthetase and ATP into glutamine. Glutamine
can lead to the formation of biomass (plant growth), and it can
also inhibit expression of the nif operon. Afterwards, a targeted
genetic variation can be introduced using methods including, but
not limited to, conjugation and recombination, chemical
mutagenesis, adaptive evolution, and gene editing. Derivative
microbes are used to inoculate crops. The crops with the best
phenotypes are selected.
Example 9: Field Trials with Microbes of the Disclosure--Summer
2016
[0346] In order to evaluate the efficacy of strains of the present
disclosure on corn growth and productivity under varying nitrogen
regimes, field trials were conducted.
[0347] Trials were conducted with (1) seven subplot treatments of
six strains plus the control--four main plots comprised 0, 15, 85,
and 100% of maximum return to nitrogen (MRTN) with local
verification. The control (UTC only) was conducted with 10 100%
MRTN plus, 5, 10, or 15 pounds. Treatments had four
replications.
[0348] Plots of corn (minimum) were 4 rows of 30 feet in length,
with 124 plots per location. All observations were taken from the
center two rows of the plots, and all destructive sampling was
taken from the outside rows. Seed samples were refrigerated until
1.5 to 2 hours prior to use.
[0349] Local Agricultural Practice:
[0350] The seed was a commercial corn without conventional
fungicide and insecticide treatment. All seed treatments were
applied by a single seed treatment specialist to assure uniformity.
Planting date, seeding rate, weed/insect management, etc. were left
to local agricultural practices. With the exception of fungicide
applications, standard management practices were followed.
[0351] Soil Characterization:
[0352] Soil texture and soil fertility were evaluated. Soil samples
were pre-planted for each replicate to insure residual nitrate
levels lower than 50 lbs/Ac. Soil cores were taken from 0 cm to 30
cm. The soil was further characterized for pH, CEC, total K and
P.
[0353] Assessments:
[0354] The initial plant population was assessed 14 days after
planting (DAP)/acre, and were further assessed for: (1) vigor (1 to
10 scale, w/10=excellent) 14 DAP & V10; (2) recordation of
disease ratings any time symptoms are evident in the plots; (3)
record any differences in lodging if lodging occurs in the plots;
(4) yield (Bu/acre), adjusted to standard moisture pet; (5) test
weight; and (6) grain moisture percentage.
[0355] Sampling Requirements:
[0356] The soil was sampled at three timepoints (prior to trial
initiation, V10-VT, 1 week post-harvest). All six locations and all
plots were sampled at 10 grams per sample (124 plots.times.3
timepoints.times.6 locations).
[0357] Colonization Sampling:
[0358] Colonization samples were collected at two timepoints (V10
and VT) for five locations and six timepoints (V4, V8, V10, VT, R5,
and Post-Harvest). Samples were collected as follows: (1) from 0%
and 100% MRTN, 60 plots per location; (2) 4 plants per plot
randomly selected from the outside rows; (3) 5 grams of root, 8
inches of stalk, and top three leaves--bagged and IDed each
separately--12/bags per plot; (4) five locations (60 plots.times.2
timepoints.times.12 bags/plot); and one location (60 plots.times.6
timepoints.times.12 bags/plot. See, FIG. 17C illustrating
colonization sampling.
[0359] Normalized difference vegetation index (NDVI) determination
was made using a Greenseeker instrument at two timepoints (V4-V6
and VT). Assessed each plot at all six locations (124 plots.times.2
timepoints.times.6 locations).
[0360] Root analysis was performed with Win Rhizo from one location
that best illustrated treatment differentiation. Ten plants per
plot were randomly sampled (5 adjacent from each outside row; V3-V4
stage plants were preferred) and gently washed to remove as much
dirt as reasonable. Ten roots were placed in a plastic bag and
labelled. Analyzed with WinRhizo Root Analysis.
[0361] Stalk Characteristics were measured at all six locations
between R2 and R5. The stalk diameter of ten plants per plot at the
6'' height were recorded, as was the length of the first internode
above the 6'' mark. Ten plants were monitored; five consecutive
plants from the center of the two inside rows. Six locations were
evaluated (124 plots.times.2 measures.times.6 locations).
[0362] The tissue nitrates were analyzed front all plots and all
locations. An 8'' segment of stalk beginning 6'' above the soil
when the corn is between one and three weeks after black layer
formation; leaf sheaths were removed. All locations and plots were
evaluated (6 locations.times.124 plots).
[0363] The following weather data was recorded for all locations
from planting to harvest: daily maximum and minimum temperatures,
soil temperature at seeding, daily rainfall plus irrigation (if
applied), and any unusual weather events such as excessive rain,
wind, cold, or heat.
[0364] Yield data across all six locations is presented in Table 8.
Nitrogen rate had a significant impact on yield, but strains across
nitrogen rates did not. However, at the lowest nitrogen rate,
strains CI006, CM029, and CI019 numerically out-yielded the UTC by
4 to 6 bu/acre. Yield was also numerically increased 2 to 4 bu/acre
by strains CM029, CI019, and CM081 at 15% MRTN.
TABLE-US-00014 TABLE 8 Yield data across all six locations Stalk
Vigor_ Vigor_ Diameter Internode NDVI_ NDVI_ MRTN % YLD (bu) E L
(mm) Length (in) Veg Rep 0 143.9 7.0 5.7 18.87 7.18 64.0 70.6 15
165.9 7.2 6.3 19.27 7.28 65.8 72.5 85 196.6 7.1 7.1 20.00 7.31 67.1
74.3 100 197.3 7.2 7.2 20.23 7.37 66.3 72.4 Stalk Vigor_ Vigor_
Diameter Internode NDVI_ NDVI_ Strain YLD (bu) E L (mm) Length (in)
Veg Rep CI006 (1) 176.6 7.2 6.6 19.56 18.78 66.1 72.3 CM029 (2)
176.5 7.1 6.5 19.54 18.61 65.4 71.9 CM038 (3) 175.5 7.2 6.5 19.58
18.69 65.7 72.8 CI019 (4) 176.0 7.1 6.6 19.51 18.69 65.5 72.9 CM081
(5) 176.2 7.1 6.6 19.57 18.69 65.8 73.1 CM029/ 174.3 7.1 6.6 19.83
18.79 66.2 72.5 CM081(6) UTC (7) 176.4 7.1 6.6 19.54 18.71 65.9
71.7 Stalk MRTN/ Vigor_ Vigor_ Diameter Internode NDVI_ NDVI_
Strain YLD (bu) E L (mm) Length (in) Veg Rep 0 1 145.6 7.0 5.6
19.07 7.12 63.5 70.3 0 2 147.0 7.0 5.5 18.74 7.16 64.4 70.4 0 3
143.9 7.0 5.5 18.83 7.37 64.6 70.5 0 4 146.0 6.9 5.7 18.86 7.15
63.4 70.7 0 5 141.7 7.0 5.8 18.82 7.05 63.6 70.9 0 6 142.2 7.2 5.8
19.12 7.09 64.7 69.9 0 7 141.2 7.0 5.8 18.64 7.32 64.0 71.4 15 1
164.2 7.3 6.1 19.09 7.21 66.1 71.5 15 2 167.3 7.2 6.3 19.32 7.29
65.5 72.7 15 3 165.6 7.3 6.3 19.36 7.23 64.8 72.5 15 4 167.9 7.3
6.4 19.31 7.51 66.1 72.3 15 5 169.3 7.2 6.2 19.05 7.32 66.0 72.8 15
6 161.9 7.1 6.3 19.45 7.20 66.2 72.2 15 7 165.1 7.3 6.4 19.30 7.18
66.0 73.3 85 1 199.4 7.3 7.2 19.70 7.32 67.2 74.0 85 2 195.1 7.1
7.2 19.99 7.09 66.5 74.4 85 3 195.0 7.0 7.0 20.05 7.26 67.3 74.6 85
4 195.6 7.2 7.1 20.04 7.29 66.4 74.4 85 5 196.4 7.2 7.0 19.87 7.39
67.3 74.5 85 6 195.1 7.0 6.9 20.35 7.34 67.4 74.4 85 7 199.5 6.9
7.2 19.97 7.48 67.4 74.1 100 1 197.1 7.2 7.3 20.38 7.68 67.5 73.4
100 2 196.5 7.0 7.1 20.11 7.21 65.3 70.2 100 3 197.6 7.5 7.3 20.08
7.42 66.3 73.4 100 4 194.6 7.1 7.1 19.83 7.40 66.1 74.1 100 5 197.4
7.2 7.3 20.53 7.36 66.2 74.3 100 6 198.1 7.2 7.4 20.40 7.16 66.6
73.6 100 7 199.9 7.2 7.2 20.26 7.39 66.2 68.1
[0365] Another analysis approach is presented in Table 9. The table
comprises the four locations where the response to nitrogen was the
greatest which suggests that available residual nitrogen was
lowest. This approach does not alter the assessment that the
nitrogen rate significantly impacted yield, which strains did not
when averaged across all nitrogen rates. However, the numerical
yield advantage at the lowest N rate is more pronounced for all
strains, particularly CI006, CM029, and CM029/CM081 where yields
were increased from 8 to 10 bu/acre. At 15% MRTN, strain CM081
outyielded UTC by 5 bu.
TABLE-US-00015 TABLE 9 Yield data across four locations 4 Location
Average - SGS, AgIdea, Bennett, RFR Stalk Internode YLD Diameter
Length (bu) Vigor_E Vigor_L (mm) (in) Table 16 MRTN % 0 137.8 7.3
5.84 18.10 5.36 15 162.1 7.5 6.63 18.75 5.40 85 199.2 7.4 7.93
19.58 5.62 100 203.5 7.5 8.14 19.83 5.65 Strain CI006 (1) 175.4 7.5
7.08 19.03 5.59 CM029 (2) 176.1 7.4 7.08 19.09 5.39 CM038 (3) 175.3
7.5 7.05 19.01 5.59 CI019 (4) 174.8 7.5 7.16 19.02 5.45 CM081 (5)
176.7 7.4 7.16 19.00 5.53 CM029/ 175.1 7.4 7.17 19.33 5.46 CM081
(6) UTC (7) 176.0 7.3 7.27 18.98 5.55 MRTN/Strain 0 1 140.0 7.3
5.69 18.32 5.28 0 2 140.7 7.4 5.69 18.19 5.23 0 3 135.5 7.3 5.63
17.95 5.50 0 4 138.8 7.3 5.81 17.99 5.36 0 5 136.3 7.3 6.06 18.05
5.34 0 6 141.4 7.5 6.00 18.43 5.30 0 7 131.9 7.3 6.00 17.75 5.48 15
1 158.0 7.6 6.44 18.53 5.34 15 2 164.1 7.5 6.56 19.13 5.42 15 3
164.3 7.6 6.63 18.68 5.51 15 4 163.5 7.6 6.81 18.84 5.34 15 5 166.8
7.5 6.63 18.60 5.39 15 6 156.6 7.4 6.56 18.86 5.41 15 7 161.3 7.5
6.81 18.62 5.42 85 1 199.4 7.6 8.00 19.15 5.63 85 2 199.0 7.4 8.09
19.49 5.46 85 3 198.2 7.4 7.75 19.88 5.69 85 4 196.8 7.4 8.00 19.65
5.60 85 5 199.5 7.4 7.75 19.26 5.70 85 6 198.7 7.3 7.81 19.99 5.61
85 7 202.8 7.2 8.13 19.66 5.65 100 1 204.3 7.4 8.19 20.11 6.10 100
2 200.6 7.3 8.00 19.53 5.46 100 3 203.3 7.7 8.19 19.55 5.67 100 4
200.2 7.6 8.00 19.59 5.49 100 5 203.9 7.4 8.19 20.08 5.68 100 6
203.8 7.5 8.31 20.05 5.52 100 7 208.1 7.4 8.13 19.90 5.63
[0366] The results from the field trial are also illustrated in
FIGS. 21-27. The results indicate that the microbes of the
disclosure are able to increase plant yield, which points to the
ability of the taught microbes to increase nitrogen fixation in an
important agricultural crop, i.e. corn.
[0367] The field based results further validate the disclosed
methods of non-intergenerially modifying the genome of selected
microbial strains, in order to bring about agriculturally relevant
results in a field setting when applying said engineered strains to
a crop.
[0368] FIG. 18 depicts the lineage of modified strains that were
derived from strain CI006 (WT Kosakonia sacchari). The field data
demonstrates that an engineered derivative of the CI006 WT strain,
i.e. CM029, is able to bring about numerically relevant results in
a field setting. For example, Table 8 illustrates that at 0% MRTN
CM029 yielded 147.0 bu/acre compared to untreated control at 141.2
bu/acre (an increase of 5.8 bu/acre). Table 8 also illustrates that
at 15% MRTN CM029 yielded 167.3 bu/acre compared to untreated
control at 165.1 bu/acre (an increase of 2.2 bu/acre). Table 9 is
supportive of these conclusions and illustrates that at 0% MRTN
CM029 yielded 140.7 bu/acre compared to untreated control at 131.9
bu/acre (an increase of 8.8 bu/acre). Table 9 also illustrates that
at 15% MRTN CM029 yielded 164.1 bu/acre compared to untreated
control at 161.3 bu/acre (an increase of 2.8 bu/acre).
[0369] FIG. 19 depicts the lineage of modified strains that were
derived from strain CI019 (WT Rahnella aquatilis). The field data
demonstrates that an engineered derivative of the CI019 WT strain,
i.e. CM081, is able to bring about numerically relevant results in
a field setting. For example, Table 8 illustrates that at 15% MRTN
CM081 yielded 169.3 bu/acre compared to untreated control at 165.1
bu/acre (an increase of 4.2 bu/acre). Table 9 is supportive of
these conclusions and illustrates that at 0% MRTN CM081 yielded
136.3 bu/acre compared to untreated control at 131.9 bu/acre (an
increase of 4.4 bu/acre). Table 9 also illustrates that at 15% MRTN
CM081 yielded 166.8 bu/acre compared to untreated control at 161.3
bu/acre (an increase of 5.5 bu/acre).
[0370] Further, one can see in Table 9 that the combination of
CM029/CM081 at 0% MRTN yielded 141.4 bu/acre compared to untreated
control at 131.9 bu/acre (an increase of 9.5 bu/acre).
Example 10: Field Trials with Microbes of the Disclosure
[0371] A diversity of nitrogen fixing bacteria can be found in
nature, including in agricultural soils. However, the potential of
a microbe to provide sufficient nitrogen to crops to allow
decreased fertilizer use may be limited by repression of
nitrogenase genes in fertilized soils as well as low abundance in
close association with crop roots. Identification, isolation and
breeding of microbes that closely associate with key commercial
crops might disrupt and improve the regulatory networks linking
nitrogen sensing and nitrogen fixation and unlock significant
nitrogen contributions by crop-associated microbes. To this end,
nitrogen fixing microbes that associate with and colonize the root
system of corn were identified.
[0372] Root samples from corn plants grown in agronomically
relevant soils were collected, and microbial populations extracted
from the rhizosphere and endosphere. Genomic DNA from these samples
was extracted, followed by 16S amplicon sequencing to profile the
community composition. A Kosakonia sacchari microbe (strain PBC6.1)
was isolated and classified through 16S rRNA and whole genome
sequencing. This is a particularly interesting nitrogen fixer
capable of colonizing to nearly 21% abundance of the
root-associated microbiota (FIG. 30). To assess strain sensitivity
to exogenous nitrogen, nitrogen fixation rates in pure culture were
measured with the classical acetylene reduction assay (ARA) and
varying levels of glutamine supplementation. The species exhibited
a high level of nitrogen fixing activity in nitrogen-free media,
yet exogenous fixed nitrogen repressed nif gene expression and
nitrogenase activity (Strain PBC6.1, FIGS. 28C and 28D).
Additionally, when released ammonia was measured in the supernatant
of PBC6.1 grown in nitrogen-fixing conditions, very little release
of fixed nitrogen could be detected (FIG. 28E).
[0373] We hypothesized that PBC6.1 could be a significant
contributor of fixed nitrogen in fertilized fields if regulatory
networks controlling nitrogen metabolism were rewired to allow
optimal nitrogenase expression and ammonia release in the presence
of fixed nitrogen. Sufficient genetic diversity should exist within
the PBC6.1 genome to enable broad phenotypic remodeling without the
insertion of transgenes or synthetic regulatory elements. The
isolated strain has a genome of at least 5.4 Mbp and a canonical
nitrogen fixation gene cluster. Related nitrogen metabolism
pathways in PBC6.1 are similar to those of the model organism for
nitrogen fixation, Klebsiella oxytoca m5al.
[0374] Several gene regulatory network nodes were identified which
may augment nitrogen fixation and subsequent transfer to a host
plant, particularly in high exogenous concentrations of fixed
nitrogen (FIG. 28A). The niFL operon directly regulates the rest of
the nif cluster through transcriptional activation by NifA and
nitrogen- and oxygen-dependent repression of NifA by NifL.
Disruption of tuff, can abolish inhibition of NifA and improve nif
expression in the presence of both oxygen and exogenous fixed
nitrogen. Furthermore, expressing nifA under the control of a
nitrogen-independent promoter may decouple nitrogenase biosynthesis
from regulation by the NtrB/NtrC nitrogen sensing complex. The
assimilation of fixed nitrogen by the microbe to glutamine by
glutamine synthetase (GS) is reversibly regulated by the two-domain
adenylyltransferase (ATase) enzyme GlnE through the adenylylation
and deadenylylation of GS to attenuate and restore activity,
respectively. Truncation of the GlnE protein to delete its
adenylyl-removing (AR) domain may lead to constitutively
adenylylated glutamine synthetase, limiting ammonia assimilation by
the microbe and increasing intra- and extracellular ammonia.
Finally, reducing expression of AmtB, the transporter responsible
for uptake of ammonia, could lead to greater extracellular ammonia.
To generate rationally designed microbial phenotypes without the
use of transgenes, two approaches were employed: creating
markerless deletions of genomic sequences encoding protein domains
or whole genes, and rewiring regulatory networks by intragenomic
promoter rearrangement. Through an iterative mutagenesis process,
several non-transgenic derivative strains of PBC6.1 were generated
(Table 10).
TABLE-US-00016 TABLE 10 List of isolated and derivative K. sacchari
strains used in this work. Prm, promoter sequence derived from the
PBC6.1 genome; .DELTA.glnE.sub.AR1 and .DELTA.glnE.sub.AR2,
different truncated versions of glnE gene removing the
adenylyl-removing domain sequence. Strain ID Genotype PBC6.1 WT
PBC6.14 .DELTA.nifL::Prm1 PBC6.15 .DELTA.nifL::Prm5 PBC6.22
.DELTA.nifL::Prm3 PBC6.37 .DELTA.nifL::Prm1 .DELTA.glnE.sub.AR2
PBC6.38 .DELTA.nifL::Prm1 .DELTA.glnE.sub.AR1 PBC6.93
.DELTA.nifL::Prm1 .DELTA.glnE.sub.AR2 .DELTA.amtB PBC6.94
.DELTA.nifL::Prm1 .DELTA.glnE.sub.AR1 .DELTA.amtB
[0375] Several in vitro assays were performed to characterize
specific phenotypes of the derivative strains. The ARA was used to
assess strain sensitivity to exogenous nitrogen, in which PBC6.1
exhibited repression of nitrogenase activity at high glutamine
concentrations (FIG. 28D). In contrast, most derivative strains
showed a derepressed phenotype with varying levels of acetylene
reduction observed at high glutamine concentrations.
Transcriptional rates of nifA in samples analyzed by qPCR
correlated well with acetylene reduction rates (FIG. 31),
supporting the hypothesis that nifL disruption and insertion of a
nitrogen-independent promoter to drive nifA can lead to nil cluster
derepression. Strains with altered GlnE or AmtB activity showed
markedly increased ammonium excretion rates compared to wild type
or derivative strains without these mutations (FIG. 28E),
illustrating the effect of these genotypes on ammonia assimilation
and reuptake.
[0376] Two experiments were performed to study the interaction of
PBC6.1 derivatives with corn plants and quantify incorporation of
fixed nitrogen into plant tissues. First, rates of microbial
nitrogen fixation were quantified in a greenhouse study using
isotopic tracers. Briefly, plants are grown with 15N labeled
fertilizer, and diluted concentrations of 15N in plant tissues
indicate contributions of fixed nitrogen from microbes. Corn
seedlings were inoculated with selected microbial strains, and
plants were grown to the V6 growth stage. Plants were subsequently
deconstructed to enable measurement of microbial colonization and
gene expression as well as measurement of 15N/14N ratios in plant
tissues by isotope ratio mass spectrometry (IRMS). Analysis of the
aerial tissue showed a small, nonsignificant contribution by
PBC6.38 to plant nitrogen levels, and a significant contribution by
PBC6.94 (p=0.011). Approximately 20% of the nitrogen found in
above-ground corn leaves was produced by PBC6.94, with the
remainder coming from the seed, potting mix, or "background"
fixation by other soilborne microbes (FIG. 29C). This illustrates
that our microbial breeding pipeline can generate strains capable
of making significant nitrogen contributions to plants in the
presence of nitrogen fertilizer. Microbial transcription within
plant tissues was measured, and expression of the nif gene cluster
was observed in derivative strains but not the wild type strain
(FIG. 29B), showing the importance of nif derepression for
contribution of BNF to crops in fertilized conditions. Root
colonization measured by qPCR demonstrated that colonization
density is different for each of the strains tested (FIG. 29A). A
50 fold difference in colonization was observed between PBC6.38 and
PBC6.94. This difference could be an indication that PBC6.94 has
reduced fitness in the rhizosphere relative to PBC6.38 as a result
of high levels of fixation and excretion.
Methods
Media
[0377] Minimal medium contains (per liter) 25 g Na.sub.2HPO.sub.4,
0.1 g CaCL.sub.2-2H.sub.2O, 3 g KH.sub.2PO.sub.4, 0.25 g
MgSO.sub.4.7H.sub.2O, 1 g NaCl, 2.9 mg FeCl.sub.3, 0.25 mg
Na.sub.2MoO.sub.4.2 H.sub.2O, and 20 g sucrose. Growth medium is
defined as minimal medium supplemented with 50 ml of 200 mM
glutamine per liter.
Isolation of Diazotrophs
[0378] Corn seedlings were grown from seed (DKC 66-40, DeKalb,
Ill.) for two weeks in a greenhouse environment controlled from
22.degree. C. (night) to 26.degree. C. (day) and exposed to 16 hour
light cycles in soil collected from San Joaquin County, Calif.
Roots were harvested and washed with sterile deionized water to
remove bulk soil. Root tissues were homogenized with 2 mm stainless
steel beads in a tissue lyser (TissueLyser II, Qiagen P/N 85300)
for three minutes at setting 30, and the samples were centrifuged
for 1 minute at 13,000 rpm to separate tissue from root-associated
bacteria. Supernatants were split into two fractions, and one was
used to characterize the microbiome through 16S rRNA amplicon
sequencing and the remaining fraction was diluted and plated on
Nitrogen-free Broth (NfB) media supplemented with 1.5% agar. Plates
were incubated at 30.degree. C. for 5-7 days. Colonies that emerged
were tested for the presence of the nifH gene by colony PCR with
primers Ueda19f and Ueda406r. Genomic DNA from strains with a
positive nifH colony PCR was isolated (QIAamp DNA Mini Kit, Cat No.
51306, QIAGEN, Germany) and sequenced (Illumina MiSeq v3, SeqMatic,
Fremont, Calif.). Following sequence assembly and annotation, the
isolates containing nitrogen fixation gene clusters were utilized
in downstream research.
Microbiome Profiling of Isolation Seedlings
[0379] Genomic DNA was isolated from root-associated bacteria using
the ZR-96 Genomic DNA I Kit (Zymo Research P/N D3011), and 16S rRNA
amplicons were generated using nextera-barcoded primers targeting
799f and 1114r. The amplicon libraries were purified and sequenced
with the Illumina MiSeq v3 platform (SeqMatic, Fremont, Calif.).
Reads were taxonomically classified using Kraken using the
minikraken database (FIG. 30).
Acetylene Reduction Assay (ARA)
[0380] A modified version of the Acetylene Reduction Assay was used
to measure nitrogenase activity in pure culture conditions. Strains
were propagated from single colony in SOB (RPI, P/N S25040-1000) at
30.degree. C. with shaking at 200 RPM for 24 hours and then
subcultured 1:25 into growth medium and grown aerobically for 24
hours (30.degree. C., 200 RPM). 1 ml of the minimal media culture
was then added to 4 ml of minimal media supplemented with 0 to 10
mM glutamine in air-tight Hungate tubes and grown anaerobically for
4 hours (30.degree. C., 200 RPM). 10% headspace was removed then
replaced by an equal volume of acetylene by injection, and
incubation continued for 1 hr. Subsequently, 2 ml of headspace was
removed via gas tight syringe for quantification of ethylene
production using an Agilent 6850 gas chromatograph equipped with a
flame ionization detector (FED).
Ammonium Excretion Assay
[0381] Excretion of fixed nitrogen in the form of ammonia was
measured using batch fermentation in anaerobic bioreactors. Strains
were propagated from single colony in 1 ml/well of SOB in a 96 well
DeepWell plate. The plate was incubated at 30.degree. C. with
shaking at 200 RPM for 24 hours and then diluted 1:25 into a fresh
plate containing 1 ml/well of growth medium. Cells were incubated
for 24 hours (30.degree. C., 200 RPM) and then diluted 1:10 into a
fresh plate containing minimal medium. The plate was transferred to
an anaerobic chamber with a gas mixture of >98.5% nitrogen,
1.2-1.5% hydrogen and <30 ppM oxygen and incubated at 1350 RPM,
room temperature for 66-70 hrs. Initial culture biomass was
compared to ending biomass by measuring optical density at 590 nm.
Cells were then separated by centrifugation, and supernatant from
the reactor broth was assayed for free ammonia using the Megazyme
Ammonia Assay kit (P/N K-AMIAR) normalized to biomass at each
timepoint.
Extraction of Root-Associated Microbiome
[0382] Roots were shaken gently to remove loose particles, and root
systems were separated and soaked in a RNA stabilization solution
(Thermo Fisher P/N AM7021) for 30 minutes. The roots were then
briefly rinsed with sterile deionized water. Samples were
homogenized using bead beating with 1/2-inch stainless steel ball
bearings in a tissue lyser (TissueLyser Qiagen P/N 85300) in 2 ml
of lysis buffer (Qiagen P/N 79216). Genomic DNA extraction was
performed with ZR-96 Quick-gDNA kit (Zymo Research P/N D3010), and
RNA extraction using the RNeasy kit (Qiagen P/N 74104).
Root Colonization Assay
[0383] Four days after planting, 1 ml of a bacterial overnight
culture (approximately 10.sup.9 cfu) was applied to the soil above
the planted seed. Seedlings were fertilized three times weekly with
25 ml modified Hoagland's solution supplemented with 0.5 mM
ammonium nitrate. Four weeks after planting, root samples were
collected and the total genomic DNA (gDNA) was extracted. Root
colonization was quantified using qPCR with primers designed to
amplify unique regions of either the wild type or derivative strain
genome. QPCR reaction efficiency was measured using a standard
curve generated from a known quantity of gDNA from the target
genome. Data was normalized to genome copies per g fresh weight
using the tissue weight and extraction volume. For each experiment,
the colonization numbers were compared to untreated control
seedlings.
In Planta Transcriptomics
[0384] Transcriptional profiling of root-associated microbes was
measured in seedlings grown and processed as described in the Root
Colonization Assay. Purified RNA was sequenced using the Illumina
NextSeq platform (SeqMatic, Fremont, Calif.). Reads were mapped to
the genome of the inoculated strain using bowtie2 using
`--very-sensitive-local` parameters and a minimum alignment score
of 30. Coverage across the genome was calculated using samtools.
Differential coverage was normalized to housekeeping gene
expression and visualized across the genome using Circos and across
the nif gene cluster using DNAplotlib. Additionally, the in planta
transcriptional profile was quantified via targeted Nanostring
analysis. Purified RNA was processed on an nCounter Sprint (Core
Diagnostics, Hayward, Calif.).
15N Dilution Greenhouse Study
[0385] 15N fertilizer dilution experiment was performed to assess
optimized strain activity in planta. A planting medium containing
minimal background N was prepared using a mixture of vermiculite
and washed sand (5 rinses in DI 1120). The sand mixture was
autoclaved for 1 hour at 122.degree. C. and approximately 600 g
measured out into 40 cubic inch (656 mL) pots, which were saturated
with sterile DI H.sub.2O and allowed to drain 24 hours before
planting. Corn seeds (DKC 66-40) were surface sterilized in 0.625%
sodium hypochlorite for 10 minutes, then rinsed five times in
sterile distilled water and planted 1 cm deep. The plants were
maintained under fluorescent lamps for four weeks with 16-hour day
length at room temperatures averaging 22.degree. C. (night) to
26.degree. C. (day).
[0386] Five days after planting, seedlings were inoculated with a 1
ml suspension of cells drenched directly over the emerging
coleoptile. Inoculum was prepared from 5 ml overnight cultures in
SOB, which were spun down and resuspended twice in 5 ml PBS to
remove residual SOB before final dilution to OD of 1.0
(approximately 10.sup.9 CFU/ml). Control plants were treated with
sterile PBS, and each treatment was applied to ten replicate
plants.
[0387] Plants were fertilized with 25 ml fertilizer solution
containing 2% 15N-enriched 2 mM KNO.sub.3 on 5, 9, 14, and 19 days
after planting, and the same solution without KNO.sub.3 on 7, 12,
16, and 18 days after planting. The fertilizer solution contained
(per liter) 3 mmol CaCl.sub.2), 0.5 mmol KH.sub.2PO.sub.4, 2 mmol
MgSO.sub.4, 17.9 .mu.mol FeSO.sub.4, 2.86 mg H.sub.3BO.sub.3, 1.81
mg MnCl.sub.2.4H.sub.2O, 0.22 mg ZnSO.sub.4.7H.sub.2O, 51 .mu.g
CuSO.sub.4.5H.sub.2O, 0.12 mg Na.sub.2MoO.sub.4.2H.sub.2O, and 0.14
nmol NiCl.sub.2. All pots were watered with sterile DI H.sub.2O as
needed to maintain consistent soil moisture without runoff.
[0388] At four weeks, plants were harvested and separated at the
lowest node into samples for root gDNA and RNA extraction and
aerial tissue for IRMS. Aerial tissues were wiped as needed to
remove sand, placed whole into paper bags and dried for at least 72
hours at 60.degree. C. Once completely dry, total aerial tissue was
homogenized by bead beating and 5-7 mg samples were analyzed by
isotope ratio mass spectrometry (IRMS) for .delta.15N by the MBL
Stable Isotope Laboratory (The Ecosystems Center, Woods Hole,
Mass.). Percent NDFA was calculated using the following formula: %
NDFA=(.delta.15N of UTC average--.delta.15N of sample)/(.delta.15N
of UTC average).times.100.
Example 11: Field Trials with Microbes of the Disclosure--Summer
2017
[0389] In order to evaluate the efficacy of strains of the present
disclosure on corn growth and productivity under varying nitrogen
regimes, field trials were conducted. The below field data
demonstrates that the non-intergeneric microbes of the disclosure
are able to fix atmospheric nitrogen and deliver said nitrogen to a
plant--resulting in increased yields--in both a nitrogen limiting
environment, as well as a non-nitrogen limiting environment.
[0390] Trials were conducted at seven locations across the United
states with six geographically diverse Midwestern locations. Five
nitrogen regimes were used for fertilizer treatments: 100% of
standard agricultural practice of the site/region, 100% minus 25
pounds, 100% minus 50 pounds, 100% minus 75 pounds, and 0%; all per
acre. The pounds of nitrogen per acre for the 100% regime depended
upon the standard agricultural practices of the site/region. The
aforementioned nitrogen regimes ranged from about 153 pounds per
acre to about 180 pounds per acre, with an average of about 164
pounds of nitrogen per acre.
[0391] Within each fertilizer regime there were 14 treatments. Each
regime had six replications, and a split plot design was utilized.
The 14 treatments included: 12 different microbes, 1 UTC with the
same fertilizer rate as the main plot, and 1 UTC with 100%
nitrogen. In the 100% nitrogen regime the 2.sup.nd UTC is 100 plus
25 pounds.
[0392] Plots of corn, at a minimum, were 4 rows of 30 feet in
length (30 inches between rows) with 420 plots per location. All
observations, unless otherwise noted, were taken from the center
two rows of the plants, and all destructive sampling was taken from
the outside rows. Seed samples were refrigerated until 1.5 to 2
hours prior to use.
[0393] Local Agricultural Practice:
[0394] The seed was a commercial corn applied with a commercial
seed treatment with no biological co-application. The seeding rate,
planting date, weed/insect management, harvest times, and other
standard management practices were left to the norms of local
agricultural practices for the regions, with the exception of
fungicide application (if required).
[0395] Microbe Application:
[0396] The microbes were applied to the seed in a seed treatment
over seeds that had already received a normal chemical treatment.
The seed were coated with fermentation broth comprising the
microbes.
[0397] Soil Characterization:
[0398] Soil texture and soil fertility were evaluated. Standard
soil sampling procedures were utilized, which included soil cores
of depths from 0-30 cm and 30-60 cm. The standard soil sampling
included a determination of nitrate nitrogen, ammonium nitrogen,
total nitrogen, organic matter, and CEC. Standard soil sampling
further included a determination of pH, total potassium, and total
phosphorous. To determine the nitrogen fertilizer levels, preplant
soil samples from each location were taken to ensure that die
0-12'' and potentially the 12'' to 24'' soil regions for nitrate
nitrogen.
[0399] Prior to planting and fertilization, 2 ml soil samples were
collected from 0 to 6-12'' from the UTC. One sample per replicate
per nitrogen region was collected using the middle of the row. (5
fertilizer regimes.times.6 replicates=thirty soil samples).
[0400] Post-planting (V4-V6), 2 ml soil samples were collected from
0 to 6-12'' from the UTC. One sample per replicate per nitrogen
region was collected using the middle of the row. (5 fertilizer
regimes.times.6 replicates=thirty soil samples).
[0401] Post-harvest (V4-V6), 2 ml soil samples were collected from
0 to 6-12'' from the UTC. One sample per replicate per nitrogen
region was collected using the middle of the row. Additional
post-harvest soil sample collected at 0-12'' from the UTC and
potentially 12-24'' from the UTC (5 fertilizer regimes.times.6
replicates=thirty soil samples).
[0402] A V6-V10 soil sample from each fertilizer regime (excluding
the treatment of 100% and 100%+25 lbs [in the 100% block] for all
fertilizer regimes at 0-12'' and 12-24''. (5 fertilizer
regimes.times.2 depths=10 samples per location).
[0403] Post-harvest soil sample from each fertilizer regime
(excluding the treatment of 100% and 100%+25 lbs [in the 100%
block] for all fertilizer regimes at 0-12'' and 12-24''. (5
fertilizer regimes.times.2 depths=10 samples per location).
[0404] Assessments:
[0405] The initial plant population was assessed at .about.50% UTC
and the final plant population was assessed prior to harvest.
Assessment included (1) potentially temperature (temperature
probe); (2) vigor (1-10 scale with 10=excellent) at V4 and V8-V10;
(3) plant height at V8-V10 and V14; (4) yield (bushels/acre)
adjusted to standard moisture percentage; (5) test weight; (6)
grain moisture percentage; (7) stalk nitrate tests at black layer
(420 plots.times.7 locations); (8) colonization with 1 plant per
plot in zip lock bag at 0% and 100% fertilizer at V4-V6 (1
plant.times.14 treatments.times.6 replicates.times.2 fertilizer
regimes=168 plants); (9) transcriptomics with 1 plant per plot in
zip lock bag at 0% and 100% fertilizer at V4-V6 (1 plant.times.14
treatments.times.6 replicates.times.2 fertilizer regimes=168
plants); (10) Normalized difference vegetative index (NDVI) or
normalized difference red edge (NDRE) determination using a
Greenseeker instrument at two time points (V4-V6 and VT) to assess
each plot at all 7 locations (420 plots.times.2 time points.times.7
locations=5,880 data points); (11) stalk characteristics measured
at all 7 locations between R2 and R5 by recording the stalk
diameter of 10 plants/plot at 6'' height, record length of first
internode above the 6'' mark, 10 plants monitored (5 consecutive
plants from center of two inside rows) (420 plots.times.10
plants.times.7 locations=29,400 data points).
[0406] Monitoring Schedule:
[0407] Practitioners visited all trials at V3-V4 stage to assess
early-season response to treatments and during reproductive growth
stage to monitor maturity. Local cooperator visited research trial
on an on-going basis.
[0408] Weather Information:
[0409] Weather data spanning from planting to harvest was collected
and consisted of daily minimum and maximum temperatures, soil
temperature at seeding, daily rainfall plus irrigation (if
applied), and unusual weather events such as excessive wind, rain,
cold, heat.
[0410] Data Reporting:
[0411] Including the data indicated above, the field trials
generated data points including soil textures; row spacing; plot
sizes; irrigation; tillage; previous crop; seeding rate; plant
population; seasonal fertilizer inputs including source, rate,
timing, and placement; harvest area dimensions, method of harvest,
such as by hand or machine and measurement tools used (scales,
yield monitor, etc.)
[0412] Results:
[0413] Select results from the aforementioned field trial are
reported in FIG. 32 and FIG. 33.
[0414] In FIG. 32, it can be seen that a microbe of the disclosure
(i.e. 6-403) resulted in a higher yield than the wild type strain
(WT) and a higher yield than the untreated control (UTC). The "-25
lbs N" treatment utilizes 25 lbs less N per acre than standard
agricultural practices of the region. The "100% N" UTC treatment is
meant to depict standard agricultural practices of the region, in
which 100% of the standard utilization of N is deployed by the
farmer. The microbe "6-403" was deposited as NCMA 201708004 and can
be found in Table A. This is a mutant Kosakonia sacchari (also
called CM037) and is a progeny mutant strain from CI006 WT.
[0415] In FIG. 33, the yield results obtained demonstrate that the
microbes of the disclosure perform consistently across locations.
Furthermore, the yield results demonstrate that the microbes of the
disclosure perform well in both a nitrogen stressed environment
(i.e. a nitrogen limiting environment), as well as an environment
that has sufficient supplies of nitrogen (i.e. a
non-nitrogen-limiting condition). The microbe "6-881" (also known
as CM094, PBC6.94), and which is a progeny mutant Kosakonia
sacchari strain from CI006 WT, was deposited as NCMA 201708002 and
can be found in Table A. The microbe "137-1034," which is a progeny
mutant Klebsiella variicola strain from CI137 WI, was deposited as
NCMA 201712001 and can be found in Table A. The microbe "137-1036,"
which is a progeny mutant Klebsiella variicola strain from CI137
WT, was deposited as NCMA 201712002 and can be found in Table A.
The microbe "6-404" (also known as CM38, PBC6.38), and which is a
progeny mutant Kosakonia sacchari strain from CI006 WT, was
deposited as NCMA 201708003 and can be found in Table A.
Example 12: Genus of Non-Intergeneric Microbes Beneficial for
Agricultural Systems
[0416] The microbes of the present disclosure were evaluated and
compared against one another for the production of nitrogen
produced in an acre across a season. See FIG. 20, FIG. 40, and FIG.
41
[0417] It is hypothesized by the inventors that in order for a
population of engineered non-intergeneric microbes to be beneficial
in a modern row crop agricultural system, then the population of
microbes needs to produce at least one pound or more of nitrogen
per acre per season.
[0418] To that end, the inventors have surprisingly discovered a
functional genus of microbes that are able to contribute, inter
alia, to: increasing yields in non-leguminous crops; and/or
lessening a farmer's dependence upon exogenous nitrogen
application; and/or the ability to produce at least one pound of
nitrogen per acre per season, even in non-nitrogen-limiting
environments, said genus being defined by the product of
colonization ability.times.mmol of N produced per microbe per hour
(i.e. the line partitioning FIGS. 20, 40, and 41).
[0419] With respect to FIGS. 20, 40, and 41, certain data utilizing
microbes of the disclosure was aggregated, in order to depict a
heatmap of the pounds of nitrogen delivered per acre-season by
microbes of the disclosure, which are recorded as a function of
microbes per g-fresh weight by mmol of nitrogen/microbe-hr. Below
the thin line that transects the larger images are the microbes
that deliver less than one pound of nitrogen per acre-season, and
above the line are the microbes that deliver greater than one pound
of nitrogen per acre-season.
[0420] Field Data & Wild Type Colonization Heatmap:
[0421] The microbes utilized in the FIG. 20 heatmap were assayed
for N production in corn. For the WT strains CI006 and CI019, corn
root colonization data was taken from a single field site. For the
remaining strains, colonization was assumed to be the same as the
WT field level. N-fixation activity was determined using an in
vitro ARA assay at 5 mM glutamine. The table below the heatmap in
FIG. 20 gives the precise value of mmol N produced per microbe per
hour (mmol N/Microbe hr) along with the precise CFU per gram of
fresh weight (CFU/g fw) for each microbe shown in the heatmap.
[0422] Field Data Heatmap:
[0423] The data utilized in the FIG. 40 heatmap is derived from
microbial strains assayed for N production in corn in field
conditions. Each point represents lb N/acre produced by a microbe
using corn root colonization data from a single field site.
N-fixation activity was determined using in vitro ARA assay at 5 mM
N in the form of glutamine or ammonium phosphate. The below Table C
gives the precise value of mmol N produced per microbe per hour
(mmol N/Microbe hr) along with the precise CFU per gram of fresh
weight (CFU/g fw) for each microbe shown in the heatmap of FIG.
40.
[0424] Greenhouse & Laboratory Data Heatmap:
[0425] The data utilized in the FIG. 41 heatmap is derived from
microbial strains assayed for N production in corn in laboratory
and greenhouse conditions. Each point represents lb N/acre produced
by a single strain. White points represent strains in which corn
root colonization data was gathered in greenhouse conditions. Black
points represent mutant strains for which corn root colonization
levels are derived from average field corn root colonization levels
of the wild-type parent strain. Hatched points represent the wild
type parent strains at their average field corn root colonization
levels. In all cases, N-fixation activity was determined by in
vitro ARA assay at 5 mM N in the form of glutamine or ammonium
phosphate. The below Table D gives the precise value of mmol N
produced per microbe per hour (mmol N/Microbe hr) along with the
precise CFU per gram of fresh weight (CFU/g fw) for each microbe
shown in the heatmap of FIG. 41.
TABLE-US-00017 TABLE C FIG. 40 - Field Data Heatmap Peak Activity
Coloniza- (mmol tion N Pro- Strain N/Mi- (CFU/ duced/acre Taxonomic
Name crobe hr) g fw) season Designation CI006 3.88E-16 1.50E+07
0.24 Kosakonia sacchari 6-404 1.61E-13 3.50E+05 2.28 Kosakonia
sacchari 6-848 1.80E-13 2.70E+05 1.97 Kosakonia sacchari 6-881
1.58E-13 5.00E+05 3.20 Kosakonia sacchari 6-412 4.80E-14 1.30E+06
2.53 Kosakonia sacchari 6-403 1.90E-13 1.30E+06 10.00 Kosakonia
sacchari CI019 5.33E-17 2.40E+06 0.01 Rahnella aquatilis 19-806
6.65E-14 2.90E+06 7.80 Rahnella aquatilis 19-750 8.90E-14 2.60E+05
0.94 Rahnella aquatilis 19-804 1.72E-14 4.10E+05 0.29 Rahnella
aquatilis CI137 3.24E-15 6.50E+06 0.85 Klebsiella variicola
137-1034 1.16E-14 6.30E+06 2.96 Klebsiella variicola 137-1036
3.47E-13 1.30E+07 182.56 Klebsiella variicola 137-1314 1.70E-13
1.99E+04 0.14 Klebsiella variicola 137-1329 1.65E-13 7.25E+04 0.48
Klebsiella variicola 63 3.60E-17 3.11E+05 0.00 Rahnella aquatilis
63-1146 1.90E-14 5.10E+05 0.39 Rahnella aquatilis 1021 1.77E-14
2.69E+07 19.25 Kosakonia pseudosacchari 728 5.56E-14 1445240.09
3.25 Klebsiella variicola
TABLE-US-00018 TABLE D FIG. 41 Greenhouse & Laboratory Data
Heatmap N Activity Peak Produced/ Strain (mmol N/ Colonization acre
Name Microbe hr) (CFU/g fw) season Taxonomic Designation CI006
3.88E-16 1.50E+07 0.24 Kosakonia sacchari 6-400 2.72E-13 1.79E+05
1.97 Kosakonia sacchari 6-397 1.14E-14 1.79E+05 0.08 Kosakonia
sacchari CI137 3.24E-15 6.50E+06 0.85 Klebsiella variicola 137-1586
1.10E-13 1.82E+06 8.10 Klebsiella variicola 137-1382 4.81E-12
1.82E+06 354.60 Klebsiella variicola 1021 1.77E-14 2.69E+07 19.25
Kosakonia pseudosacchari 1021-1615 1.20E-13 2.69E+07 130.75
Kosakonia pseudosacchari 1021-1619 3.93E-14 2.69E+07 42.86
Kosakonia pseudosacchari 1021-1612 1.20E-13 2.69E+07 130.75
Kosakonia pseudosacchari 1021-1623 4.73E-17 2.69E+07 0.05 Kosakonia
pseudosacchari 1293 5.44E-17 8.70E+08 1.92 Azospirillum lipoferum
1116 1.05E-14 1.37E+07 5.79 Enterobacter sp. 1113 8.05E-15 4.13E+07
13.45 Enterobacter sp. 910 1.19E-13 1.34E+06 6.46 Kluyvera
intermedia 910-1246 2.16E-13 1.34E+06 11.69 Kluyvera intermedia 850
7.2301E-16 1.17E+06 0.03 Achromobacter spiritinus 852 5.96E-16
1.07E+06 0.03 Achromobacter marplatensis 853 6.42E-16 2.55E+06 0.07
Microbacterium murale
[0426] Conclusions:
[0427] The data in FIGS. 20, 40, 41, and Tables C and D,
illustrates more than a dozen representative members of the
described genus (i.e. microbes to the right of the line in the
figures). Further, these numerous representative members come from
a diverse array of taxonomic genera, which can be found in the
above Tables C and D. Further still, the inventors have discovered
numerous genetic attributes that depict a structure/function
relationship that is found in many of the microbes. These genetic
relationships can be found in the numerous tables of the disclosure
setting forth the genetic modifications introduced by the
inventors, which include introducing at least one genetic variation
into at least one gene, or non-coding polynucleotide, of the
nitrogen fixation or assimilation genetic regulatory network.
[0428] Consequently, the newly discovered genus is supported by:
(I) a robust dataset, (2) over a dozen representative members, (3)
members from diverse taxonomic genera, and (4) classes of genetic
modifications that define a structure/function relationship, in the
underlying genetic architecture of the genus members.
Example 13: Methods and Assays for Detection of Non-Intergeneric
Engineered Microbes
[0429] The present disclosure teaches primers, probes, and assays
that are useful for detecting the microbes utilized in the various
aforementioned Examples. The assays are able to detect the
non-natural nucleotide "junction" sequences in the derived/mutant
non-intergeneric microbes. These non-naturally occurring nucleotide
junctions can be used as a type of diagnostic that is indicative of
the presence of a particular genetic alteration in a microbe.
[0430] The present techniques are able to detect these
non-naturally occurring nucleotide junctions via the utilization of
specialized quantitative PCR methods, including uniquely designed
primers and probes. The probes can bind to the non-naturally
occurring nucleotide junction sequences. That is, sequence-specific
DNA probes consisting of oligonucleotides that are labelled with a
fluorescent reporter, which permits detection only after
hybridization of the probe with its complementary sequence can be
used. The quantitative methods can ensure that only the
non-naturally occurring nucleotide junction will be amplified via
the taught primers, and consequently can be detected via either a
non-specific dye, or via the utilization of a specific
hybridization probe. Another aspect of the method is to choose
primers such that the primers flank either side of a junction
sequence, such that if an amplification reaction occurs, then said
junction sequence is present.
[0431] Consequently, genomic DNA can be extracted from samples and
used to quantify the presence of microbes of the disclosure by
using qPCR. The primers utilized in the qPCR reaction can be
primers designed by Primer Blast
(https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify
unique regions of the wild-type genome or unique regions of the
engineered non-intergeneric mutant strains. The qPCR reaction can
be carried out using the SYBR GreenER qPCR SuperMix Universal
(Thermo Fisher P/N 11762100) kit, using only forward and reverse
amplification primers; alternatively, the Kapa Probe Force kit
(Kapa Biosystems P/N KK4301) can be used with amplification primers
and a TaqMan probe containing a FAM dye label at the 5' end, an
internal ZEN quencher, and a minor groove binder and fluorescent
quencher at the 3' end (Integrated DNA Technologies).
[0432] Certain primer, probe, and non-native junction
sequences--which can be used in the qPCR methods--are listed in the
below Table E. Specifically, the non-native junction sequences can
be found in SEQ ID NOs: 372-405.
TABLE-US-00019 TABLE E Microbial Detection up/down SEQ 100 bp SEQ
100 bp SEQ Junction SEQ F R base Junction stream ID upstream ID
downstream ID ''/'' indicating Junction primer primer Probe CI Name
junction NO of junction NO junction NO junction des. SEQ SEQ SEQ
1021 ds1131 up 304 TGGTGTCCGGGC 338 TTCTTGGTTCTCT 372 5'- disrupted
N/A N/A N/A GAACGTCGCCAG GGAGCGCTTTAT TGGTGTCCGGGC nifL gene/
GTGGCACAAATT CGGCATCCTGAC GAACGTCGCCAG PinfC GTCAGAACTACG
TGAAGAATTTGC GTGGCACAAATT ACACGACTAACC AGGCTTCTTCCCA GTCAGAACTACG
GACCGCAGGAGT ACCTGGCTTGCA ACACGACTAACC GTGCGATGACCC CCCGTGCAGGTA
GACCGCAGGAGT TGAATATGATGA GTTGTGATGAAC GTGCGATGACCC TGGA AT
TGAATATGATGA TGGA/ TTCTTGGTTCTCT GGAGCGCTTTAT CGGCATCCTGAC
TGAAGAATTTGC AGGCTTCTTCCCA ACCTGGCTTGCA CCCGTGCAGGTA GTTGTGATGAAC
AT-3' 1021 ds1131 down 305 CGGAAAACGAGT 339 GCGATAGAACTC 373 5'-
PinfC/ N/A N/A N/A TCAAACGGCGCG ACTTCACGCCCC CGGAAAACGAGT disrupted
TCCCAATCGTATT GAAGGGGGAAGC TCAAACGGCGCG nifL gene AATGGCGAGATT
TGCCTGACCCTAC TCCCAATCGTATT CGCGCCACGGAA GATTCCCGCTATT AATGGCGAGATT
GTTCGCTTAACAG TCATTCACTGACC CGCGCCACGGAA GTCTGGAAGGCG GGAGGTTCAAAA
GTTCGTTAACA AGCACCTTGGTA TGACCCAGCGAA GGTCTGGAAGGC TT C
GAGCAGCTTGGT ATT/ GCGATAGAACTC ACTTCACGCCCC GAAGGGGGAAGC
TGCCTGACCCTA CGATTCCCGCTAT TTCATTCACTGAC CGGAGGTTCAAA ATGACCCAGCGA
AC-3' 1021 ds1133 N/A 306 CGCCAGAGAGTT 340 TCCCTGTGCGCCG 374 5'- 5'
UTR N/A N/A N/A GAAATCGAACAT CGTCGCCGATGG CGCCAGAGAGTF and ATG/
TTCCGTAATACCG TGGCCAGCCAAC GAAATCGAACAT truncated CCATTACCCAGG
TGGCGCGCTACC TTCCGTAATACC glnE gene AGCCGTTCTGGTT CGATCCTGCTCG
GCCATTACCCAG GCACAGCGGAAA ATGAACTGCTCG GAGCCGTTCTGG ACGTTAACGAAA
ACCCGAACACGC TTGCACAGCGGA GGATATTTCGCAT TCTATCAACCGA AAACGTTAACGA G
CGG AAGGATATTTCG CATG/ TCCCTGTGCGCC GCGTCGCCGATG GTGGCCAGCCAA
CTGGCGCGCTAC CCGATCCTGCTC GATGAACTGCTC GACCCGAACACG CTCTATCAACCG
ACGG-3' 1021 ds1145 up 307 CGGGCGAACGTC 341 CGTTCTGTAATAA 375 5'-
disrupted N/A N/A N/A GCCAGGTGGCAC TAACCGGACAAT CGGGCGAACGTC nifL
gene/ AAATTGTCAGAA TCGGACTGATTA GCCAGGTGGCAC Prm1 CTACGACACGAC
AAAAAGCGCCCT AAATTGTCAGAA TAACCGACCGCA CGCGGCGCTTTTT CTACGACACGAC
GGAGTGTGCGAT TTATATTCTCGAC TAACCGACCGCA GACCCTGAATAT TCCATTTAAAATA
GGAGTGTGCGAT GATGATGGATGC AAAAATCCAATC GACCCTGAATAT CAGC
GATGATGGATGC CAGC/ CGTTCTGTAATA ATAACCGGACAA TFCGGACTGATT
AAAAAAGCGCCC TCGCGGCGCTTTT TTTATATTCTCGA CTCCATTTAAAAT AAAAAATCCAAT
C-3' 1021 ds1145 down 308 TCAACCTAAAAA 342 AACTCACTTCAC 376 5'-
Prm1/ N/A N/A N/A AGTTTGTGTAATA GCCCCGAAGGGG TCAACCTAAAAA disrupted
CTTGTAACGCTAC GAAGCTGCCTGA AGTTTGTGTAAT nifL gene ATGGAGATTAAC
CCCTACGATTCCC ACTTGTAACGCT TCAATCTAGAGG GCTATTTCATTCA ACATGGAGATTA
GTATTAATAATG CTGACCGGAGGT ACTCAATCTAGA AATCGTACTAAA TCAAAATGACCC
GGGTATTAATAA CTGGTACTGGGC AGCGAACCGAGT TGAATCGTACTA GC CG
AACTGGTACTGG GCGC/ AACTCACTTCAC GCCCCGAAGGGG GAAGCTGCCTGA
CCCTACGATTCCC GCTATTTCATTCA CTGACCGGAGGT TCAAAATGACCC AGCGAACCGAGT
CG-3' 1021 ds1148 up 309 CGGGCGAACGTC 343 CGCGTCAGGTTG 377 5'-
disrupted N/A N/A N/A GCCAGGTGGCAC AACGTAAAAAAG CGGGCGAACGTC nifL
gene/ AAATTGTCAGAA TCGGTCTGCGCA GCCAGGTGGCAC Prm7 CTACGACACGAC
AAGCACGTCGTC AAATTGTCAGAA TAACCGACCGCA GTCCGCAGTTCTC CTACGACACGAC
GGAGTGTGCGAT CAAACGTTAATT TAACCGACCGCA GACCCTGAATAT GGTTTCTGCTFCG
GGAGTGTGCGAT GATGATGGATGC GCAGAACGATTG GACCCTGAATAT CAGC GC
GATGATGGATGC CAGC/ CGCGTCAGGTTG AACGTAAAAAAG TCGGTCTGCGCA
AAGCACGTCGTC GTCCGCAGTTCTC CAAACGTTAATT GGTTTCTGCTTCG GCAGAACGATTG
GC-3' 1021 ds1148 down 310 AATTTTCTGCCCA 344 AACTCACTTCAC 378 5'-
Prm4/ N/A N/A N/A AATGGCTGGGAT GCCCCGAAGGGG AATGGCTGCCCA disrupted
TGTTCATTTTTTG GAAGCTGCCFGA AATGGCTGGGAT nifL gene TTTGCCTTACAAC
CCCTACGATTCCC TGTTCATTTTTTG GAGAGTGACAGT GCTATTTCATTCA
TTTGCCTTACAAC ACGCGCGGGTAG CTGACCGGAGGT GAGAGTGACAGT TTAACTCAACATC
TCAAAATGACCC ACGCGCGGGTAG TGACCGGTCGAT AGCGAACCGAGT TTAACTCAACAT CG
CTGACCGGTCGA T/ AACTCACTTCAC GCCCCGAAGGGG GAAGCTGCCTGA
CCCTACGATTCCC GCTATTTCATTCA CTGACCGGAGGT TCAAAATGACCC AGCGAACCGAGT
CG-3' CI006 ds126 N/A 311 GTAACCAATAAA 345 CCGATCCCCATC 379 5'- 5'
UTR up N/A N/A N/A GGCCACCACGCC ACTGTGTGTCTTG GTAACCAATAAA to ATG-
AGACCACACGAT TATTACAGTGCC GGCCACCACGCC 4 bp of AGTGATGGCAAC
GCTTCGTCGGCTT AGACCACACGAT amtB gene/ ACTTTCCAGCTGC CGCCGGTACGAA
AGTGATGGCAAC disrupted ACCAGCACCTGA TACGAATGACGC ACTTTCCAGCTGC amtB
gene TGGCCCATGGTC GTTGCAGCTCAG ACCAGCACCTGA ACACCTTCAGCG
CAACGAAAATTT TGGCCCATGGTC AAA TG ACACCTTCAGCG AAA/ CCGATCCCCATC
ACTGTGTGTCTTG TATTACAGTGCC GCTTCGTCGGCTT CGCCGGTACGAA TACGAATGACGC
CAACGAAAATTT TG-3' CI019 ds172 down 312 TGGTATTGTCAGT 346
CCGTCTCTGAAG 380 5'- Pnn1.2/ SEQ SEQ N/A CTGAATGAAGCT CTCTCGGTGAAC
TGGTATTGTCAGT disrupted ID ID CTTGAAAAAGCT ATTGTTGCGAGG
CTGAATGAAGCT nifL gene NO: NO: GAGGAAGCGGGC CAGGATGCGAGC
CTTGAAAAAGCT 406 407 GTCGATTTAGTAG TGGTTGTGTMG GAGGAAGCGGGC CAAG
TGCC AAATCAGTCCGA ACATTACCGATA GTCGATTTAGTA AAGT TCGC ATGCCGAGCCGC
ATGTGCCGCGTG GAAATCAGTCCG TCGC AACA CAGTTTGTCGAAT AACGGGTGCGTT
AATGCCGAGCCG CTCA ATGT C ATG CCAGTTTGTCGA CAGG TCAC ATC/
CCGTCTCTGAAG CTCTCGGTGAAC ATTGTTGCGAGG CAGGATGCGAGC TGGTTGTGTTTTG
ACATTACCGATA ATGTGCCGCGTG AACGGGTGCGTT ATG-3' CI019 ds172 up 313
ACCGATCCGCAG 347 TGAACATCACTG 381 5'- disrupted N/A N/A N/A
GCGCGCATTTGTT ATGCACAAGCTA ACCGATCCGCAG nifL gene/ ATGCCAATCCGG
CCTATGTCGAAG GCGCGCATTTGTT Prm1.2 CATTCTGCCGCCA AATTAACTAAAA
ATGCCAATCCGG GACGGGTTTTGC AACTGCAAGATG CATTCTGCCGCC ACTTGAGACACTT
CAGGCATTCGCG AGACGGGTTTTG TTGGGCGAGAAC TTAAAGCCGACT CACTTGAGACAC
CACCGTCTGCTGG TGAGAAATGAGA TTTTGGGCGAGA AGAT ACCACCGTCTGC TGG/
TGAACATCACTG ATGCACAAGCTA CCTATGTCGAAG AATTAACTAAAA AACTGCAAGATG
CAGGCATTCGCG TTAAAGCCGACT TGAGAAATGAGA AGAT-3' CI019 ds175 down 314
CGGGAACCGGTG 348 CCGTCTCTGAAG 382 5'- Prm3.1/ SEQ SEQ SEQ
TTATAATGCCGCG CTCTCGGTGAAC CGGGAACCGGTG disrupted ID ID ID
CCCTCATATTGTG ATTGTTGCGAGG TTATAATGCCGC nifL gene NO: NO: NO:
GGGATTTCTTAAT CAGGATGCGAGC GCCCTCATATTGT 408 409 410 GACCTATCCTGG
TGGTTGTGTTTTG GGGGATTTCTTA CGCC GGCA /56- GTCCTAAAGTTGT
ACATTACCGATA ATGACCTATCCT CTCA TAAC FAM/ AGTTGACATTAG ATGTGCCGCGTG
GGGTCCTAAAGT TATT GCAC TA CGGAGCACTAAC AACGGGTGCGTT TGTAGTTGACATT
GTGG CCGT ACC ATG AGCGGAGCACTA GGAT TCA CGT AC/ C/ CCGTCTCTGAAG
ZEN/T CTCTCGGTGAAC CTG ATTGTTGCGAGG AAG CAGGATGCGAGC CTC
TGGTTGTGTTTTG TCG ACATTACCGATA GT/ ATGTGCCGCGTG 3IABkFQ/
AACGGGTGCGTT ATG-3' CI019 ds175 up 315 ACCGATCCGCAG 349
TACAGTAGCGCC 383 5c disrupted N/A N/A N/A GCGCGCATTTGTT
TCTCAAAAATAG ACCGATCCGCAG nifL gene/ ATGCCAATCCGG ATAAACGGCTCA
GCGCGCATTTGTT Prm3.1 CATTCTGCCGCCA TGTACGTGGGCC ATGCCAATCCGG
GACGGGTTTTGC GTTTATTTTTTT CATTCTGCCGCC ACTTGAGACACTT ACCCATAATCGG
AGACGGGTTTTG TTGGGCGAGAAC GAACCGGTGTTA CACTTGAGACAC CACCGTCTGCTGG
TAATGCCGCGCC TTTTGGGCGAGA CTC ACCACCGTCTGC TGG/ TACAGTAGCGCC
TCTCAAAAATAG ATAAACGGCTCA TGTACGTGGGCC GTTTATTTTTTCT ACCCATAATCGG
GAACCGGTGTTA TAATGCCGCGCC CTC-3' CI006 ds20 down 316 TCAACCTAAAAA
350 AACTCACTTCAC 384 5'- Prm1/ SEQ SEQ SEQ AGTTTGTGTAATA
ACCCCGAAGGGG TCAACCTAAAAA disrupted ID ID ID CTTGTAACGCTAC
GAAGTTGCCTGA AGTTTGTGTAAT nifL gene NO: NO: NO: ATGGAGATTAAC
CCCTACGATTCCC ACTTGTAACGCT 411 412 413 TCAATCTAGAGG GCTATTTCATTCA
ACATGGAGATTA TAAA CAAA 56- GTATTAATAATG CTGACCGGAGGT ACTCAATCTAGA
CTGG TCGA FAM AATCGTACTAAA TCAAAATGACCC GGGTATTAAXAA TACT AGCG AAG
CTGGTACTGGGC AGCGAACCGAGT TGAATCGTACTA GGGC CCAG TTGC GC CG
AACTGGTACTGG GCAA ACGG CT/ GCGC/ CT TAT ZEN/G AACTCACTTCAC ACC
ACCCCGAAGGGG CTAC GAAGTTGCCTGA GATT CCCTACGATTCCC CCC/
GCTATTTCATTCA 3IABkFQ/ CTGACCGGAGGT TCAAAATGACCC AGCGAACCGAGT CG-3'
CI006 ds20 up 317 GGGCGACAAACG 351 CGTCCTGTAATA 385 5'- disrupted
N/A N/A N/A GCCTGGTGGCAC ATAACCGGACAA GGGCGACAAACG nifL gene/
AAATTGTCAGAA TTCGGACTGATTA GCCTGGTGGCAC Prm1 CTACGACACGAC
AAAAAGCGCCCT AAATTGTCAGAA TAACTGACCGCA TGTGGCGCTTTTT CTACGACACGAC
GGAGTGTGCGAT TTATATTCCCGCC TAACTGACCGCA GACCCTGAATAT TCCATTTAAAATA
GGAGTGTGCGAT GATGATGGATGC AAAAATCCAATC GACCCTGAATAT CGGC
GATGATGGATGC CGGC/ CGTCCTGTAATA ATAACCGGACAA TTCGGACTGATT
AAAAAAGCGCCC TTGTGGCGCTTTT TTTATATTCCCGC CTCCATTTAAAAT AAAAAATCCAAT
C-3' CI006 ds24 up 318 GGGCGACAAACG 352 GGACATCATCGC 386 5'-
disrupted SEQ SEQ SEQ GCCTGGTGGCAC GACAAACAATAT GGGCGACAAACG nifL
gene/ ID ID ID AAATTGTCAGAA TAATACCGGCAA GCCTGGTGGCAC Prm5 NO: NO:
NO: CTACGACACGAC CCACACCGGCAA AAATTGTCAGAA 414 415 416 TAACTGACCGCA
TTTACGAGACTG CTACGACACGAC GGTG GCGC /56- GGAGTGTGCGAT CGCAGGCATCCT
TAACTGACCGCA CACT AGTC FAM/ GACCCTGAATAT TTCTCCCGTCAAT GGAGTGTGCGAT
CTTT TCGT CA GATGATGGATGC TTCTGTCAAATAA GACCCTGAATAT GCAT AAAT GGA
CGGC AG GATGATGGATGC GGTT TGCC GTG CGGC/ T/ GGACATCATCGC ZEN/G
GACAAACAATAT CGA TAATACCGGCAA TGA CCACACCGGCAA CCC TTTACGAGACTG TGA
CGCAGGCATCCT AT/ TTCTCCCGTCAAT 3IABkFQ TTCTGTCAAATA AAG-3' CI006
ds24 down 319 TAAGAATTATCTG 353 AACTCACTTCAC 387 5'- Prm5/ N/A N/A
N/A GATGAATGTGCC ACCCCGAAGGGG TAAGAATTATCT disrupted ATTAAATGCGCA
GAAGTTGCCTGA GGATGAATGTGC nifL gene GCATAATGGTGC CCCTACGATTCCC
CATTAAATGCGC GTTGTGCGGGAA GCTATTTCATTCA AGCATAATGGTG AACTGCTTTTTTT
CTGACCGGAGGT CGTTGTGCGGGA TGAAAGGGTTGG TCAAAATGACCC AAACTGCTTTTTT
TCAGTAGCGGAA AGCGAACCGAGT TTGAAAGGGTTG AC CG GTCAGTAGCGGA AAC/
AACTCACTTCAC ACCCCGAAGGGG GAAGTTGCCTGA CCCTACGATTCCC GCTATTTCATTCA
CTGACCGGAGGT TCAAAATGACCC AGCGAACCGAGT CG-3' CI006 ds30 N/A 320
CGCCAGAGAGTC 354 TTTAACGATCTGA 388 5'- 5' UTR N/A N/A N/A
GAAATCGAACAT TTGGCGATGATG CGCCAGAGAGTC and ATG/ TTCCGTAATACCG
AAACGGATTCGC GAAATCGAACAT truncated CGATTACCCAGG CGGAAGATGCGC
TTCCGTAATACC glnE gene AGCCGTTCTGGTT TTTCTGAGAGCTG GCGATTACCCAG
GCACAGCGGAAA GCGCGAATTGTG GAGCCGTTCTGG ACGTTAACGAAA GCAGGATGCGTT
TTGCACAGCGGA GGATATTTCGCAT GCAGGAGGAGGA AAACGTTAACGA G TT
AAGGATATTTCG CATG/ TTTAACGATCTG ATTGGCGATGAT GAAACGGATTCG
CCGGAAGATGCG CTTTCTGAGAGCT GGCGCGAATTGT GGCAGGATGCGT TGCAGGAGGAGG
ATT-3' CI006 ds31 N/A 321 CGCCAGAGAGTC 355 GCACTGAAACAC 389 5'- 5'
UTR N/A N/A N/A GAAATCGAACAT CTCATTTCCCTGT CGCCAGAGAGTC and ATG/
TTCCGTAATACCG GTGCCGCGTCGC GAAATCGAACAT truncated CGATTACCCAGG
CGATGGTTGCCA TTCCGTAATACC glnE gene AGCCGTTCTGGTT GTCAGCTGGCGC
CFCGATTACCCAG GCACAGCGGAAA GCTACCCGATCCT GAGCCGTTCTGG ACGTTAACGAAA
GCTTGATGAATT TTGCACAGCGGA GGATATTTCGCAT GCTCGACCCGAA AAACGTTAACGA G
TA AAGGATATTTCG CATG/ GCACTGAAACAC CTCATTTCCCTGT GTGCCGCGTCGC
CGATGGTTGCCA GTCAGCTGGCGC GCTACCCGATCC TGCTTGATGAATT GCTCGACCCGAA
TA-3' CI019 ds34 N/A 322 GATGATGGATGC 356 GCGCTCAAACAG 390 5'- 5'
UTR N/A N/A N/A TTTCTGGTTAAAC TTAATCCGTCTGT GATGATGGATGC and ATG/
GGGCAACCTCGT GTGCCGCCTCGC TTTCTGGTTAAAC truncated TAACTGACTGACT
CGATGGTCGCGA GGGCAACCTCGT glnE gene AGCCTGGGCAAA CACAACTTGCAC
TAACTGACTGAC CTGCCCGGGCTTT GTCATCCTTTATT TAGCCTGGGCAA TTTTTGCAAGGAA
GCTCGATGAACT ACTGCCCGGGCT TCTGATTTCATG GCTCGACCCGCG TTTTTTTGCAAGG
CA AATCTGATTTCAT G/ GCGCTCAAACAG TTAATCCGTCTGT GTGCCGCCTCGC
CGATGGTCGCGA CACAACTTGCAC GTCATCCTTTATT GCTCGATGAACT GCTCGACCCGCG
CA-3' CI019 ds70 up 323 ACCGATCCGCAG 357 AGTCTGAACTCA 391 5'-
disrupted N/A N/A N/A GCGCGCATTTGTT TCCTGCGGCAGT ACCGATCCGCAG nifL
gene/ ATGCCAATCCGG CGGTGAGACGTA GCGCGCATTTGTT Prm4 CATTCTGCCGCCA
TTTTTGACCAAAG ATGCCAATCCGG GACGGGTTTTGC AGTGATCTACAT CATTCTGCCGCC
ACTTGAGACACTT CACGGAATTTTGT AGACGGGTTTTG TTGGGCGAGAAC GGTTGTTGCTGCT
CACTTGAGACAC CACCGTCTGCTGG TAAAAGGGCAAA TTTTGGGCGAGA T ACCACCGTCTGC
TGG/ AGTCTGAACTCA TCCTGCGGCAGT CGGTGAGACGTA TTTTTGACCAAA
GAGTGATCTACA TCACGGAATTTT GTGGTTGTTGCTG CTTAAAAGGGCA AAT-3' CI019
ds70 down 324 CATCGGACACCA 358 CCGTCTCTGAAG 392 5'- Prm4/ N/A N/A
N/A CCAGCTTACAAA CTCTCGGTGAAC CATCGGACACCA disrupted TTGCCTGATTGCG
ATTGTTGCGAGG CCAGCTTACAAA nifL gene GCCCCGATGGCC CAGGATGCGAGC
TTGCCTGATTGCG GGTATCACTGAC TGGTTGTGTTTTG GCCCCGATGGCC CGACCATTTCGTG
ACATTACCGATA GGTATCACTGAC CCTTATGTCATGC ATGTGCCGCGTG CGACCATTTCGT
GATGGGGGCTGG AACGGGTGCGTT GCCTTATGTCATG G ATG CGATGGGGGCTG GG/
CCGTCTCTGAAG CTCTCGGTGAAC ATTGTTGCGAGG CAGGATGCGAGC TGGTTGTGTTTTG
ACATTACCGATA ATGTGCCGCGTG AACGGGTGCGTT ATG-3' 137 ds799 down 325
TCTTCAACAACTG 359 GCCATTGAGCTG 393 5'- PinfC/ SEQ SEQ SEQ
GAGGAATAAGGT GCTTCCCGACCG TCTTCAACAACT disrupted ID ID ID
ATTAAAGGCGGA CAGGGCGGCACC GGAGGAATAAGG nifL gene NO: NO: NO:
AAACGAGTTCAA TGCCTGACCCTGC TATTAAAGGCGG 417 418 419 ACGGCACGTCCG
GTTTCCCGCTGTT AAAACGAGTTCA CTCG AGGG /56- AATCGTATCAAT TAACACCCTGAC
AACGGCACGTCC GCAG TGTT FAM/ GGCGAGATTCGC CGGAGGTGAAGC GAATCGTATCAA
CATG AAAC AA GCCCTGGAAGTT ATGATCCCTGAA TGGCGAGATTTCG GACG AGCG CGG
CGC TC CGCCCTGGAAGT TAA GGAA CAC TCGC/ A G/ GCCATTGAGCTG ZEN/T
GCTTCCCGACCG CCG CAGGGCGGCACC AAT TGCCTGACCCTG CGT CGTTTCCCGCTGT
ATC TTAACACCCTGA AA/ CCGGAGGTGAAG 3IABkFQ/ CATGATCCCTGA ATC-3' 137
ds799 up 326 TCCGGGTTCGGCT 360 AGCGTCAGGTAC 394 5'- disrupted N/A
N/A N/A TACCCCGCCGCGT CGGTCATGATTC TCCGGGTTCGGC nifL gene/
TTTGCGCACGGTG ACCGTGCGATTCT TTACCCCGCCGC PinfC TCGGACAATTTGT
CGGTTCCCTGGA GTTTTGCGCACG CATAACTGCGAC GCGCTTCATTGGC GTGTCGGACAAT
ACAGGAGTTTGC ATCCTGACCGAA TTGTCATAACTGC GATGACCCTGAA GAGTTCGCTGGC
GACACAGGAGTT TATGATGCTCGA TTCTTCCCAACCT TGCGATGACCCT G GAATATGATGCT
CGA/ AGCGTCAGGTAC CGGTCATGATTC ACCGTGCGATTC TCGGTTCCCTGG
AGCGCTTCATTG GCATCCTGACCG AAGAGTTCGCTG GCTTCTTCCCAAC CTG-3' 137
ds809 N/A 327 ATCGCAGCGTCTT 361 GCGCTGAAGCAC 395 5'- 5' UTR SEQ SEQ
SEQ TGAATATTTCCGT CTGATCACGCTCT ATCGCAGCGTCT and ATG/ ID ID ID
CGCCAGGCGCTG GCGCGGCGTCGC TTGAATATTTCCG truncated NO: NO: NO:
GCTGCCGAGCCG CGATGGTCGCCA TCGCCAGGCGCT glnE gene 420 421 422
TTCTGGCTGCATA GCCAGCTGGCGC GGCTGCCGAGCC GAGC GCCG /56- GTGGAAAACGAT
GCCACCCGCTGC GTTCTGGCTGCAT CGTT TCGG FAM AATTTCAGGCCA TGCTGGATGAGC
AGTGGAAAACGA CTGG CTGA TTAT GGGAGCCCTTAT TGCTGGATCCCA TAATTTCAGGCC
CTGC TAGA GGC G ACA AGGGAGCCCTTA ATAG GG GC/ TG/ ZEN/T
GCGCTGAAGCAC GAA CTGATCACGCTCT GCA GCGCGGCGTCGC CCTG CGATGGTCGCCA
ATC GCCAGCTGGCGC A/ GCCACCCGCTGC 3IABkFQ/ TGCTGGATGAGC TGCTGGATCCCA
ACA-3' 137 ds843 up 328 TCCGGGTTCGGCT 362 GCCCGCTGACCG 396 5'-
disrupted N/A N/A N/A TACCCCGCCGCGT ACCAGAACTTCC TCCGGGTTCGGC nifL
gene/ TTTGCGCACGGTG ACCTTGGACTCG TTACCCCGCCGC Prm1.2 TCGGACAATTTGT
GCTATACCCTTGG GTTTTGCGCACG CATAACTGCGAC CGTGACGGCGCG GTGTCGGACAAT
ACAGGAGTTTGC CGATAACTGGGA TTGTCATAACTGC GATGACCCTGAA CTACATCCCCATT
GACACAGGAGTT TATGATGCTCGA CCGGTGATCTTAC TGCGATGACCCT C GAATATGATGCT
CGA/ GCCCGCTGACCG ACCAGAACTTCC ACCTTGGACTCG GCTATACCCTTG
GCGTGACGGCGC GCGATAACTGGG ACTACATCCCCA TTCCGGTGATCTT ACC-3' 137
ds843 down 329 TCACTTTTTAGCA 363 GCCATTGAGCTG 397 5'- Prm1.2/ N/A
N/A N/A AAGTTGCACTGG GCTTCCCGACCG TCACTTTTCAGCA disrupted
ACAAAAGGTACC CAGGGCGGCACC AAGTTGCACTGG nifL gene ACAATTGGTGTA
TGCCTGACCCTGC ACAAAAGGTACC CTGATACTCGAC GTTTCCCGCTGTT ACAATTGGTGTA
ACAGCATTAGTG TAACACCCTGAC CTGATACTCGAC TCGATTTTTCATA CGGAGGTGAAGC
ACAGCATTAGTG TAAAGGTAATTTT ATGATCCCTGAA TCGATTTTTCATA G TC
TAAAGGTAATTT TG/ GCCATTGAGCTG GCTTCCCGACCG CAGGGCGGCACC
TGCCTGACCCTG CGTTTCCCGCTGT TTAACACCCTGA CCGGAGGTGAAG CATGATCCCTGA
ATC-3' 137 ds853 up 330 TCCGGGTTCGGCT 364 GCTAAAGTTCTC 398 5'-
disrupted N/A N/A N/A TACCCCGCCGCGT GGCTAATCGCTG TCCGGGTTCGGC nifL
gene/ TTTGCGCACGGTG ATAACATTTGAC TTACCCCGCCGC Prm6.2 TCGGACAATTTGT
GCAATGCGCAAT GTTTTGCGCACG CATAACTGCGAC AAAAGGGCATCA GTGTCGGACAAT
ACAGGAGTTTGC TTTGATGCCCTTT TTGTCATAACTGC GATGACCCTGAA TTGCACGCTTTCA
GACACAGGAGTT TATGATGCTCGA TACCAGAACCTG TGCGATGACCCT GC GAATATGATGCT
CGA/ GCTAAAGTTCTC GGCTAATCGCTG ATAACATTDGAC GCAATGCGCAAT
AAAAGGGCATCA TTTGATGCCCTTT TTGCACGCTTTCA TACCAGAACCTG GC-3' 137
ds853 down 331 GTTCTCCTTTGCA 365 GCCATTGAGCTG 399 5'- Prm6.2/ N/A
N/A N/A ATAGCAGGGAAG GCTTCCCGACCG GTTCTCCTTTGCA disrupted
AGGCGCCAGAAC CAGGGCGGCACC ATAGCAGGGAAG nifL gme CGCCAGCGTTGA
TGCCTGACCCTGC AGGCGCCAGAAC AGCAGTTTGAAC GTTTCCCGCTGTT CGCCAGCGTTGA
GCGTTCAGTGTAT TAACACCCTGAC AGCAGTTTGAAC AATCCGAAACTT CGGAGGTGAAGC
GCGTTCAGTGTA AATTTCGGTTTGG ATGATCCCTGAA TAATCCGAAACT A TC
TAATTTCGGTTTG GA/ GCCATTGAGCTG GCTTCCCGACCG CAGGGCGGCACC
TGCCTGACCCTG CGTTTCCCGCTGT TTAACACCCTGA CCGGAGGTGAAG CATGATCCCTGA
ATC-3' 137 ds857 up 332 TCCGGGTTCGGCT 366 CGCCGTCCTCGC 400 5'-
disrupted N/A N/A N/A TACCCCGCCGCGT AGTACCATTGCA TCCGGGTTCGGC nifL
gene/ TTTGCGCACGGTG ACCGACTTTACA TTACCCCGCCGC Prm8.2 TCGGACAATTTGT
GCAAGAAGTGAT GTTTTGCGCACG CATAACTGCGAC TCTGGCACGCAT GTGTCGGACAAT
ACAGGAGTTTGC GGAACAAATTCT TTGTCATAACTGC GATGACCCTGAA TGCCAGTCGGGC
GACACAGGAGTT TATGATGCTCGA TTTATCCGATGAC TGCGATGACCCT GAA
GAATATGATGCT CGA/ CGCCGTCCTCGC ACTACCATTGCA ACCGACTTTACA
GCAAGAAGTGAT TCTGGCACGCAT GGAACAAATTCT TGCCAGTCGGGC TTTATCCGATGAC
GAA-3' 137 ds857 down 333 GATATGCCTGAA 367 GCCATTGAGCTG 401 5'-
Prm8.2/ N/A N/A N/A GTATTCAATTACT GCTTCCCGACCG GATATGCCTGAA
disrupted TAGGCATTTACTT CAGGGCGGCACC GTATTCAATTACT nifL gene
AACGCAGGCAGG TGCCTGACCCTGC TAGGCATTTACTT CAATTTTGATGCT
GTTTCCCGCTGTT AACGCAGGCAGG GCCTATGAAGCG TAACACCCTGAC CAATTTTGATGCT
TTTGATTCTGTAC CGGAGGTGAAGC GCCTATGAAGCG TTGAGCTTGATC ATGATCCCTGAA
TTTGATTCTGTAC TC TTGAGCTTGATC/ GCCATTGAGCTG GCTTCCCGACCG
CAGGGCGGCACC TGCCTGACCCTG CGTTTCCCGCTGT TTAACACCCTGA CCGGAGGTGAAG
CATGATCCCTGA ATC-3' 63 ds908 down 334 TGGTATTGTCAGT 368
TCTTTAGATCTCT 402 5'- PinfC/ SEQ SEQ N/A CTGAATGAAGCT CGGTCCGCCCTG
TGGTATTGTCAGT disrupted ID ID CTTGAAAAAGCT ATGGCGGCACCT
CTGAATGAAGCT nifL gene NO: NO: GAGGAAGCGGGC TGCTGACGTTAC
CTTGAAAAAGCT 423 424 GTCGATTTAGTAG GCCTGCCGGTAC GAGGAAGCGGGC GGAA
GGGC AAATCAGTCCGA AGCAGGTTATCA GTCGATTTAGTA AACG GGAC ATGCCGAGCCGC
CCGGAGGCTTAA GAAATCAGTCCG AGTT CGAG CAGTTTGTCGAAT AATGACCCAGTT
AATGCCGAGCCG CAAC AGAT C ACC CCAGTTTGTCGA CGGC CTAA ATC/
TCTTTAGATCTCT CGGTCCGCCCTG ATGGCGGCACCT TGCTGACGTTAC GCCTGCCGGTAC
AGCAGGTTATCA CCGGAGGCTTAA AATGACCCAGTT ACC-3' 63 ds908 up 335
TGCAAATTGCAC 369 TGAATATCACTG 403 5'- disrupted N/A N/A N/A
GGTTATTCCGGGT ACTCACAAGCTA TGCAAATTGCAC nifL gene/ GAGTATATGTGT
CCTATGTCGAAG GGTFATTCCGGG PinfC GATTTGGGTTCCG AATTAACTAAAA
TGAGTATATGTG GCATTGCGCAAT AACTGCAAGATG TGATTTGGGTTCC AAAGGGGAGAAA
CAGGCATTCGCG GGCATTGCGCAA GACATGAGCATC TTAAAGCCGACT TAAAGGGGAGAA
ACGGCGTTATCA TGAGAAATGAGA AGACATGAGCAT GC AGAT CACGGCGTTATC AGC/
TGAATATCACTG ACTCACAAGCTA CCTATGTCGAAG AATTAACTAAAA AACTGCAAGATG
CAGGCATTCGCG TTAAAGCCGACT TGAGAAATGAGA AGAT-3' 910 ds960 up 336
TCAGGGCTGCGG 370 CTGGGGTCACTG 404 5'- disrupted N/A N/A N/A
ATGTCGGGCGTTT GAGCGCTTTATC TCAGGGCTGCGG nifL gene/ CACAACACAAAA
GGCATCCTGACC ATGTCGGGCGTT PinfC TGTTGTAAATGCG GAAGAATTTGCC
TCACAACACAAA ACACAGCCGGGC GGTTTCTTCCCGA ATGTTGTAAATG CTGAAACCAGGA
CCTGGCTGGCCC CGACACAGCCGG GCGTGTGATGAC CTGTTCAGGTTGT GCCTGAAACCAG
CTTTAATATGATG GGTGATGAATAT GAGCGTGTGATG C CA ACCTTTAATATG ATGC/
CTGGGGTCACTG GAGCGCTTTATC GGCATCCTGACC GAAGAATTTGCC GGTTTCTTCCCGA
CCTGGCTGGCCC CTGTTCAGGTTGT GGTGATGAATAT CA-3' 910 ds960 down 337
CGGAAAACGAGT 371 GCAATAGAACTA 405 5'- PinfC/ N/A N/A N/A
TCAAACGGCACG ACTACCCGCCCT CGGAAAACGAGT disrupted TCCGAATCGTATC
GAAGGCGGTACC TCAAACGGCACG nifL gene AATGGCGAGATT TGCCTGACCCTGC
TCCGAATCGTAT CGCGCCCAGGAA GATTCCCGTTATT CAATGGCGAGAT GTTCGCTTAACTG
TCATTCACTGACC TCGCGCCCAGGA GTCTGGAAGGTG GGAGGCCCACGA AGTTCGCTTAACT
AGCAGCTGGGTA TGACCCAGCGAC GGTCTGGAAGGT TT C GAGCAGCTGGGT ATT/
GCAATAGAACTA ACTACCCGCCCT GAAGGCGGTACC TGCCTGACCCTG CGATTCCCGTTAT
TTCATTCACTGAC CGGAGGCCCACG CC-3'
TABLE-US-00020 TABLE F Engineered Non-intergeneric Microbes Strain
Name Genotype SEQ ID NO CI006 16S rDNA-contig 5 62 CI006 16S
rDNA-contig 8 63 CI019 16S rDNA 64 CI006 nifH 65 CI006 nifD 66
CI006 nifK 67 CI006 nifL 63 CI006 nifA 69 CI019 nifH 70 CI019 nifD
71 CI019 nifK 72 CI019 nifL 73 CI019 nifA 74 CI006 Prm5 with 500 bp
75 flanking regions CI006 nifLA operon-upstream 76 intergenic
region plus nifL and nifA CDSs CI006 nifL (Amino Acid) 77 CI006
nifA (Amino Acid) 78 CI006 glnE 79 CI006 glnE_KO1 80 CI006 glnE
(Amino Acid) 81 CI006 glnE_KO1 (Amino Acid) 82 CI006 GME ATase
domain 83 (Amino Acid) CM029 Prm5 inserted into nifL 84 region
TABLE-US-00021 TABLE G Engineered Non-intergeneric Microbes
Associated Novel Strain SEQ ID Junction If Strain ID NO Genotype
Description Applicable CI63; 63 SEQ ID 16S N/A N/A CI063 NO 85
CI63; 63 SEQ ID nifH N/A N/A CI063 NO 86 CI63; 63 SEQ ID nifD1 1 of
2 unique genes annotated as nifD N/A CI063 NO 87 in 63 genome CI63;
63 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A CI063 NO
88 in 63 genome CI63; 63 SEQ ID nifK1 1 of 2 unique genes annotated
as nifK N/A CI063 NO 89 in 63 genome CI63; 63 SEQ ID nifK2 2 of 2
unique genes annotated as nifK N/A CI063 NO 90 in 63 genome CI63;
63 SEQ ID nifL N/A N/A CI063 NO 91 CI63; 63 SEQ ID nifA N/A N/A
CI063 NO 92 CI63; 63 SEQ ID glnE N/A N/A CI063 NO 93 CI63; 63 SEQ
ID amtB N/A N/A CI063 NO 94 CI63; 63 SEQ ID PinfC 500 bp
immediately upstrea of the ATG N/A CI063 NO 95 start codon of the
infC gene CI137 137 SEQ ID 16S N/A N/A NO 96 CI137 137 SEQ ID nifH1
1 of 2 unique genes annotated as nifH N/A NO 97 in 137 genome CI137
137 SEQ ID nifH2 2 of 2 unique genes annotated as nifH N/A NO 98 in
137 genome CI137 137 SEQ ID nifDl 1 of 2 unique genes annotated as
nifD N/A NO 99 in 137 genome CI137 137 SEQ ID nifD2 2 of 2 unique
genes annotated as nifD N/A NO 100 in 137 genorne CI137 137 SEQ ID
nifK1 1 of 2 unique genes annotated as nifK N/A NO 101 in 137
genome CI137 137 SEQ ID nifK2 2 of 2 unique genes annotated as nifK
N/A NO 102 in 137 genome CI137 137 SEQ ID nifL N/A N/A NO 103 CI137
137 SEQ ID nifA N/A N/A NO 104 CI137 137 SEQ ID glnE N/A N/A NO 105
CI137 137 SEQ ID PinfC 500 bp immediately upstream of the N/A NO
106 TTG start codon of infC CI137 137 SEQ ID amtB N/A N/A NO 107
CI137 137 SEQ ID Prm8.2 internal promoter located in nfpI gene; N/A
NO 108 299 bp starting at 81 bp after the A of the ATG of the nlpI
gene CI137 137 SEQ ID Prm6.2 300 bp upstream of the secE gene N/A
NO 109 starting at 57 bp upstream of the A of the ATG of secE CI137
137 SEQ ID Prm1.2 400 bp immediately upstream of the N/A No 110 ATG
of cspE gene none 728 SEQ ID 16S N/A N/A No 111 none 728 SEQ ID
nifH N/A N/A NO 112 none 728 SEQ ID nifD1 1 of 2 unique genes
annotated as nifD N/A NO 113 in 728 genome none 728 SEQ ID nifD2 2
of 2 unique genes annotated as nifD N/A NO 114 in 728 genome none
728 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A NO 115
in 728 genome none 728 SEQ ID nifK2 2 of 2 unique genes annotated
as nifK N/A NO 116 in 728 genome none 728 SEQ ID nifL N/A N/A NO
117 none 728 SEQ ID nifA N/A N/A NO 118 none 728 SEQ ID glnE N/A
N/A NO 119 none 728 SEQ ID amtB N/A N/A NO 120 none 850 SEQ ID 16S
N/A N/A NO 121 none 852 SEQ ID 16S N/A N/A NO 122 none 853 SEQ ID
16S N/A N/A NO 123 none 910 SEQ ID 16S N/A N/A NO 124 none 910 SEQ
ID nifH N/A N/A NO 125 none 910 SEQ ID Dinitrogenase iron- N/A N/A
NO 126 molybdenum cofactor CDS none 910 SEQ ID nifD1 N/A N/A NO 127
none 910 SEQ ID nifD2 N/A N/A NO 128 none 910 SEQ ID nifK1 N/A N/A
NO 129 none 910 SEQ ID nifK2 N/A N/A NO 130 none 910 SEQ ID nifL
N/A N/A NO 131 none 910 SEQ ID nifA N/A N/A NO 132 none 910 SEQ ID
glnE N/A N/A NO 133 MC 910 SEQ ID amtB N/A N/A NO 134 none 910 SEQ
ID PinfC 498 bp immediately upstream of the N/A NO 135 ATG of the
infC gene none 1021 SEQ ID 16S N/A N/A NO 136 none 1021 SEQ ID nifH
N/A N/A NO 137 none 1021 SEQ ID nifD1 1 of 2 unique genes annotated
as nifD N/A NO 138 in 910 genome none 1021 SEQ ID nifD2 2 of 2
unique genes annotated as nifD N/A NO 139 in 910 genome none 1021
SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A NO 140 in
910 genome none 1021 SEQ ID nifK2 2 of 2 unique genes annotated as
nifK N/A NO 141 in 910 genome none 1021 SEQ ID nifL N/A N/A NO 142
none 1021 SEQ ID nifA N/A N/A NO 143 none 1021 SEQ ID glnE N/A N/A
NO 144 none 1021 SEQ ID amtB N/A N/A NO 145 none 1021 SEQ ID PinfC
500 bp immediately upstream of the N/A NO 146 ATG start codon of
the infC gene none 1021 SEQ ID Prm1 348 bp includes the 319 bp
immediately N/A NO 147 upstream of the ATG start codon of the lpp
gene and the first 29 bp of the lpp gene none 1021 SEQ ID Prm7 339
bp upstream of the sspA gene, N/A NO 148 ending at 46 bp upstream
of the ATG of the sspA gene none 1113 SEQ ID 16S N/A N/A NO 149
none 1113 SEQ ID nifH N/A N/A NO 150 none 1113 SEQ ID nifD1 1 of 2
unique genes annotated as nifD N/A NO 151 in 1113 genome none 1113
SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 152 in
1113 genome none 1113 SEQ ID nifK N/A N/A NO 153 none 1113 SEQ ID
nifL N/A N/A NO 154 none 1113 SEQ ID nifA partial gene due to a gap
in the sequence assembly, N/A NO 155 we can only identify a partial
gene from the 1113 genome none 1113 SEQ ID glnE N/A N/A NO 156 none
1116 SEQ ID 16S N/A NO 157 none 1116 SEQ ID nifH N/A NO 158 none
1116 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A NO 159
in 1116 genome none 1116 SEQ ID nifD2 2 of 2 unique genes annotated
as nifD N/A NO 160 in 1116 genome none 1116 SEQ ID nifK1 1 of 2
unique genes annotated as nifK N/A NO 161 in 1116 genome none 1116
SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A NO 162 in
1116 genome none 1116 SEQ ID nifL N/A N/A NO 163 none 1116 SEQ ID
nifA N/A N/A NO 164 none 1116 SEQ ID glnE N/A N/A NO 165 none 1116
SEQ ID amtB N/A N/A NO 166 none 1293 SEQ ID 16S N/A N/A NO 167 none
1293 SEQ ID nifH N/A N/A NO 168 none 1293 SEQ ID nifD1 1 of 2
unique genes annotated as nifD N/A NO 169 in 1293 genome none 1293
SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A NO 170 in
1293 genome none 1293 SEQ ID nifK 1 of 2 unique genes annotated as
nifK N/A NO 171 in 1293 genome none 1293 SEQ ID nifK1 2 of 2 unique
genes annotated as nifK N/A NO 172 in 1293 genome none 1293 SEQ ID
nifA N/A N/A NO 173 none 1293 SEQ ID glnE N/A N/A NO 174 none 1293
SEQ ID amtB1 1 of 2 unique genes annotated as amtB N/A NO 175 in
1293 genome none 1293 SEQ ID amtB2 2 of 2 unique genes annotated as
amtB N/A NO 176 in 1293 genome none 1021-1612 SEQ ID
.DELTA.nifL::PinfC starting at 24 bp after the A of the ATG ds1131
NO 177 start codon, 1375 bp of nifL, have been deleted and replaced
with the 1021 PinfC promoter sequence none 1021-1612 SEQ ID
.DELTA.nifL::PinfC with starting at 24 bp after the A of the ATG
ds1131 NO 178 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the 1021 PinfC promoter sequence; 500 bp
flanking the nifL gene upstream and downstream are included none
1021-1612 SEQ ID glnE.DELTA.AR-2 glnE gene with 1673 bp immediately
ds1133 NO 179 downstream of the ATG start codon deleted, resulting
in a truncated glnE protein lacking the adenylyl-removing (AR)
domain none 1021-1612 SEQ ID glnE.DELTA.AR-2 with glnE gene with
1673 bp immediately ds1133 NO 180 500 bp flank downstream of the
ATG start codon deleted, resulting in a truncated glnE protein
lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE
gene upstream and downstream are included none 1021-1615 SEQ ID
.DELTA.nifL::Prm1 starting at 24 bp after the A of the ATG ds1145
NO 181 start codon, 1375 bp of nifL have been deleted and replaced
with the 1021 Prm1 promoter sequence none 1021-1615 SEQ ID
.DELTA.nifL:Prm1 with starting at 24 bp after the A of the ATG
ds1145 NO 182 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the 1021 rm1 promoter sequence; 500 bp
flanking the nifL gene upstream and downstream are included none
1021-1615 SEQ ID glnE.DELTA.AR-2 glnE gene with 1673 bp immediately
ds1133 NO 183 downstream of the ATG start codon deleted, resulting
in a truncated glnE protein lacking the adenylyl-removing (AR)
domain none 1021-1615 SEQ ID glnE.DELTA.AR-2 with glnE gene with
1673 bp immediately ds1133 NO 184 500 bp flank downstream of the
ATG start codon deleted, resulting in a truncated glnE protein
lacking the adenylyl-removing (AR) domain; 500 bp flanking the
glnE
gene upstream and downstream are included none 1021-1619 SEQ ID
.DELTA.nifL::Prm1 starting at 24 bp after the A of the ATG ds1145
NO 185 start codon, 1375 bp of nifL have been deleted and replaced
with the 1021 Prm1 promoter sequence none 1021-1619 SEQ ID
.DELTA.nifL:Prm1 with starting at 24 bp after the A of the ATG
ds1145 NO 186 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the 1021 rm1 promoter sequence; 500 bp
flanking the nifL gene upstream and downstream are included none
1021-1623 SEQ ID glnE.DELTA.AR-2 glnE gene with 1673 bp immediately
ds1133 NO 187 downstream of the ATG start codon deleted, resulting
in a truncated glnE, protein lacking the adenylyl-removing (AR)
domain none 1021-1623 SEQ ID glnE.DELTA.AR-2 with glnE gene with
1673 bp immediately ds1133 NO 188 500 bp flank downstream of the
ATG start codon deleted, resulting in a truncated glnE protein
lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE
gene upstream and downstream are included none 1021-1623 SEQ ID
.DELTA.nifL::Prm7 starting at 24 bp after the A of the ATG ds1148
NO 189 start codon, 1375 bp of nifL have been deleted and replaced
with the 1021 Prm7 promoter sequence none 1021-1623 SEQ ID
.DELTA.nifL::Prm7 with starting at 24 bp after the A of the ATG
ds1148 NO 190 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the 1021 rm7 promoter sequence; 500 bp
flanking the nifL gene upstream and downstream are included none
137-1034 SEQ ID glnE.DELTA.AR-2 glnE gene with 1290 bp immediately
ds809 NO 191 downstream of the ATG start codon deleted, resulting
in a truncated glnE protein lacking the adenylyl-removing (AR)
domain none 137-1034 SEQ ID glnE.DELTA.AR-2 with glnE gene with
1290 bp immediately ds809 NO 192 500 bp flank downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene
upstream and downstream are included none 137-1036 SEQ ID
.DELTA.nifL::PinfC starting at 24 bp after the A of the ATG ds799
NO 193 start codon, 1372 bp of nifL have been deleted and replaced
with the 137 PinfC promoter sequence none 137-1036 SEQ ID
.DELTA.nifL::PinfC with starting at 24 bp after the A of the ATG
ds799 NO 194 500 bp flank start codon, 1372 bp of nifL have been
deleted and replaced with the 137 PinfC promoter sequence; 500 bp
flanking the nifL gene upstream and downstream are included none
137-1314 SEQ ID glnE.DELTA.AR-2 36 bp glnE gene with 1290 bp
immediately none NO 195 deletion downstream of the ATG start codon
deleted AND 36 bp deleted beginning at 1472 bp downstream of the
start codon, resulting in a truncated glnE protein lacking the
adenylyl-removing (AR) domain none 137-1314 SEQ ID glnE.DELTA.AR-2
36 bp glnE gene with 1290 bp immediately none NO 196 deletion
downstream of the ATG start codon deleted AND 36 bp deleted
beginning at 1472 bp downstream of the start codon, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain;
500 bp flanking the nifL gene upstream and downstream are included
none 137-1314 SEQ ID .DELTA.nifL::Prm8.2 starting at 24 bp after
the A of the ATG ds857 NO 197 start codon, 1372 bp of nifL have
been deleted and replaced with the 137 Prm8.2 promotor sequence
none 137-1314 SEQ ID .DELTA.nifL::Prm8.2 with starting at 24 bp
after the A of the ATG ds857 NO 198 500 bp flank start codon, 1372
bp of nifL have been deleted and replaced with the 137 Prm8.2
promoter sequence: 500 bp flanking the nifL gene upstream and
downstream are included none 137-1329 SEQ ID glnE.DELTA.AR-2 36 bp
glnE gene with 1290 bp immediately none NO 199 deletion downstream
of the ATG start codon deleted AND 36 bp deleted beginning at 1472
bp downstream of the start codon resulting in a truncated gluE
protein lacking the adenylyl-removing (AR) domain none 137-1329 SEQ
ID glnE.DELTA.AR-2 36 bp glnE gene with 1290 bp immediately none NO
200 deletion downstream of the ATG start codon deleted AND 36 bp
deleted beginning at 1472 bp downstream of the start codon,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the nifL gene upstream and downstream
are included none 137-1329 SEQ ID .DELTA.nifL::Prm6.2 starting at
24 bp after the A of the ATG ds853 NO 201 start codon, 1372 bp of
nifL have been deleted and replaced with the 137 Prm6.2 promoter
sequence none 137-1329 SEQ ID .DELTA.nifL::Prm6.2 with starting at
24 bp after the A of the ATG ds853 NO 202 500 bp flank start codon,
1372 bp of nifL have been deleted and replaced with the 137 Prm6.2
promoter sequence; 500 bp flanking the nifL gene upstream and
downstream are included none 137-1382 SEQ ID .DELTA.nifL::Prm1.2
starting at 24 bp after the A of the ATG NO 203 start codon, 1372
bp of nifL have been ds843 deleted and replaced with the 137 Prm1.2
promoter sequence none 137-1382 SEQ ID .DELTA.nifL::Prm1.2 with
starting at 24 bp after the A of the ATG ds843 NO 204 500 bp flank
start codon, 1372 bp of nifL have been deleted and replaced with
the 137 Prm1.2 promoter sequence; 500 bp flanking the nifL gene
upstream and downstream are included none 137-1382 SEQ ID
glnE.DELTA.AR-2 36 bp glnE gene with 1290 bp immediately none NO
205 deletion downstream of the ATG start codon deleted AND 36 bp
deleted beginning at 1472 bp downstream of the start codon,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain none 137-1382 SEQ ID glnE.DELTA.AR-2 36 bp glnE gene
with 1290 bp immediately none NO 206 deletion downstream of the ATG
start codon deleted AND 36 bp deleted beginning at 1472 bp
downstream of the start codon, resulting in a truncated glnE
protein lacking the adenylyl-removing (AR) domain; 500 bp flanking
the nifL gene upstream and downstream are included none 137-1586
SEQ ID .DELTA.nifL::PinfC starting at 24 bp after the A of the ATG
ds799 NO 207 start codon, 1372 bp of nifL have been deleted and
replaced with the 137 PinfC promoter sequence none 137-1586 SEQ ID
.DELTA.nifL::PinfC with starting at 24 bp after the A of the ATG
ds799 NO 208 500 bp flank start codon, 1372 bp of nifL have been
deleted and replaced with the 137 PinfC promoter sequence; 500 bp
flanking the nifL gene upstream and downstream are included none
137-1586 SEQ ID glnE.DELTA.AR-2 glnE gene with 1290 bp immediately
NO 209 downstream of the ATG start codon deleted, resulting in a
truncated glnE ds809 protein lacking the adenylyl-removing (AR)
domain none 137-1586 SEQ ID glnE.DELTA.AR-2 with glnE gene with
1290 bp immediately ds809 NO 210 500 bp flank downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene
upstream and downstream are included none 19-594 SEQ ID
glnE.DELTA.AR-2 glnE gene with 1650 bp immediately ds34 NO 211
downstream of the ATG start codon deleted, resulting in a truncated
glnE protein lacking the adenylyl-removing (AR) domain none 19-594
SEQ ID glnE.DELTA.AR-2 with glnE gene with 1650 bp immediately ds34
NO 212 500 bp flank downstream of the ATG start codon deleted,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE gene upstream and downstream
are included none 19-594 SEQ ID .DELTA.nifL::Prm6.1 starting at 221
bp after the A of the ds180 NO 213 ATG start codon, 845 bp of nifL
have been deleted and replaced with the CI019 Prm6.1 promoter
sequence none 19-594 SEQ ID .DELTA.nifL::Prm6.1 with starting at
221 bp after the A of the ds180 NO 214 500 bp flank ATG start
codon, 845 bp of nifL have been deleted and replaced with the CI019
Prm6.1 promoter sequence; 500 bp flanking the nifL gene upstream
and downstream are included none 19-714 SEQ ID .DELTA.nifL::Prm6.1
starting at 221 bp after the A of the ds180 NO 215 ATG start codon,
845 bp of nifL have been deleted and replaced with the CI019 Prm6.1
promoter sequence none 19-714 SEQ ID .DELTA.nifL::Prm6.1 with
starting at 221 bp after the A of the ds180 NO 216 500 bp flank ATG
start codon, 845 bp of nifL have been deleted and replaced with the
CI019 Prm6.1promoter sequence; 500 bp flanking the nifL gene
upstream and downstream are included none 19-715 SEQ ID
.DELTA.nifL::Prm7.1 starting at 221 bp after the A of the ds181 NO
217 ATG start codon, 845 bp of nifL have been deleted and replaced
with the CI019 Prm7.1 promoter sequence none 19-715 SEQ ID
.DELTA.nifL::Prm7.1 with starting at 221 bp after the A of the
ds181 NO 218 500 bp flank ATG start codon, 845 bp of nifL have been
deleted and replaced with the CI019 Prm76.1promoter sequence; 500
bp flanking the nifL gene upstream and downstream are included
19-713 19-750 SEQ ID .DELTA.nifL::Prm1.2 starting at 221 bp after
the A of the ds172 NO 219 ATG start codon, 845 bp of nifL have been
deleted and replaced with the CI019 Prm1.2 promoter sequence 19-713
19-750 SEQ ID .DELTA.nifL::Prm1.2 with starting at 22l bp after the
A of the ds172 NO 220 500 bp flank ATG start codon, 845 bp of nifL
have been deleted and replaced with the CI019 Prm1.2 promoter
sequence; 500 bp flanking the nifL gene upstream and downstream are
included 17-724 19-804 SEQ ID .DELTA.nifL::Prm1.2 starting at 221
bp after the A of the ds172 NO 221 ATG start codon, 845 bp of nifL
have been deleted and replaced with the CI019 Prm1.2 promoter
sequence 17-724 19-804 SEQ ID .DELTA.nifL::Prm1.2 with starting at
221 bp after the A of the ds172 NO 222 500 bp flank ATG start
codon, 845 bp of nifL have been deleted and replaced with the CI019
Prm1.2 promoter sequence;
500 bp flanking the nifL gene upstream and downstream are included
17-724 19-804 SEQ ID glnE.DELTA.AR-2 glnE gene with 1650 bp
immediately ds34 NO 223 downstream of the ATG start codon deleted,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain 17-724 19-804 SEQ ID glnE.DELTA.AR-2 with glnE gene
with 1650 bp immediately ds34 NO 224 500 bp flank downstream of the
ATG start codon deleted, resulting in a truncated glnE protein
lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE
gene upstream and downstream are included 19-590 19-806 SEQ ID
.DELTA.nifL::Prm3.1 starting at 22l bp after the A of the ds175 NO
225 ATG start codon, 845 bp of nifL have been deleted and replaced
with the CI019 Prm3.1 promoter sequence 19-590 19-806 SEQ ID
.DELTA.nifL::Prm3.1 with starting at 221 bp after the A of the
ds175 NO 226 500 bp flank ATG start codon, 845 bp of nifL have been
deleted and replaced with the CI019 Prm3.1 promoter sequence; 500
bp flanking the nifL gene upstream and downstream are included
19-590 19-806 SEQ ID glnE.DELTA.AR-2 glnE gene with 1650 bp
immediately ds34 NO 227 downstream of the ATG start codon deleted,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain 19-590 19-806 SEQ ID glnE.DELTA.AR-2 with glnE gene
with 1650 bp immediately ds34 NO 228 500 bp flank downstream of the
ATG start codon deleted, resulting in a truncated glnE protein
lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE
gene upstream and downstream are included none 63-1146 SEQ ID
.DELTA.nifL::PinfC starting at 24 bp after the A of the ATG ds908
NO 229 start codon, 1375 bp of nifL have been deleted and replaced
with the 63 PinfC promoter sequence none 63-1146 SEQ ID
.DELTA.nifL::PinfC with starting at 24 bp after the A of the ATG
ds908 NO 230 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the 63 PinfC 500 bp flank sequence; 500
bp flanking the nifL gene upstream and downstream are included
CM015; 6-397 SEQ ID .DELTA.nifL::Prm5 starting at 31 bp after the A
of the ATG ds24 PBC6.15 NO 231 start codon, 1375 bp of nifL have
been deleted and replaced with the CI006 Prm5 promoter sequence
CM015; 6-397 SEQ ID .DELTA.nifL::Prm5 with starting at 31 bp after
the A of the ATG ds24 PBC6.15 NO 232 500 bp flank start codon, 1375
bp of nifL have been deleted and replaced with the CI006 Prm5
promoter sequence; 500 bp flanking the nifL gene upstream and
downstream are included CM014 6-400 SEQ ID .DELTA.nifL:Prm1
starting at 31 bp after the A of the ATG ds20 NO 233 start codon,
1375 bp of nifL have been deleted and replaced with the CI006 Prm1
promoter sequence CM014 6-400 SEQ ID .DELTA.nifL::Prm1 with
starting at 31 bp after the A of the ATG ds20 NO 234 500 bp flank
start codon, 1375 bp of nifL have been deleted and replaced with
the CI006 Prm1 promoter sequence; 500 bp flanking the nifL gene
upstream and downstream are included CM037; 6-403 SEQ ID
.DELTA.nifL::Prm1 starting at 31 bp after the A of the ATG ds20
PBC6.37 NO 235 start codon, 1375 bp of nifL have been deleted and
replaced with the CI006 Prm1 promoter sequence CM037; 6-403 SEQ ID
.DELTA.nifL::Prm1 with starting at 3l bp after the A of the ATG
ds20 PBC6.38 NO 236 500 bp flank start codon, 1375 bp of nifL have
been deleted and replaced with the CI006 Prm1 promoter sequence:
500 bp flanking the nifL gene upstream and downstream are included
CM037; 6-403 SEQ ID glnE.DELTA.AR-2 glnE gene with 1644 bp
immediately ds31 PBC6.3-9 NO 237 downstream of the ATG start codon
deleted, resulting in a truncated glnE protein lacking the
adenylyl-removing (AR) domain CM037; 6-403 SEQ ID glnE.DELTA.AR-2
with glnE gene with 1644 bp immediately ds31 PBC6.40 NO 238 500 bp
flank downstream of the ATG start codon deleted, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain;
500 bp flanking the glnE gene upstream and downstream are included
CM038; 6-404 SEQ ID glnE.DELTA.AR-1 glnE gene with 1287 bp
immediately ds30 PBC6.38 NO 239 downstream of the ATG start codon
deleted, resulting in a truncated glnE protein lacking the
adenylyl-removing (AR) domain CM038; 6-404 SEQ ID .DELTA.nifL::Prm1
starting at 31 bp after the A of the ATG ds20 PBC6.38 NO 240 start
codon, 1375 bp of nifL have been deleted and replaced with the
CI006 Prm1 promoter sequence CM038; 6-404 SEQ ID .DELTA.nifL:Prm1
with starting at 31 bp after the A of the ATG ds20 PBC6.38 NO 241
500 bp flank start codon, 1375 bp of nifL have been deleted and
replaced with the CI006 Prm1 promoter sequence; 500 bp flanking the
nifL gene upstream and downstream are included CM038; 6-404 SEQ ID
glnE.DELTA.AR4 with glnE gene with 1287 bp immediately ds30 PBC6.38
NO 242 500 bp flank downstream of the ATG start codon deleted,
resulting in a truncated glnE protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE gene upstream and downstream
are included CM029; 6-412 SEQ ID glnE.DELTA.AR-1 glnE gene with
1287 bp immediately ds30 PBC6.29 No 243 downstream of the ATG start
codon deleted, resulting in a truncated glnE protein lacking the
adenylyl-removing (AR) domain CM029; 6-412 SEQ ID glnE.DELTA.AR-1
with glnE gene with 1287 bp immediately ds30 PBC6.29 NO 244 500 bp
flank downstream of the ATG start codon deleted, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain;
500 bp flanking the glnE gene upstream and downstream are included
CM029; 6-412 SEQ ID .DELTA.nifL::Prm5 starting at 31 bp after the A
of the ATG ds24 PBC6.29 NO 245 start codon, 1375 bp of nifL have
been deleted and replaced with the CI006 Prm5 promoter sequence
CM029; 6-412 SEQ ID .DELTA.nifL::Prm5 with starting at 31 bp after
the A of the ATG ds24 PBC6.29 NO 246 500 bp flank start codon, 1375
bp of nifL have been deleted and replaced with the CI006 Prm5
promoter sequence; 500 bp flanking the nifL gene upstream and
downstream are included CM093; 6-848 SEQ ID .DELTA.nifL::Prm1
starting at 31 bp after the A of the ATG ds20 PBC6.93 NO 247 start
codon, 1375 bp of nifL have been deleted and replaced with the
CI006 Prm1 promoter sequence CM093; 6-848 SEQ ID .DELTA.nifL::Prm1
with starting at 31 bp after the A of the ATG ds20 PBC6.93 NO 248
500 bp flank start codon, 1375 bp of nifL have been deleted and
replaced with the CI006 Prm1 promoter sequence; 500 bp flanking the
nifL gene upstream and downstream are included CM093; 6-848 SEQ ID
glnE.DELTA.AR-2 glnE gene with 1644 bp immediately ds31 PBC6.93 NO
249 downstream of the ATG start codon deleted, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain
CM093; 6-848 SEQ ID glnE.DELTA.AR-2 with glnE gene with 1644 bp
immediately ds31 PBC6.93 NO 250 500 bp flank downstream of the ATG
start codon deleted, resulting in a truncated glnE protein lacking
the adenylyl-removing (AR) domain; 500 bp flanking the glnE gene
upstream and downstream are included CM093; 6-848 SEQ ID
.DELTA.amtB First 1088 bp of amtB gene and 4 bp ds126 PBC6.93 NO
251 upstream of start codon deleted; 199 bp of gene remaining lacks
a start endow no amtB protein is translated CM093; 6-848 SEQ ID
.DELTA.amtB with 500 bp First 1088 bp of amtB gene and 4 bp ds126
PBC6.93 NO 252 flank upstream of start codon deleted; 199 bp of
gene remaining lacks a start codon; no amtB protein is translated
CM094; 6-881 SEQ ID glnE.DELTA.AR-1 glnE gene with 1287 bp
immediately ds30 PBC6.94 NO 253 downstream of the ATG start codon
deleted, resulting in a truncated glnE protein lacking the
adenylyl-removing (AR) domain CM094; 6-881 SEQ ID glnE.DELTA.AR-1
with glnE gene with 1287 bp immediately ds30 PBC6.94 NO 254 500 bp
flank downstream of the ATG start codon deleted, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain;
500 bp flanking the gthE gene upstream and downstream are included
CM094; 6-881 SEQ ID .DELTA.nifL::Prm1 starting at 31 bp after the A
of the ATG ds20 PBC6.94 NO 255 start codon, 1375 bp of nifL have
been deleted and replaced with the CI006 Prm1 promoter sequence
CM094; 6-881 SEQ ID .DELTA.nifL::Prm1 with starting at 31 bp after
the A of the ATG ds20 PBC6.94 NO 256 500 bp flank start codon, 1375
bp of nifL have been deleted and replaced with the CI006 Prm1
promoter sequence; 500 bp flanking the nifL gene upstream and
downstream are included CM094; 6-881 SEQ ID .DELTA.amtB First 1088
bp of amtB gene and 4 bp ds126 PBC6.94 NO 257 upstream of start
codon deleted; 199 bp of gene remaining lacks a start codon; no
amtB protein is translated CM094; 6-881 SEQ ID .DELTA.amtB with 500
bp First 1088 bp of amtB gene and 4 bp ds126 PBC6.94 NO 258 flank
upstream of start codon deleted; 199 bp of gene remaining lacks a
start codon; no amtB protein is translated none 910-1246 SEQ ID
.DELTA.nifL::PinfC starting at 20 bp after the A of the ATG ds960
NO 259 start codon, 1379 bp of nifL have been deleted and replaced
with the 910 PinfC promoter sequence none 910-1246 SEQ ID
.DELTA.nifL::PinfC with starting at 20 bp after the A of the ATG
ds960 NO 260 500 bp flank start codon, 1379 bp of nifL have been
deleted and replaced with the 910 PinfC promoter sequence; 500 bp
flanking the nifL gene upstream and downstream are included PBC6.1,
CI006 SEQ ID 16S-1 1 of 3 unique 16S rDNA genes in the N/A 6, CI6
NO 261 CI006 genome PBC6.1, CI006 SEQ ID 16S-2 2 of 3 unique 16S
rDNA genes in the N/A 6, CI6 NO 262 CI006 genome PBC6.1, CI006 SEQ
ID nifH N/A N/A 6, CI6 NO 263 PBC6.1, CI006 SEQ ID nifD2 2 of 2
unique genes annotated as nifD N/A 6, CI6 NO 264 in CI006 genome
PBC6.1, CI006 SEQ ID nifK2 2 of 2 unique genes annotated as nifK
N/A 6, CI6 NO 265 in CI006 genome PBC6.1, CI006 SEQ ID nifL N/A
N/A
6, CI6 NO 266 PBC6.1, CI006 SEQ ID nifA N/A N/A 6, CI6 NO 267
PBC6.1, CI006 SEQ ID glnE N/A N/A 6, CI6 NO 268 PBC6.1, CI006 SEQ
ID 16S-3 3 of 3 unique 16S rDNA genes in the N/A 6, CI6 NO 269
CI006 genome PBC6.1, CI006 SEQ ID nifD1 1 of 2 unique genes
annotated as nifD N/A 6, CI6 NO 270 in CI006 genome PBC6.1, CI006
SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A 6, CI6 NO
271 in CI006 genome PBC6.1, CI006 SEQ ID amtB N/A N/A 6, CI6 NO 272
PBC6.1, CI006 SEQ ID Prm1 348 bp includes the 319 bp immediately
N/A 6, CI6 NO 273 upstream of the ATG start codon of the lpp gene
and the first 29 bp of the lpp gene PBC6.1, CI006 SEQ ID Prm5 313
bp starting at 432 bp upstream of the N/A 6, CI6 NO 274 ATG start
codon of the ompX gene and ending 119 bp upstream of the ATG start
codon of the ompX gene 19, CI19 CI019 SEQ ID nifL N/A N/A NO 275
19, CI19 CI019 SEQ ID nifA N/A N/A NO 276 19, CI19 CI019 SEQ ID
16S-1 1 of 7 unique 16S rDNA genes in the N/A NO 277 CI019 genome
19, CI19 CI019 SEQ ID 16S-2 2 of 7 unique 16S rDNA genes in the N/A
NO 278 CI019 genome 19, CI19 CI019 SEQ ID 16S-3 3 of 7 unique 16S
rDNA genes in the N/A NO 279 CI019 genome 19, CI19 CI019 SEQ ID
16S-4 4 of 7 unique 16S rDNA genes in the N/A NO 280 CI019 genome
19, CI19 CI019 SEQ ID 16S-5 5 of 7 unique 16S rDNA genes in the N/A
NO 281 CI019 genome 19, CI19 CI019 SEQ ID 16S-6 6 of 7 unique 16S
rDNA genes in the N/A NO 282 CI019 genome 19, CI19 CI019 SEQ ID
16S-7 7 of 7 unique 16S rDNA genes in the N/A NO 283 CI019 genome
19, CI19 CI019 SEQ ID nifH1 1 of 2 unique genes annotated as nifH
N/A NO 284 in CI019 genome 19, CI19 CI019 SEQ ID nifH2 2 of 2
unique genes annotated as nifH N/A NO 285 in CI019 genome 19, CI19
CI019 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A NO 286
in CI019 genome 19, CI19 CI019 SEQ ID nifD2 2 of 2 unique genes
annotated as nifD N/A NO 287 in CI019 genome 19, CI19 CI019 SEQ ID
nifK1 1 of 2 unique genes annotated as nifK N/A NO 288 in CI019 19,
CI19 CI019 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A
NO 289 in CI019 genome 19, CI19 CI019 SEQ ID glnE N/A N/A NO 290
19, CI19 CI019 SEQ ID prm4 449 bp immediately upstream of the N/A
NO 291 ATG of the dscC 2 gene 19, CI19 CI019 SEQ ID Prm1.2 500 bp
immediately upstream of the N/A NO 292 TTG start codon of the infC
gene 19, CI19 CI019 SEQ ID Prm3.1 170 bp immediately upstream of
the N/A NO 293 ATG start codon of the rplN gene 19, CI20 CI020 SEQ
ID Prm6.1 142 bp immediately upstream of the N/A NO 294 ATG of a
highly-expressed hypothetical protein (annotated as PROKKA_00662 in
CI019 assembly 82) 19, CI21 CI021 SEQ ID Prm7.1 293 bp immediately
upstream of the N/A NO 295 ATG of the lpp gene 19-375, CM67 SEQ ID
glnE.DELTA.AR-2 glnE gene with 1650 bp immediately ds34 19-417, NO
296 downstream of the ATG start codon CM067 deleted, resulting in a
truncated glnE protein lacking the adenylyl-removing (AR) domain
19-375, CM67 SEQ ID glnE.DELTA.AR-2 with glnE gene with 1650 bp
immediately ds34 19-417, NO 297 500 bp flank downstream of the ATG
start codon CM067 deleted, resulting in a truncated g1nE protein
lacking the adenylyl-removing (AR) domain; 500 bp flanking the glnE
gene upstream and downstream are included 19-375, CM67 SEQ ID
.DELTA.nifL::null-v1 starting at 221 bp after the A of the none
19-417, NO 298 ATG start codon, 845 bp of nifL have CM067 been
deleted and replaced with the 31 bp sequence
"GGAGTCTGAACTCATCCTGCGA TGGGGGCTG" 19-375, CM467 SEQ ID
.DELTA.nifL::null-v1 with starting at 221 bp after the A of the
none 19-417, NO 299 500 bp flank ATG start codon, 845 bp of nifL
have CM067 been deleted and replaced with the 31 bp sequence
"GGAGTCTGAACTCATCCTGCGA TGGGGGCTG"; 500 bp flanking the nifL gene
upstream and downstream are included 19-377, CM69 SEQ ID
.DELTA.nifL::null-v2 starting at 221 bp after the A of the none
CM069 NO 300 ATG start codon, 845 bp of nifL have been deleted and
replaced with the 5 bp sequence "TTAAA" 19-377 CM69 SEQ ID
.DELTA.nifL::null-v2 with starting at 221 bp after the A of the
none CM069 NO 301 500 bp flank ATG start codon, 845 bp of nifL have
been deleted and replaced with the 5 bp sequence "TTAAA"; 500 bp
flanking the nifL gene upstream and downstream are included 19-389,
CM81 SEQ ID .DELTA.nifL::Prm4 starting at 221 bp after the A of the
ds70 19-418, NO 302 ATG start codon, 845 bp of nifL have CM081 been
deleted and replaced with the CI19 Prm4 sequence 19-389, CM81 SEQ
ID .DELTA.nifL::Prm4 with starting at 221 bp after the A of the
ds70 19-418, NO 303 500 bp flank ATG start codon, 845 bp of nifL
have CM081 been deleted and replaced with the CI19 Prm4 sequence;
500 bp flanking the nifL gene upstream and downstream are
included
TABLE-US-00022 TABLE H SEQ ID NO: Sequence 61
atgttttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcg-
aattgtggcaggatgcgttgcaggagga
ggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgatttt-
cgcaaagagttggataaacgcacc
attggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacg-
atgcgccagtaccgctgtcacgcc
tgacgccgctgctcaccggaattattacccgcaccacttaccttgagagctaagtgaatttcccggcgcactg-
aaacacctcatttccctgtgtgccgc
gtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctc-
tatcaaccgacggcgatgaatgccta
tcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcgg-
cagtttaagcaggcgcagttgct
gcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcg-
gaagcgattattgatgcggtggtg
cagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggtt-
ttgcggtggtcggttatggcaag
ctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactgcccgatggatgtga-
tgaccgatggcgagcgtgaaatcg
atggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcat-
cctttatgaagttgatgcgcgtctgcgt
ccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctgga-
cgtgggaacatcaggcgctggcc
cgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatga-
cgcctcgcgacggcgcaacgctgc
aaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatct-
gaaagccgatgaaggcggtatca
ccgacatcgagtttatcgcccaatatctggtgctgugctttgcccatgacaagccgaaactgacgcgctggtc-
ggataatgcgcattctcgaagggc
tggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatga-
gctgcaccacctggcgctgcaa
gagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagt-
ggctggtggaaccgtgcgccccggc gtaa 62
ttgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagca-
cagagagcttgctctcgggtgacgagt
ggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaatacc-
gcataacgtcgcaagaccaaaga
gggggaccttcgggcctcttgccatcagaggcccagatgggattagctagtaggtggggtaacggctcaccta-
ggcgacgatccctagctggtag
agaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgc-
acaatgggcgcaagcctgatgc
agccatgccgcggtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaagggagtaaggttaa-
taaccttattcattgacgttacccg
cagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattac-
tgggcgtaaagcgcacgcaggc
ggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcatccgaaactggcaggcttgagtct-
cgtagagggaggtagaattccagg
tgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactgacgc-
tcaggtgcgaaagcgtgggga
gcaaacaggattagataccctggtagtccacgccgtaaacgatgtctatttggaggttgtgcccttgaggcgt-
ggcttccggagctaacgcgttaaata
gaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggcgagcat-
gtggtttaattcgatgcaacgc
gaagaaccttacctggtcttgacatccacagaactttccagagatggattggtgccttcgggaactgtgagac-
aggtgctgcatggctgtcgtcagctc
gtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggccgcg-
aactcaaaggagactgccagtgataa
actggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggc-
gcatacaaagagaagcgacctcg
cgagagtaagcggacctcataaagtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcgga-
atcgctagtaatcgtggatcagaat
gccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagta-
ggtagcttaaccttcgggagggc
gcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatc-
acctcctt 63
ttgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagca-
cagagagcttgctctcgggtgacgagt
ggcggacgggtgagtaatgtcgggaaactgcctgatggagggggataactactggaaacggtagctaataccg-
cataacgtcgcaagaccaaaga
gggggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctcacc-
taggcgacgatccctagctggtctg
agaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgc-
acaatgggcgcaagcctgatgc
agccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaggnantanggttaa-
taacctgtgttnattgacgttacc
cgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaatt-
actgggcgtaaagcgcacgcag
gcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggactgcatccgaaactggcaggcttgagtc-
tcgtagagggaggtagaattcca
ggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactgac-
gctcaggtgcgaaagcgtggg
gagcaaacaggattagataccctggtagtccacgccgtaaacgatgtctatttggaggttgtgcccttgaggc-
gtggcttccggagctaacgcgttaaa
tagaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggag-
catgtggtttaattcgatgcaac
gcgaagaaccttacctggtcttgacatccacagaacttagcagagatgctttggtgccttcgggaactgtgag-
acaggtgctgcatggctgtcgtcagc
tcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggttaggccg-
ggaactcaaaggagactgccagtgat
aaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccacggctacacacgtgctacaatg-
gcgcatacaaagagaagcgacct
cgcgagagtaagcggacctcataaagtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcg-
gaatcgctagtaatcgtggatcaga
atgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaag-
taggtagcttaaccttcgggaggg
cgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggat-
cacctcctt 64
attgaagagtttgatcatggctcagattgaacgctggcggtcaggcctaacacatgcaagtcgagcggcan-
cgggaagtagcttgctactttgccggc
gagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaa-
taccgcatgacctcgaaagagc
aaagtggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggtaatggct-
nacctaggcgacgatccctagct
ggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaa-
tattgcacaatgggcgcaagc
ctgatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaggcanca-
nacttaatacgtgtgntgattgac
gttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatc-
ggaattactgggcgtaaagcgca
cgcaggcggtttgttaagtcagatgtgaaatccccgagcttaacttcgggaactgcatttgaaactggcaagc-
tagagtcttgtagaggggggtagaatt
ccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagact-
gacgctcaggtgcgaaagcgt
ggggagcaattcaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttg-
aggcgtggcttccggagctaacgcgt
taagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggt-
ggagcatgtggtttaattcgatgc
aacgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaactct-
gagacaggtgctgcatggctgcgt
cagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtna-
tggtgggaactcaaaggagactgccg
gtgataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgcta-
caatggcatatacaaagagaagc
gaactcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtagcaactcgactccatgaa-
gtcggaatcgctagtaacgtaga
tcagaatgctacggtgaatacgttccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaa-
gaagtaggtagcttaaccttcggg
agggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggtt-
ggatcacctcctt 65
atgaccatgcgtcaatgcgccatttacggcaaaggtgggatcggcaaatcgaccaccacacagaacctggt-
cgccgcgctggcggagatgggtaaa
aaagtcatgattgtcggctgtgacccgaaagccgattccacgcgtttgatcctgcatgcgaaagcgcagaaca-
ccattatggagatggctgctgaagt
cggctccgtggaagacctggagttagaagacgtgctgcaaatcggttacggcggcgtgcgctgcgcagagtcc-
ggcggcccggagccaggcgtg
ggctgtgccggtcgcggggtgatcaccgcgattaacttcctcgaagaagaaggcgcttacgtgccggatctcg-
attttgttttctacgacgtgctgggc
gacgtggtatgcggtggtttcgccatgccgattcgtgaaaacaaagcgcaggagatctacatcgtttgctctg-
gcgaaatgatggcgatgtacgccgc
caacaacatctccaaaggcatcgtgaaatacgccaaatccggtaaagtgcgcctcggcgggctgatttgtaac-
tcgcgccagaccgaccgtgaagat
gaactgatcattgcgctggcagaaaaactcggcacgcagatgatccactttgttcccgcgacaacattgtgca-
gcgtgcggaaatccgccgtatgac
ggttatcgaatatgacccgacctgcaatcaggcgaacgaatatcgcagccttgccagcaaaatcgtcaacaac-
accaaaatggtggtgcccaccccc
tgcaccatggatgaactggaagaactgctgatggagttcggcattatggatgtggaagacaccagcatcattg-
gtaaaaccgccgccgaagaaaacg ccgtctga 66
atgagcaatgcaacaggcgaacgcaacctggagataatcgagcaggtgctcgaggttttcccggagaagac-
gcgcaaagaacgcagaaaacacat
gatggtgacggacccggagcaggaaagcgtcggtaagtgcatcatctctaaccgcaaatcgcagccaggcgtg-
atgaccgtgcgcggctgctcgta
tgccggttcgaaaggggtggtatttgggccaatcaaggatatggcgcatatctcgcatggcccaatcggctgc-
ggccaatactcccgcgccgggcgg
cggactactacaccggcgtcagcggcgtggacagcttcggcacgctcaacttcacctccgattttcaggagcg-
cgacatcgtgtttggcggcgataa
aaagctcgccaaactgattgaagagctggaagagctgttcccgctgaccaaaggcatttcgattcagtcggaa-
tgcccggtcggcctgattggcgatg
acattgaggccgtcgcgaacgccagccgcaaagccatcaacaaaccggttattccggtgcgttgcgaaggctt-
tcgcggcgtgtcgcaatccctcgg
tcaccatattgccaacgatgtgatccgcgactgggtgctggataaccgcgaaggcaaaccgttcgaatccacc-
ccttacgatgtggcgatcatcggcg
attacaacatcggcggcgatgcctgggcttcgagcattttgctcgaagagatgggcttgcgggtggtggcaca-
gtggtctggcgacggtacgctggt
ggagatggaaaacacgccgttcgtcaaactgaacctggtgcattgttaccgctcaatgaactacatctcgcgc-
catatggaggagaagcacggtattc
cgtggatggaatacaacttcttggtccgacgaaaatcgcggaatcgctgcgcaaaatcgccgaccagtttgac-
gacaccattcgcgccaacgccga
agcggtgatcgccagataccaggcgcaaaacgacgccattatcgccaaatatcgcccgcgtctggaggggcgc-
aaagtgctgctttatatgggcgg
gctgcgtccgcgccatgtgattggcgcctatgaagacctgggaatggagatcatcgctgccggttatgagttc-
ggtcataacgatgattacgaccgca
ccttgccggatctgaaagagggcacgctgctgtttgatgatgccagcagttatgagaggaggcgttcgtcaac-
gcgctgaaaccggatctcatcggtt
ccggcatcaaagagaagtacatctttcagaaaatgggcgtgccgtttcgccagatgcactcctgggattactc-
cggcccgtaccacggctatgacggc
ttcgccatcttcgcccgcgatatggatatgacgctcaacaaccccgcgtggggccagttgaccgcgccgtggc-
tgaaatccgcctga 67
atgagccagactgctgagaaaatacagaattgccatcccctgtttgaacaggatgcttaccagacgctgtt-
tgccggtaaacgggcactcgaagaggc
gcactcgccggagcgggtgcaggagtgtttcaatggaccactaccccggaatatgaagcgctgaactttaaac-
gcgaagcgctgactatccaccc
ggcaaaagcctgccagccgctgggcgcggtgctctgttcgctggggtttgccaataccctaccgtatgtgcac-
gcttcacaggttgcgtggcctattt
ccgcacgtactttaaccgccactttaaagaaccggtggcctgcgtgtcggattcaatgacggaagacgcggcg-
gtgttcggcgggaataacaacctc
aacaccggcttacaaaacgccagcgcgctgtataaaccggagattatcgccgtctctaccacctgtatggcgg-
aagtgatcggtgatgatttgcaggc
ctttatcgccaacgccaaaaaagatggttttctcgatgccgccatccccgtgccctacgcgcacacccccagt-
tttatcggcagccatatcaccggctg
ggataacatgtttgaaggttttgcccggacctttacggagaccatgaagctcagcccggcaaactttcacgca-
tcaacctggtgaccgggtttgaaac
ctatctcggcaatttccgcgtgctgaaacgcatgatggaacaaatggaggtgccggcgagtggctctccgatc-
cgtcggaagtgctgcatactcccg
ccaacgggcattaccagatgtacgcgggcgggacgacgcagcaagagatgcgcgaggcgccggatgctatcga-
caccctgttcctgcagccctg
gcaactggtgaaaagcaaaaaagtggtgcaggagatgtggaatcagcccgccaccgaggtttctgttcccgtt-
gggctggcaggaacacacgaact
gttgatggcgattagccagttaaccggcaaggccattcccgattcactggcgctggagcgccggcggctggtc-
gatatgatgctcgattcccacacct
ggttgcacggtaaaaaattcggcctgaggcgatccggattttgtcatgggattgacccgtttcctgctggagc-
tgggctgcgaaccgaccgttatcctc
tgccacaacggtaacaaacgctggcagaaagcaatgaagaaaatgcttgacgcctcgccgtacggccaggaga-
gcgaagtttatcaactgcgatt
tgtggcatttccgctcgctgatgtttacccgccagccggattttatgattggcaactcgtacggcaagttcat-
tcagcgcgacaccttagccaaaggcga
gcagtttgaagttccgctgatccgcctcggttttccccttcgaccgccaccatctgcaccgccagaccacctg-
gggctacgagggcgccatgagca
ttctcactacccttgtgaatgcggtactggagaaagtggacaaagagaccatcaagctcggcaaaaccgacta-
cagcttcgatcttatccgttaa 68
atgaccctgaatatgatgatggatgccggcgcgcccgaggcaatccccggtgcgctttcgcgacaccatcc-
tgggctgttttttaccatcgttgaagaa
gcgcccgtcgccatttcgctgactgatgccgacgcacgcattgtctatgccaacccggctttctgccgccaga-
ccggctatgaactagaagcgttgttg
cagcaaaatccccgcctgatgcaagtcgccaaaccccacgggaaatctatcaggatatgtggcacaccttgtt-
acaacgccgaccgtggcgcgggc
aattgattaaccgccaccgcgacggcagcctgatctggtcgagatcgatatcaccccggtgattaacccgttt-
ggcgaactggaacactacctggca
atgcagcgcgatatcagcgccagttatgcgctggagcagcggttgcccaatcacatgacgctgaccgaagcgg-
tgctgaataacattccggcggcg
gtggttgtagtggatgaacgcgatcatgtggttatggataaccttgcctacaaaacgttctgtgccgactgcg-
gcggaaaagagctcctgagcgaactc
aatttttcagcccgaaaagcggagctggcaaacggccagctcttaccggtggtgctgcgcggtgaggtgcgct-
ggttgtcggtgacctgctgggcgc
tgccgcgcgtcagcgaagaagccagcgctactttattgataacaggctgacgcgcacgctggggtgatcaccg-
acgacacccaacaacgccagc
agcaggaacagggccgacttgaccgccttaaacagcagatgaccaacggcaaactactggcagcgatccgcga-
agcgcttgacgccgcgctgatc
cagcttaactgccccatcaatatgctggcggcggcgcgacgtttaaacggcagtgataacaacaatgtggcgc-
tcgacgccgcgtggcgcgaaggt
gaagaggcgatggcgcggctgaaacgttgccgcccgtcgctggaactggaaaggcggccgtctggccgctgca-
acccattttgacgatctgcgc
gcgctttatcttcacccgctacgagcaggggaaaaatttgcaggtcacgctggattcccatcatctggtggga-
tttggtcagcgtacgcaactgttagcct
gcctgagtctgtggctcgatcgcacgctggatattgccgccgggctgggtgatttcaccgcgcaaacgcagat-
ttacgcccgcgaagaagagggctg
gctactttgtatatcactgacaatgtgccgagatcccgctgcgccacacccactcgccggatgcgcttaacgc-
tccgggaaaaggcatggagctgc
gcctgatccagacgctggtggcacaccaccacggcgcaatagaactcacttcacaccccgaagggggaagttg-
cctgaccctacgattcccgctatt tcattcactgaccgcaggttcaaaatga 69
atgacccagcgaaccgagtcgggtaataccgtctggcgatcgatttgtcccagcagttcactgcgatgcag-
cgcataaggtggtactcagccggg
cgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgcacaatgacgcctttttgcagcacggcat-
gatctgctgtacgacagccagcag
gcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatccccggcagctcgcaaatccgctatc-
gtccgggcgaagggctggtcggga
cggtgctttcgcagggccaatcattaggctggcgcgcgttgctgacgatcagcgctttcttgaccggctcggg-
ttgtatgattacaacctgccgtttatc
gccgtgccgctgatagggccagatgcgcagactttcggtgtgagacggcacaacccatggcgcgttacgaaga-
gcgattacccgcctgcacccgc
tttctggaaacggtcgctaacctggtcgcgcaaaccgtgcgtttgatggcaccaccggcagtgcgcccttccc-
cgcgcgccgccataacacaggccg
ccagcccgattatcctgcacggcctcacgcgcatttggttttgaaaatatggtcggtaacagtccggcgatgc-
gccagaccatggagattatccgcag
gtttcgcgctgggacaccaccgttctggtacgcggcgagagtggcaccggcaaggagctgattgccaacgcca-
tccaccaccattcgccgcgtgcc
ggtgcgccatttgtgaaattcaactgtgcggcgctgccggacacactgctggaaagcgaattgttcggtcacg-
agaaaggggcatttaccggcgcgg
tacgccagcgtaaaggccgttttgagctggccgatggcggcacgctgtttcttgacgagatcggcgagagtag-
cgcctcgtttcaggctaagctgctg
cgcattttgcaggaaggcgaagggaacgcgtcggcggcgacgagacattgcaagtgatttgtgcgcattattg-
ccgcgacgaaccgcaatcttgaa
gatgaagtccggctggggcactttcgcgaagatctctattatcgcctgaatgtgatgcccatcgccctgccgc-
cactacgcgaacgccaggaggacat
tgccgagctggcgcactactggtgcgtaaaatcgcccataaccagagccgtacgctgcgcattagcgagggcg-
ctatccgcctgctgatgagctaca
actggcccggtaatgtgcgcgaactggaaaactgcatgagcgctcagcggtgatgtcggagaacggtctgatc-
gatcgggatgtgattttgtttaatc
atcgcgaccagccagccaaaccgccagttatcaggtctcgcatgatgataactggctcgataacaaccttgac-
gagcgccagcggctgattgcggc
gctggaaaaagcgggatgggtacaagccaaagccgcgcgcttgctggggatgacgccgcgccaggtcgcctat-
cgtattcagacgatggatataac cctgccaaggctataa 70
atggcaatgcgtcaatgtgcaatctacgggaaagggggtattggtaaatccaccactacccaaaaccttgt-
agcggctctggccgaaatgaataagaa
ggtcatgatcgtcggctgtgaccctaaggctgattcaacccgactcattctgcatgcgaaagcacagaacacc-
atcatggaaatggccgctgaagtgg
gctccgtggtagatctggagctggaagatgtgatgcaaatcggctatggcggcgtgcgctggcggaatcaggc-
ggccctgagcctggtgtgggtt
gtgccggacgcgggggtgatcaccgccatcaacttcctcgaagaagaaggcgcgtatgtgccggatctggatt-
tcgtgttttacgacgtattgggcgat
gtggtctgtggcggtttcgcgatgccaattcgcgaaaacaaagcgcaggaaatctacatcgtatgctccggtg-
aaatgatggcgatgtatgccgccaa
caacatttccaaaggcatcgtgaaatacgcgaaatcgggcaaagttcgcctggccgggctgatctgtaactcc-
cgccagacggatcgcgaagatgaa
ctgatcatcgcgaggctgaaaaacttggcacgcaaatgatccacttcgtgccgcgtgacaacattgtgcaacg-
cgctgaaatccgccgcatgacggt
catcgaatacgacccgacttgtgcgcaggcagatcagtcgtgcactggcgaacaaaatcgtcaacaacaccaa-
aatggtggtgccgacaccggtc
accatggatgagctggaagccctgttaatggaatttggcattatggaagaagaagacctgaccatcgtcggtc-
gtaccgccgccgaagaggcgtga 71
atgaccagtgaaacacgcgaacgtaacgaggcattgatccaggaagtgctggagatcttccccgagaaggc-
gcttaaagatcgtaagaaacacatg
atgaccaccgacccggcgatggaatctgtcggcaaggtattgtctcaaaccgcaaatcacagccgggcgtgat-
gaccgtgcgaggctgcgcttacg
ccggttccaaaggcgtggtctttggcccgatcaaagacatggcgcatatctcccacggcccggttggttgcgg-
ccagtattctcgtgccggacgccgt
aactattacaccggctggagggcgtgaacagctttggcaccctcaacttcaccagtgattttcaggaacggga-
catcgtatttggcggcgataaaaag
ctcgacaaactgatcgacgaactggagatgttgtcccgctgaccaaaggcatttcggtacagtcggaatgtcc-
ggtcggtctgatcggcgatgacatt
tctgccgtcgccaaagccagcagcgccaaaatcggtaagccggtcgtgccggtacgctgcgaggggttccgcg-
gtgtgtcgcaatcgctcggccat
cacattgctaacgatgtcatccgcgactgggtgctggataaccgcgaaggcaatgaatttgaaaccacgcctt-
acgacgtggcgattatcggcgacta
caacatcggcggtgacgcctgggcctcacgtattctgctcgaagaaatggggctgcgtgtggtggcgcagtgg-
tccggcgacggcacgctggtgga
gatggaaaacaccccgaaagtcgcactcaatctggtgcactgctaccgctcgatgaactacatctcccgcata-
tggaagaaaaacacggcattccgt
ggatggaatacaacttctttggcccgtccaaaattgcggaatctctgcgcgaaatcgcggcgcgttttgacga-
taccatccggaaaaacgccgaagc
ggtgattgaaaaatatcaggcgcaaacgcaggcggtgatcgacaaataccgtccgcgtaggaaggcaaaaagg-
tgctgttgtatctcggcggtttac
gtccgcgccacatcatcggggcgtatgaagatctgggaatggaaatcatcggtaccggctatgaattcggtca-
taacgatgattacgaccgcaccttac
cgatgctcaaagaaggcacgttgctgttcgatgacctgtgcagttatgagctggaagcgttcgttaaagcgct-
gaaaccggatcttgtcgggtcaggc
atcaaagaaaaatacattttccacaaaatgggcgtgccgttccgccagatgcactcctgggattattccggcc-
cttatcacggctacgacggtttcggca
tttttgcccgtgacatggacatgacgctgaacaatccgggctggagtcagctgaccgccccctggttgaaatc-
ggcctga 72
atgagtcaagatcttggcaccccaaaatcctgtttcccgctgacgagcaggatgaataccagagtatgttt-
acccacaaacgcgcgaggaagaagca
cacggcgaggcgatagtgcgggaagtgtttgaatggaccaccacgcaggaatatcaggatctgaacttctcgc-
gtgaagcgctgaccgtcgacccg
gcgaaagcctgccagccgttaggcgcggtactttgcgcgctgggttttgccaacacgttgccgtatgtccacg-
gttcacaaggctgtgtggcgtatttc
cgtacctattttaatcctcatttcaaagagccggtggcctgtgtttccgactcaatgaccgaagatgccgccg-
tttttggcggaaataacaacatgaatgtc
ggtctggaaaacgccagcgcgctgtacaagccgganattattgctgtctccaccacctgtatggcggaagtga-
tcggtgatgacctgcaggcttttatc
gccaacgccaaaaaagacggatttgtggatgccgggatgccaatcccgtatgcccatacaccgagtttctggg-
cagtcatgtcaccggctgggacaa
catgtttgaaggcttcgcccgtacctttaccaccgacgccacgcgggaatatcagccgggcaaacttgccaaa-
ctgaacgtggtgaccggttttgaaa
cttatctcggcaactaccgggttattcaccgcatgatgagccagatgggggtcgaatgcagcgtcttgtccga-
tccgtctgaagtgctcgacaccccgg
ctgacggccaataccgcatgtatgccggcggcaccacgcaaaccgaaatgcgtgatgcaccggatgccatcga-
caccttgctgctgcaaccgtggc
aattgcagaaaaccaaaaaagtggtgcagggcgactggaatcagccgggcaccgaagtcagtgtaccgattgg-
cctggcggcgaccgatgccttg
ctgatgacggtangcgaactgaccggcaaaccgatagctgacacgctggcgactgaacgtggccgtctggtgg-
acatgatgctcgattcccacacct
ggctgcatggcaagcgtttcggtctctacggtgacccggattttgtgatgggcatgaccgcattcctgctgga-
actgggctgtgaaccgaccaccattct
cagccataacggcaacaaacgctggcagaaagccatgaagaaaatgctggctgattcgccttacgggcaggac-
agcgaagtgtattgtgaactgcga
tctgtggcatttccgctcgctgatgtttacccgtaaaccggactttatgatcggcaactcaacggaaaattca-
ttcagcgtgacacgctggccaaaggcg
aacagttcgaagtgccgctgatccgcatcggttttccgatttttgaccggcaccatttgcaccgtcagaccac-
ctggggatacgaaggggcgatgagc
atactgacgcaactggtgaatgccgtacttgaacaactggatcgcgaaaccatgaagctcggcaaaaccgact-
acaacttcgtcctgttccgctaa 73
atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatcc-
ttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcanctgccgccagacggg-
ttttgcacttgagacacttttgggcg
agaaccaccgtctgctggccagccagcagacgccgaaacatatctatgacgaaatgtggcgcactttgagcag-
ggcaaatcctggaacggccaact
gatcaaccggcgtaataaccgttcgctttatctggcggatgtcactatcacgcctgttttaggcgggacgggc-
aggtggagcattacctcggcatgca
caaagatatcagcgagaaatacgcgctggaacagcggttgcgcaaccacatcaccttgttcacggaggtgctg-
aacaatattcccgccgccgtggtg
gtggtggatgagcaggacaatgtggtgatggacaatctggcctacaaaaccctttgcgcggactgcggcggca-
aagagctgctggctgaaatgggc
tatccgcaactcaaagagatgctcancagtggcgaaccggtgccggtttccatgcgcggcaacgtacgctggt-
tttctttcggtcaatggttattcagg
gcgttaatgattgaggccagccgcttttttaccggcattaccgcgccgggaaaactgattgttctgaccgact-
gcaccgatcagcatcaccggcagcag
cagggttatcttgaccggcttaagcaaaaactcaccaacggcaaattattggcggccatccgtgagtcgctcg-
atgccgcgcttatccagctcaacgg
gccaatcaatatgctggcggctccgcgtcgtcttaacggcgaagaaggcaacaacatggcgctggaattcgcc-
tggcgcgaaggcgagcaggcgg
tgagtcgcttacaggcctgccgtccgtcgctggattttgagccgcaggcagaatggccggtcagtgaattctt-
tgacgatctgagcgcgctgtacgaca
gccattttctcagtgacggtgaattgcgttacgtggtcatgccatctgatctgcacgctgtcgggcaacgaac-
gcaaatccttaccgcgctgagcttatg
gattgatcacacgctgtcacaggcgcaggccatgccgtctctgaagctctcggtgaacattgcgcgaggcagg-
atgcgagctggttgtgttttgacatt
accgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccggggaatggca-
tggagctgcgccttatccagacgc
tgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcct-
gccggtacagcaggttatcaccggag gcttaaatga 74
atgacccagttcctaccgcgggcccggttatccggcgctttgatatgtctgcccagtttacggcgctttat-
cgcatcagcgtggcgctgagtcaggaa
agcaacaccgggcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggcatgg-
tgtgtctgtttgataaagaacgcaat
gcactctttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttaccgca-
tgggggaaggcgtgatcggcgcg
gtgatgagccagcgtcaggcgctcgtgttaccgcgcatttcagacgatcagcgttttctcgaccgcctgaata-
tttacgattacagccttccgttgattgg
cgtgcccgatccccggtgcggataatcagccatcgggcgtgctggtggcacagccgatggcgttgcacgaaga-
ccggctgactgccagtacgcggtt
tttagaaatggtcgccaatctcatcagccagccactgcgttctgccacgcccccggaatcattgcctgctcaa-
acgccggtccggtgcagtgttccgcg
ccagtttggtttcgagcagatggtcgggaaaagtcaggcgatgcgccagacgatggacattttacggcaggtt-
tccaaatgggataccacggttctggt
gcgtggtgaaagcggcaccggcaaggaacttatcgccaatgccattcattacaactcaccccgtgcggccgcg-
ccatttgtgaaattcaactgcgccg
cgctgccggataacctgctggaaagcgaactgttcggtcatgaaaaaggggccttcaccggcgctatccgtac-
ccgtaaaggccgctttgaactggc
ggacgggggcacgttattcctcgatgaaatcggcgaatcgagcgcgtcgtttcaggccaaattgctgcgcatt-
ttgcaggaaggtgaaatggaacgg
gtcggcggcgataccacgctgaaagttgatgtgcgcattattgctgccaccaaccgtaatcttgaagaggaag-
tgcgtgccgggaattttcgcgaaga
cctgtattatcgcctgaacgtgatgccggtttcgctgcctgcactgcgtgaaaggctggatgatatcgccgat-
ctggcgccgtttaggtcaattaagatt
gcgctgcgtcaggggcgggaactgcgcatcagcgacggtgcggtgcgtctgctgatgacctacagctggccag-
gcaacgtgcgtgaactggaaaa
ctgtctcgaacgggcgtcggtaatgaccgatgaagggctgatcgaccgcgacgtgatcctgttcaatcaccat-
gaatccccggcgctgtccgtcaaac
ccggcctgccgctcgcgacagatgaaagctggctggatcaggaactcgacgaacgccagcgggtgattgccgc-
actggagaaaaccggctgggt
gcaggccaaagcggcccgactgctgggcatgacaccgcgccagattgcctaccgtatccagattatggacatc-
aacatgcaccgtatctga 75
aaaactaccgccgcaattaatgaacccaacgctactgttgccgggccatgctcttccccggcgcgctgccc-
ggaaaggatatagattgcccagcacg
cgccagcaccaagcgcgaacgccgcgccagtgagatcaacatgtgaaacattttcgcccagcggcagcagata-
caagaggccaagtaccgccag
gatcacccagatgaaatccaccgggcggcgtgaggcaaaaagcgccaccgccagcgggccggtaaattccagc-
gccaccgcaacgccgagcgg
tatcgtctggatcgataaatagaacatatagttcatggcgccgagcgacaggccataaaacagcagtggcagg-
cgttgttcacgggtaaaatgtaaac
gccagggcttgaacactacgaccaaaataagggtgccaagtgcgagacgcagcgcggtgacgccgggtgcgcc-
aacaatcggaaacagtgatttc
gccagcgacgcgcctccctgaatggacatcatcgcgacaaacaatattaataccggcaaccacaccggcaatt-
tacgagactgcgcaggcatccttt
ctcccgtcaatttctgtcaaataaagtaaaagaggcagtctacttgaattacccccggctggttgagcgttgt-
tgaaaaaaagtaactgaaaaatccgta
gaatagcgccactctgatggttaattaacctattcaattaagaattatctggatgaatgtgccattaaatgcg-
cagcataatggtgcgttgtgcgggaaaa
ctgcttttttttgaaagggttggtcagtagcggaaactttctgttacatcaaatggcgctttagaccccaatt-
cccgcaaagagtttcttaactaattttgatat
atttaaacgcgtaggacgtaggatttacttgaagcacatttgaggtggattatgaaaaaaattgcatgtcttt-
cagcactggccgcacttctggcggtttct
gcaggttccgcagtagcagcaacttcaaccgtaactggcggctacgctcagagcgacgctcagggtattgcta-
acaaaactaacggtttcaacctgaa
atatcgctacgagcaggacaacaacccgctgggtgttatcggttcctttacttacactgaaaaagatcgcacc-
gaaagcagcgtttataacaaagcgca
gtactacggcatcaccgcaggcccggcttaccgcatcaacgactgggcgagcatctacggtgttgtgggtgta-
ggttacggtaaattccagcagattg tagacaccgctaaagtgtctgacaccagcgactacg 76
aacacacgctcctgttganaaagagatcccgccgggaaatgcggtgancgtgtctgatattgcgaagagtg-
tgccagttttggcgcgggcaaaacct
gcaccagtttggtattaatgcaccagtctggcgctttttttcgcgagtttctcctcgctaatgcccgccaggc-
gcggctttggcgctgatagcgcgctg
aataccgatctggatcaaggttttgtcgggttatcagccaaaaggtgcactctttgcatggttatacgtgcct-
gacatgttgtccgggcgacaaacggcct
ggtggcacaaattgtcagaactacgacacgactaactgaccgcaggagtgtgcgatgaccctgaatatgatga-
tggatgccggcgcgcccgaggca
atcgccggtgcgctttcgcgacaccatcctgggctgttttttaccatcgttgaagaagcgcccgtcgccattt-
cgctgactgatgccgacgcacgcattg
tctatgccaacccggctttctgccgccagaccggctatgaactagaagcgttgttgcagcaaaatccccgcct-
gcttgcaagtcgccaaaccccacgg
gaaatctatcaggatatgtggcacaccattgttacaacgccgaccgtggcgcgggcaattgattaaccgccac-
cgcgacggcagcctgtatctggtcga
gatcgatatcaccccggtgattaacccgtttggcgaactggaacactacctggcaatgcagcgcgatatcagc-
gccagttatgcgctggagcagggt
tgcgcaatcacatgacgctgaccgaagcggtgctgaataacauccggcggcggtggngtagtggatgaacgcg-
atcatgtggttatggataaccttg
cctacaaaacgttctgtgccgactgcggcggaaaagagctcctgaggaactcaatttttcagcccgaaaaggg-
agctggcaaacggccaggtctt
accggtggtgctgcgcggtgaggtgcgctggttgtcggtgacctgctgggcgctgccgggcgtcagcgaagaa-
gccagttcgctactttattgataac
aggctgacgcgcacgctggtggtgatcaccgacgacacccaacaacgccagcagcaggaacagggccgacttg-
accgccttaaacagcagatga
ccaacggcaaactactggcagcgatccgcgaagcgcttgacgccgcgctgatccagataactgccccatcaat-
atgctggcggcggcgcgacgttt
aaacggcagtgataacaacaatgtggcgctcgacgccgcgtggcgcgaaggtgaagtggcgatggcgcggctg-
aaacgttgccgcccgtcgctg
gaactggaaagtgcggccgtctggccgctgcaaccctttttgacgatctgcgcgcgctttatcacacccgcta-
cgagcaggggaaaaatttgcaggt
cacgctggattcccatcatctggtgggatttggtcagcgtacgcaactgttagcctgcctgagtctgtggctc-
gatcgcacgctggatattgccgccgg
gctgggtgatttcaccgcgcaaacgcagatttacgcccgcgaagaagagggctggctctctttgtatatcact-
gacaatgtgccgctgatcccgctgcg
ccacacccactcgccggatgcgcttaacgctccgggaaaaggcatgcacctgcgcctgatccagacgctggtg-
gcacaccaccacggcgcaatag
aactcacttcacaccccgaagggggaagttgcctgaccctacgattcccgctattcattcactgaccggaggt-
tcaaatgacccagcgaaccgagt
cgggtaataccgtctggcgcttcgatttgtcccagcagttcactgcgatgcagcgcataagcgtggtactcag-
ccgggcgaccgaggtcgatcagac
gctccagcaagtgctgtgcgtattgcacaatgacgcctttttgcagcacggcatgatctgtctgtacgacagc-
cagcaggcgattttgaatattgaagcg
ttgcaggaagccgatcagcagttaatccccggcagctcgcaaatccgctatcgtccgggcgaagggctggtcg-
ggacggtgctttcgcagggccaa
tcattagtgctggcgcgcgttgctgacgatcaggctttcttgaccggctcgggttgtatgattacaacctgcc-
gtttatcgccgtgccgctgatagggcc
agatgcgcagactttcggtgtgctgacggcacaacccatggcgcgttacgaagagcgattacccgcctgcacc-
cgctttctggaaacggtcgctaac
ctggtcgcgcaaaccgtgcgtttgatggcaccaccggcagtgcgcccttccccgcgcgccgccataacacagg-
ccgccagcccgaaatcctgcac
ggcctcacgcgcatttggttttgaaaatatggtcggtaacagtccggcgatgcgccagaccatggagattatc-
cgtcaggtttcgcgctgggacaccac
cgttctggtacgcggcgagagtggcaccggcaaggagctgattgccaacgccatccaccaccattcgccgcgt-
gccggtgcgccatttgtgaaattc
aactgtgcggcgctgccggacacactgctggaaagcgaattgttcggtcacgagaaaggggcatttaccggcg-
cggtacgccagcgtaaaggccg
ttttgagctggccgatggcggcacgctgtttcttgacgagatcggcgagagtagcgcctcgtttcaggctaag-
ctgctgcgcattttgcaggaaggcga
aatggaacgcgtcggcggcgacgagacattgcaagtgaatgtgcgcattattgccgcgacgaaccgcaatctt-
gaagatgaagtccggctggcgca
ctttcgcgaagatctctattatcgcctgaatgtgatgcccatcgccctgccgccactacgcgaacgccaggag-
gacattgccgagctggcgcactttct
ggtgcgtaaaatcgcccataaccagagccgtacgctgcgcattagcgagggcgctatccgcctgctgatgagc-
tacaactggcccggtaatgtgcgc
gaactggaaaactgccttgagcgctcagcggtgatgtcggagaacggtctgatcgatcgggatgtgattttgt-
ttaatcatcgcgaccagccagccaaa
ccgccagttatcagcgtctcgcatgatgataactggctcgataacaaccttgacgagcgccagggctgattgc-
ggcgctggaaaaaggggatgg
gtacaagccaaagccgcgcgcttgctggggatgacgccgcgccaggtcgcctatcgtattcagacgatggata-
taaccctgccaaggctataa 77
MTLNMMMDAGAPEAIAGALSRHHPGLFFTIVEEAPVAISLTDADARIVYANPAFcRQTGYELE
ALLQQNPRLLASRQTPREIYQDMWHTLLQRRPWRGQLINRHRDGSLYLVEIDITPVINPFGELE
HYLAMQRDISASYALEQRLRNHMTLTEAVLNNIPAAVVVVDERDHVVMDNLAYKTFcADcG
GKELLSELNFSARKAELANGQVLPVVLRGEVRWLSVTcWALPGVSEEASRYFIDNRLTRTLVV
ITDDTQDRQQQEQGRLDRLKQQMTNGKLLAAIREALDAALIQLNcPINMLAAARRLNGSDNN
NVALDAAWREGEEAMARLKRcRPSLELESAAVWPLQPFFDDLRALYHTRYEQGKNLQVTLD
SHHLVGFGQRTQLLAcLSLWLDRTLDIAAGLGDFTAQTQIYAREEEGWLSLYITDNVPLIPLRH
THSPDALNAPGKGMELRLIQTLVAHHHGAIELTSHPEGGScLTLRFPLFHSLTGGSK 78
MTQRTESGNTVWRFDLSQQFTAMQRISVVLSRATEVDQTLQQVLcVLHNDAFLQHGMIcLYD
SQQAILNIEALQEADQQLIPGSSQIRYRPGEGLVGTVLSQGQSLVLARVADDQRFLDRLGLYDY
NLPFIAVPLIGPDAQTFGVLTAQPMARYEERLPAcTRFLETVANLVAQTVRLMAPPAVRPSPRA
AITQAASPKScTASRAFGFENMVGNSPAMRQTMEIIRQVSRWDTTVLVRGESGTGKELIANAIH
HHSPRAGAPFVKFNcAALPDTLLESELFGHEKGAFTGAVRQRKGRFELADGGTLFLDEIGESSA
SFQAKLLRILQEGEMERVGGDETLQVNVRIIAATNRNLEDEVRLGHFREDLYYRLNVMPIALPP
LRERQEDIAELAHFLVRKIAHNQSRTLRISEGAIRLLMSYNWPGNVRELENcLERSAVMSENGL
IDRDVILFNHRDQPAKPPVISVSHDDNWLDNNLDERQRLIAALEKAGWVQAKAARLLGMTPR
QVAYRIQTMDITLPRL 79
atgccgcaccacgcagcattgtcgcagcactggcaaacggtattttctcgtctgccggaatcgctcaccgc-
gcagccattgagcgcgcaggcgcagt
cagtgctcacttttagtgattttgttcaggacagcatcatcgcgcatcctgagtggctggcagagcttgaaag-
cgcgccgccgcctgcgaacgaatggc
aacactatgcgcaatggctgcaagcggcgctggatggcgtcaccgatgaagcctcgctgatgcgcgcgctgcg-
gctgtttcgccgtcgcatcatggt
gcgcatcgcctggagccaggcgttacagttggtggcggaagaagatatcctgcaacagcttagcgtgctggcg-
gaaaccctgatcgtcgccgcgcg
cgactggctttatgaggcctgctgccgtgagtggggaacgccgagcaatccacaaggcgtggcgcagccgatg-
ctggtactcggcatgggcaaact
gggtggcggcgaactcaatttctcatccgatatcgatttgattttcgcctggccggaaaatggcgcaacgcgc-
ggtggacgccgtgagctggataacg
cgcaatttttcactcgccttggtcaacggctgattaaagtcctcgaccagccaacgcaggatggctttgtcta-
ccgcgtcgatatgcgcttgcgcccgttt
ggcgacagcggcccgctggtgctgagctttgccgcgctggaagattactaccaggagcaggggcgcgattggg-
aacgctacgcgatggtgaaagc
gcgcattatgggcgataacgacggcgaccatgcgcgggagttgcgcgcaatgctgcgcccgtttgttttccgc-
cgtatatcgacttcagcgtgaaca
gtccctgcgtaacatgaaaggcatgattgcccgcgaaggtgcgtcgccgtggcctgaaggacaacattaagct-
cggcgcgggcgggatccgcgaaat
agaatttatcgtccaggttttccagagattcgcggcggtcgcgagcctgcactgcaatcgcgacactgttgcc-
gacgcttgctgccatagatcaactg
catctgctgccggatggcgacgcaacccggctgcgcgaggcgtatttgtggctgcgacggctggagaacctgc-
tgcaaagcatcaatgacgaacag
acacagacgctgccgggcgatgaactgaatcgcgcgcgcctcgcctggggaatgggcaaagatagctgggaag-
cgctctgcgaaacgctggaag
cgcatatgtcggcggtgcgtcagatatttaacgatctgattggcgatgatgaaacggattcgccggaagatgc-
gctttctgagagctggcgcgaattgt
ggcaggatgcgttgcaggaggaggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgt-
ggtggcgctgattgccgattttcg
caaagagttggataaacgcaccattggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgaca-
gcgatgtatgctcgcgcgacgat
gcgccagtaccgctgtcacgcctgacgccgctgacaccggaattattacccgcaccacttaccttgagctgct-
aagtgaatttcccggcgcactgaaa
cacctcatttccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatg-
aattgctcgacccgaatacgctctatc
aaccgacggcgatgaatgcctatcgcgatgagagcgccaatacctgctgcgcgtgccggaagatgatgaagag-
caacagcttgaggcgctgcgg
cagtttaagcaggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtga-
gcgatcacttaacctggctggcgg
aagcgattattgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatct-
gcacgatcgcgaagggcgcggtt
ttgcggtggtcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcct-
gcacgactgcccgatggatgtgatga
ccgatggcgagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttag-
cacgcgcacgtcgtccggcatccttt
atgaagttgatgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccga-
ttaccagcaaaacgaagcctggacg
tgggaacatcaggcgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgcca-
ttcgccgcgatattctgatcacgc
ctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaa-
gcataaagaccgcttcgatctga
aagccgatgaaggcgftatcaccgacatcgagtttatcgcccaatataggtgaggctttgcccatgacaagcc-
gaaactgacgcgctggtcggat
aatgtgcgcattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctgg-
cgtacaccacattgcgtgatga
gctgcaccacctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgctt-
attaaaaccagctgggacaagtggc tggtggaaccgtgcgccccggcgtaa 80
atgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcga-
attgtggcaggatgcgttgcaggagga
ggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgatttt-
cgcaaagagttggataaacgcacc
attggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacg-
atgcgccagtaccgctgtcacgcc
tgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcact-
gaaacacctcatttccctgtgtgccgc
gtcgccgaggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctct-
atcaaccgacggcgatgaatgccta
tcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcgg-
cagtttaagcaggcgcagttgct
gcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcg-
gaagcgattattgatgcggtggtg
cagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggtt-
ttgcggtggtcggttatggcaag
ctgggcggctgggagctgggttacagctccgatctggatctggattcctgcacgactgcccgatggatgtgat-
gaccgatggcgagcgtgaaatcg
atggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcat-
cctttatgaagttgatgcgcgtctgcgt
ccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctgga-
cgtgggaacatcaggcgctggcc
cgtgcgcgcgtggtgtacggcgaccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgac-
gcctcgcgacggcgcaacgctgc
aaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatct-
ganagccgatgaaggcggtatca
ccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtc-
ggataatgtgcgcattctcgaagggc
tggcgcaaaacggcatcatggaggagcaggaagcgcaggcattcacgctggcgtacaccacattgcgtgatga-
gctgcaccacctggcgctgcaa
gagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagt-
gcctggtggaaccgtgcgccccggc gtaa 81
MPHHAGLSQHWQTVFSRLPESLTAQPLSAQAQSVLTFSDFVQDSIIAHPEWLAELESAPPPANE
WQHYAQWLQAALDGVTDEASLMRALRLFRRRIMVRIAWSQALQLVAEEDILQQLSVLAETLI
VAARDWLYEAccREWGTPSNPQGVAQPMLVLGMGKLGGGELNFSSDIDLIFAWPENGATRG
GTRELDNAQFFTRLGQRLIKVLDQPTQDGFVYRVDMRLRPFGDSGPLVLSFAALEDYYQEQGR
DWERYAMVKARIMGDNDGDHARELRAMLRPFVFRRYIDFSVIQSLRNMKGMIAREVRRRGL
KDNIKLGAGGIREIEFIVQVFQLIRGGREPALQSRSLLPTLAAIDQLHLLPDGDATRLREAYLWL
RRLENLLQSINDEQTQTLPGDELNRARLAWGMGKDSWEALcETLEAHMSAVRQIFNDLIGDD
ETDSPEDALSESWRELWQDALQEEDSTPVLAHLSEDDRRRVVALIADFRKELDKRTIGPRGRQ
VLDHLMPHLLSDVcSRDDAPVPLSRLTPLLTGIITRTTYLELLSEFPGALKHLISLcAASPMVAS
QLARYPILLDELLDPNTLYQPTAMNAYRDELRQYLLRVPEDDEEQQLEALRQFKQAQLLRVA
AADIAGTLPVMKVSDHLTWLAEAIIDAVVQQAWGQMVARYGQPTHLHDREGRGFAVVGYG
KLGGWELGYSSDLDLVFLHDcPMDVMTDGEREIDGRQFYLRLAQRVMHLFSTRTSSGILYEV
DARLRPSGAAGMLVTTTESFADYQQNEAWTWEHQALARARVVYGDPQLTAEFDAIRRDILM
TPRDGATLQTDVREMREKMRAHLGNKHKDRFDLKADEGGITTDIEFIAQYLVLRFAHDKPKLT
RWSDNVRILEOLAQNGIMEEQEAQALTLAYTTLRDELHHLALQELPGHVALScFVAERALIKT
SWDKWLVEPcAPA 82
MFNDLIGDDETDSPEDALSESWRELWQDALQEEDSTPVLAHLSEDDRRRVVALIADFRKELDK
RTIGPRGRQVLDHLMPHLLSDVcSRDDAPVPLSRLTPLLTGIITRTTYLELLSEFPGALKHLISLc
AASPMVASQLARYPILLDELLDPNTLYQPTAMNAYRDELRQYLLRVPEDDEEQQLEALRQFKQ
AQLLRVAAADIAGTLPVMKVSDHLTWLAEAIIDAVVQQAWGQMVARYGQPTHLHDREGRGF
AVVGYGKLGGWELGYSSDLDLVFLHDcPMDVMTDGEREIDGRQFYLRLAQRVMHLFSTRTSS
GILYEVDARLRPSGAAGMLVTTTESFADYQQNEAWTWEHQALARARVVYGDYQLTAEFDAIR
RDILMTPRDGATLQTDVREMEREKMRAHLGNKHKDRFDLKADEGGITDIEFIAQYLVLRFAHD
KPKLTRWSDNVRILEGLAQNGIMEEQEAQALTLAYTTLRDELHHLALQELPGHVALScFVAER
ALIKTSWDKWLVEPcAPA 83
EEQQLEALRQFKQAQLLRVAAADIAGTLPVMKVSDHLTWLAEAIIDAVVQQAWGQMVARYG
QPTHLHDREGRGFAVVGYGKLGGWELGYSSDLDLVFLHDcPMDVMTDGEREIDGRQFYLRL
AQRVMHLFSTRTSSGILYEVDARLRPSGAAGMLVTTTESFADYQQNEAWTWEHQALARARV
VYGDPQLTAEFDAIRRDILMTPRDGATLQTDVREMREKMRAHLGNKHKDRFDLKADEGGITD
IEFIAQYLVLRFAHDKPKLTRWSDNVRILEGLAQNGIMEEQEAQALTLAYTTLRDELHHLALQE
LPGHVALScFVAERALIKTSWDKWLVEPcAPA 84
ccgagcgtcggggtgcctaatatcagcaccggatacgagagaaaagtgtctacatcggttcggttgatatt-
gaccggcgcatccgccagcccgccca
gtttctggtggatctgtttggcgattttgcgggtcttgccggtgtcggtgccgaaaaaaataccaatatttgc-
cataacacacgctcctgttgaaaaagag
atcccgccgggaaatgcggtgaacgtgtctgatattgcgaagaggtgccagttttatcgcgggcaaaacctgc-
accagtttggttattaatgcacca
gtctggcgctttttttcgccgagtttctcctcgctaatgcccgccaggcgcggctttggcgctgatagcgcgc-
tgaataccgatctggatcaaggttttgtc
gggttatcagccaaaaggtgcactctttgcatggttatacgtgcctgacatgttgtccgggcgacaaacggcc-
tggtggcacaaattattgtcagaactacg
acacgactaactgaccgcaggagtgtgcgatgaccctgaatatgatgatggatgccggcggacatcatcgcga-
caaacaatattaataccggcaacc
acaccggcaatttacgagactgcgcaggcatcctttctcccgtcaatttctgtcaaataaagtaaaagaggca-
gtctacttgaattacccccggctggttg
agcgtttgttgaaaaaaagtaactgaaaaatccgtagaatagcgccactctgatggttaattaacctattcaa-
ttaagaattatctggatgaatgtgccatta
aatgcgcagcataatggtgcgttgtgcgggaaaactgcttttttttgaaagggttggtcagtagcggaaacaa-
ctcacttcacaccccgaagggggaa
gttgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcg-
ggtaataccgtctggcgcttcgatttgt
cccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctcca-
gcaagtgctgtgcgtattgcaca
atgacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcgtt-
gcaggaagccgatcagcagttaatccc
cggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaatcatta-
gtgctggcgcgcgttgctgacgat
cagcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatc 85
attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggcagc-
gggaagtagatgctactttgccggc
gagcggcggacgggtgagtaatgtctgggtatctgcctgatggagggggataactactggaaacggtagctaa-
taccgcatgacctcgaaagagc
aaagtgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggtaatggc-
tcacctaggcgacgatccctagct
ggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaa-
tattgcacaatgggcgcaagc
ctgatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaaggcatc-
anacttaatacgtcggtgattgac
gttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatc-
ggaattactgggcgtaaagcgca
cgcaggcggtttgttaagtcagatgtgaaatccccgcgcttaacgtgggaactgcatttgaaactggcaagct-
agagtcttgagaggggggtagaatt
ccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagact-
gacgctcaggtgcgaaagcgt
ggggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttga-
ggcgtggcttccggagctaacgcgt
taagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggt-
ggagcatgtggtttaattcgatgc
aacgcgaagaaccttacctactcttgacatccacgcaattcgccagagatggcttagtgccttcgggaaccgt-
ganacaggtgctgcatggctgtcgt
cagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcnngtna-
tgnngggaactcaaaggagactgcc
ggtgataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgct-
acaatggcatatacaaacagaag cgaactcgcgagg 86
atggcaatgcgtcaatgcgcaatctacgggaaagggggtattgggaaatccaccactacccaaaaccttgt-
agcggctctggccgaaatgaataaga
aggtcatgatcgtcggctgtgaccctaaggctgattcaacccgcctcattctgcatgcgaaagcacagaacac-
catcatggaaatggccgctgaagtg
ggctccgtggaagatctggagctggaagatgtgatgcaaatcggctatggcggcgtgcgctgtgcggaatcag-
gcggccctgagcctggtgtgggt
tgtgccggacgcggggtgatcaccgccatcaacttcctcgaagaagaaggcgcgtatgtgccggatctggatt-
ttgtgttttacgacgtattgggcgat
gtggtctgtggcggtttcgcgatgccaattcgcgaaaacaaagcgcaggaaatctacatcgtgtgctccggtg-
aaatgatggcgatgtatgccgccaa
caacatttccaaaggcatcgtgaaatacgcgaaatcgggcaaagttcgcctggccgggctgatctgtaactcc-
cgccagacggatcgcgaagatgaa
ctgatcatcgcgctggctgaaaaacttacacgcaatttgatccacttcgtgccgcgtgacaacattgtgcaac-
gcgctgaaatccgccgcatgacggt
catcgaatacgacccgacttgtgcgcaggcagatcagtatcgtgcactggcgaacaaaatcgtcaacaacacc-
aaaatggtggtgccgacaccggtc
accatggatgagctggaagccctgttaatggaatttggcattatggaagaagaagacctcgccatcgtcggtc-
gtaccgccgccgaagaggcgtga 87
atgaaggcaaaagagattctggcgctgattgatgagccagcctgtgagcataaccacaagcagaagtcggg-
ttgcagcctgccgaaaccgggcgc
gacggcaggcggttgtgcgtttgatggcgcgcagattgcgctgctgccggtcgcggacgtcgcgcatctggtg-
cacggcccgattggctgtaccgg
cagttcatgggacaaccgtggcagccgcagttccgggccttccatcaaccgcatgggcttcaccaccgacatg-
agcgagcaggatgtgattatgggg
cgcggcgagcgacgcttatttcacgccgtgcagcacatcgtcagccattaccatccggtggcggtctttattt-
acaacacctgcgtacccgcgatggaa
ggggatgacgttgaagccgtgtgtcgcgccgcatcggccgctgccggtgtgccggttatttcagtcgatgccg-
ccggtttctacggcagcaaaaatct
cggtaaccgcattgccggggacgtgatggtcaaaaaggtgatcggccagcgcgaacccgcgccgtggccggaa-
aactcaccgatccccgccgga
caccgccacagcatcagcctgattggcgaattcaatattgccggcgagttctggcacgttctgccgctgctcg-
atgagctcgggatccgcgtgctgtgc
agcctttccggggattcccgttttgctgaaatccagactatgaaccgtggcgaagccaacatgctggtgtgct-
cgcgggcgctgatcaacgtcgcccg
aaaaatggaagagcgttaccagatcccatggtttgaaggcagtttttatggcctgcgttccatggctgattcc-
ctgcgcacgatcgccgtgctgctcaaa
gacccggatttacaggcgcgcacagaacgtctgattgagcgcgaggaggcggcgacacatcttgcgcttgcgc-
cttaccgtgcgcggctcagcgg
gcgcaaggcgctgctgtataccggtggcgtgaaatcctggtcggtggtctcggcgttacaggatttaggcatc-
acggtggtggcgaccggcacccga
aaatcaaccgaagaagacaagcagcgtattcgcgaactgatgggtgaagacgtgctgatgctcgacgaaggca-
atgccagaaccttgctcgacacc
ctctatcgtttcggcggcgacatcatgatcgccgggggccgcaacatgtataccgcgtacaaagccgcctgcc-
gttcctggatatcaatcaggagcg
cgagcatgcgtttgccggatatcacgggctggtaaatctggccgaacagttgtgtatcaccctggaaagcccg-
gtctgggcgcaggtcaaccgtctg gcgccgtggcgctaa 88
atgaccagtgaaacacgcgaacgtaacgaggcattgatccaggaagtgctggagatcttccccgagaaggc-
gcttaaagatcgtaagaaacacatg
atgaccaccgacccggcgatggaatctgtcggcaagtgtattgtctcaaaccgcaaatcacagccgggcgtga-
tgaccgtgcgaggctgcgcttacg
ccggttccaaaggcgtggtctttggcccgatcaaagacatggcgcatatctcccacggcccggttggttgcgg-
ccagtattcccgtgccggacgccgt
aactattacaccggctggagcggcgtgaacagctttggcaccctcaacttcaccagtgattttcaggaacggg-
acatcgtatttggcggcgataaaaag
ctcgacaaattgatcgatgaactggagatgttgttcccgctgagcaaaggcatttcggtgcagtcggaatgtc-
cggtcggtctgatcggcgatgacattt
ctgccgtcgccaaagccagcagcgccaaaatcggtaagccggtcgtgccggtacgctgcgaggggttccgcgg-
tgtgtcgcaatcgctcggccatc
acattgctaacgatgtcatccgcgactgggtgctggataaccgcgaaggcaatgaatttgaaaccacgcctta-
cgacgtggcgattatcggcgactac
aacatcggcggtgacgcctggccctcacgtattctgctcgaagaaatggggctgcgcgtggtggcgcagtggt-
ccggcgacggcacgctggtgga
gatggaaaacaccccgaaagtcgcgctcaatctggtgcactgctaccgctcgatgaactacatctcccgtcat-
atggaagaaaaacacggcattccgt
ggatggaatacaacttctttggcccgaccaaaattgcggaatctctgcgcgaaatcgcggcggttttgacgat-
accatccggaaaaacgccgaagc
ggtgattgaaaaatatcaggcgcaaacgcaggcggtgatcgacaaataccgtccgcgtctggaaggcaaaaag-
gtgctgttgtatctcggcggtttac
gtccgcgccacatcatcggggcgtatgaagatctgggaatggaaatcatcggtaccggctatgaattcggtca-
taacgatgattacgaccgcaccttac
cgatgctcaaagaaggcacgttgctgttcgatgacctgagcagttatgagctggaagcgttcgttaaagcgct-
gaaaccggatcttgtcgggtcaggta
tcaaagaaaaatacattttccagaaaatgggcgtgccgttccgccagatgcactcctgggattattccggccc-
ttatcacggctacgacggtttcggcat
ttttgcccgtgacatggacatgacgctgaacaatccgggctggagtcagctgaccgccccctggttgaaaagc-
ctga 89
atggctcaaattctgcgtaatgccaagccgcttgccaccacgcctgtcaaaagcgggcaaccgctcgcggc-
gatcctggccagtcaggggctggaa
aattgcatcccgctggttcacggcggcaaggttgtagcgcgttcgccaaagttttcttcatccagcattttca-
cgatccgatcccgttgcagtccacggc
gatggaatcgaccacgactatcatgggctcggatggcaacgtcagtactgcgttgaccacgttgtgtcagcgc-
agtaatccaaaagccattgtgattttg
agcaccggactgtcagaagcgcagggcagtgatttgtcgatggcgctgcgtgagtttcgcgacaaagaaccgc-
gctttaatgccatcgctattctgac
cgttaacacgccggatttttacggctcgctggaaaacggctacagcgcgctgatggaaagcgtgatcactcag-
tgggtgccggaaaagccgccgac
cggcatgcgtaacaagcgcgtgaacctgctggtgagccatctgctgacgccgggggatctggaattactgcgc-
agctatgtcgaagcctttggcctg
caaccggtgatcctgccggatttatcacagtcgctggacggacatctggcgaatggcgatttcaatccggtca-
cgcagggcggcacgtcgcaacgcc
agattgaacaaatggggcagagcctgaccaccattaccattggcagttcgctcaactgcgccgccagtctgat-
ggcgatgcgcagccgtggcatggc
gctgaacctgccgcacctgatgacgctggaaaacatggacagtctgatccgccatctgcatcaggtgtcaggc-
cgcgaggtaccggcatggattgag
cgccagcgcgggcaactgaggacgccatgatcgactgccatacctggctgcagtcacagcgtattgcgctggc-
ggcagaagcggatttgctggtg
gcgtggtgcgatttcgctcagagccagggaatgcgcgtcgggccggtgattgcgccggttaatcagcagtcac-
tggccgggctgccggtcgaacag
gtggtgatcggcgatctggaagatttacaaacccggctcgacagctacccggtttcactgctggtggcgaact-
cccacgctgcaccactggcggaaa
aaaacggtatcgcgctggtacgtgccggtttcccgctttacgaccgtctcggggaatttcgccgcgtgcggca-
gggctatgcgggtattcgcgacacc
ttgttcgaactcgcgaacctgatgaggcgcgccatcacatgctgacggcgtatcactcaccgcttaggcaggt-
gttcggcctgagcccggtaccgg aggccagtcatgaggcggctaa 90
atgagtcaagatcttggcaccccaaaatcctgtttcccgctcttccagcaggatgaataccagaatatgtt-
tacccacaaacgcgcgctggaagaagca
cacggcgaggcgaaagtgcgggaagtgtttgaatggaccaccacgcaggaatatcaggatctgaacttctcgc-
gtgaagcgctgaccgtcgacccg
gcgaaagcctgccagccgttaggcgccgtactttgcgcgctgggttttaccaacacgttgccgtatgtccatg-
gttcacaaggctgtgtggcctatttcc
gtacctattttaatcgtcatttcaaagagccggtggcctgtgtttccgactcaatgaccgaagatgccgccgt-
ttttggcggaaataacaacatgaatgtc
ggtctggaaaacgccagcgcgctgtacaagccggaaattattgcggtctccaccacctgtatggcggaagtga-
tcggtgatgacctgcaggcttttatc
gccaacgccaaaaaagacggatttgtggatgcggtatgccaatcccgtatgcccatacaccgagttttctggg-
cagtcatgtcaccggctgggacaa
catgtttgaaggcttcgcccgtacctttaccaccgacgccacgcgggaatatcagccgggcaaacttgccaaa-
ctgaacgtggtgaccggttttgaaa
cttatctcggcaactaccgggttattcaccgcatgatgagccagatgggggtcgaatgcagcgtcttgtccga-
tccgtctgaagtgctcgacaccccgg
ctgacggccaataccgcatgtatgccgacggcaccacgcaaaccgaaatgcgtgatgaccggatgccatcgac-
accttgctgctgcaaccgtggc
aattacagaaaaccaaaaaggtggtgcagggcgactggaatcagccgggcaccgaagtcagtgtaccgattgg-
cctggcggcgaccgatgccttg
ctgatgacggtaaggaactgaccggcaaaccgatagctgacgcgctggcgactgaacgtggccgtctggtgga-
catgatgctcgattctcacacct
ggctgcacggcaagcgtttcggtctctacggtgacccggattttgtgatgggcatgaccgcattcctgctgga-
actgggctgtgaaccgaccaccattc
tcagccataacggcaacaaacgctggcagaaagccatgaagaaaatgctggctgattcgccttacggacagga-
cagcgaagtgtatgtgaactgcg
atctgtggcatttccgctcgctgatgtttacccgtaaaccggactttatgatcggcaactatacggaaaattc-
attcagcgtgacacgctggccaaaggc
gaacagttcgaagtgccgctgatccgtatcggattcccgatttttgaccggcaccatttgcaccgtcagacca-
cctggggatacgagggcgcgatgag
catcctgacgcaactggtgaatgcggtgctcgaacagctggatcgcgaaaccatgaagctcggcaaaaccgac-
tacaacttcgatctgatccgctaa 91
atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagcgcttgtcgctgcaacatcct-
tcactgttttataccgtggttgaacaatca
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgg-
gttttgcacttgagacacttttgggcg
agaaccaccgtctgctggccagccagcagacgccgaaacatatctatgacgaaatgtggcgcactttgttgca-
gggctaatcctggaacggccaact
gatcaaccggcgtaataaccgttcgctttatctggcggatgtcactatcacgcctgttttaggcgcggacggg-
caggtggagcattacctcggcatgca
caaagatatcagcgagaaatacgcgctgcagcagggttgcgcaaccacatcaccttgttcacggaggtgctga-
acatttattcccgccgccgtggtg
gtggtggatgagaggacaatgtggtgatggacaatctggcctacaaaaccctgtgcgctgactgcggcgcaaa-
agagctgttggccgaaatgggc
tatccgcaactcaaagagatgctcaacagtggcgaaccggtgccggtttccatgcgcggcaacgtacgctggt-
tttctttcggtcagtggtcattgcag
ggcgttaatgaagaggccagccgcttttttaccggcattaccgcgccgggaaaactgattgttctcaccgact-
gaccgatcagcatcaccggcagca
gcagggttatcttgaccggctcaagcaaaaacttaccaacggcaaattgctggcagccatccgcgagtcgctt-
gatgccgcgctgattcagctcaacg
ggccattttaatatgctggcggctgcgcgtcgtcttaacggcgaagaaggcaacaacatggcgctggaattcg-
cctggcgcgaaggcgagcaggcg
gtgagtcgcttacaggcctgccgtccgtcgctggattttgagccgcaggcagaatggccggtcagtgaattct-
tcgacgatctgagcgcgctgtacga
cagccattttctcagtgacggtgaattgcgtacgtggtcatgccatctgatctgcacgctgtcgggcaacgaa-
cgcaaatccttaccgcgctgagcttat
ggattgatcacacgctgtcacaggcgcaggccatgccgtctctgaagctctcggtgaacattgttgcgaagca-
ggatgcgagctggttgtgttttgaca
ttaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccgggcaatgg-
catggagctgcgccttatccagac
gctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgagacgttacgcc-
tgccggtacagcaggttatcaccgga ggcttaaaatga 92
atgacccagttacctaccgcgggcccggttatccggcgctttgatatgtntgcccagtttacggcgcttta-
tcgcatcagcgtggcgctgagtcaggaa
agcaataccgcgcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggcatgg-
tgtgtctgttcgataaagaacgcaat
gcactgtttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttaccgca-
tgggggaaggcgtgatcggcgc
ggtgatgagccagcgtcaggcgctggtgtaccgcgcatttcagacgatcagcgttttctcgaccgcctgaata-
tttacgattacagcctgccgatgatt
ggtgtgccgatccccggtgcggataatcagcctgcgggtggctggtggcacagccgatggcgttgcacgaaga-
ccggctggctgccagtacgcg
gtttttagaaatggcgccaatctcatcagccagccactgcgttctgccacgcccccggaatcattgcctgctc-
aaacgccggtccggtgcagtgttccg
cgccagtttggttttgagcagatggtcgggaaagtcaggcgatgcgccagacgatggacattttacggcaggt-
ttccaaatgggataccacggttctg
gtgcgtggtgaaagcggcaccggcaaggaacttatcgccaatgccattcattacaactcaccccgtgcggccg-
cgccatttgtgaaattcaactgcgc
cgcgctgccggataacctgctggaaagcgaactgttcggtcatgaaaaaggggccttcaccggcgctatacgc-
acccgaaaaggccgctttgaact
ggcggacgggggcacgttattcctcgatgaaatcggcgaatcgagcgcgtcgtttcaggccaaattgctgcgc-
attttgcaggaaggtgaaatggaa
cgggtcggcggcgataccacgctgaaagttgatgtgcgcattattgctgccaccaaccgtaatcttgaggagg-
aagtgcgtgccgggaattttcgcga
agacctgtattatcgcctcaacgtgatgccggtttcgctgcctgcactgcgtgaaaggctggatgatatcgcc-
gatctggcgccgtttctggtcaaaaag
attgcgctgcgtcaggggcgggaactgcgcatcagtgatggtgcggtgcgtctgctgatgacctacagctggc-
caggcaacgtgcgtgaactggaa
aactgcctcgaacgggcgtcggtaatgaccgatgaagggctgatcgaccgcgacgtgatcctgttcaatcacc-
atgagtccccggcgctgtccgtca
aacccggtctgccgctcgcgacagatgaaagctggctggatcaggaactcgacgaacgccagcgggtgattgc-
tgcactggagaaaaccggctgg
gtgcaggccaaagcggcccgactgagggcatgacaccgcgccagattgcctaccgtatccagattatggacat-
caacatgcaccgtatctga 93
atgttgccactttcttctgttttgcaaagccacgcgcagagtttgcctgaacgctggcatgaacatcctga-
aaacctgcccctccccgatgatgaacagct
ggctgtgctgagcagcagtgaattcatgacggacagtttgctggcttttccgcagtggtggcatgaaattgtc-
caaaatccccctcaggcgcaggagtg
gcaactttaccgtcagtggctggatgaatcactgacgcaggtgactgacgaagccgggttaatgaaagctttg-
cgtctgttccgccgccgtattctgac
ccgcattgcgtggtcacagtccgcgcaaaccagcgaagcaaaagatacgcttcaccagctgagtgaactggcg-
gaattattgattgtcagcgcccgt
gactggctgtatgccgcttgctgtcgcgagttcggtacgccggtcaatgccgcaggggaaccgcagagaatgc-
tgatcctcgggatgggcaaactc
ggcggtggcgagctgaatttcatcggacatcgacctgacctgatttttgcttatccggaaaatggccagacac-
gcggcggtcggcgtgaactggataacgc
acaattttcacccggctcggccagcgtctgatcaaagcgctggatcagcccactatcgacggttttgtctatc-
gcgtggacatgcgtttgcgtccgttcg
gcgacagtggcccgctggtgatgagcttcccggcactggaagattattatcaggaacaggggcgcgactggga-
acgctacgcaatggtgaaagcg
cgtctgatgggcggcgcggaggacatcagcagtcaggaattgcgtaaaatgctgatgcttttgtcttccgccg-
ttatatcgatttcagtgtgatccagtc
cctgcgtaacatgaaaggcatgatcgcccgcgaagtacgccgccgtggtctgaaagacaacatcaaactcggc-
gcaggcggtattcgtgaaattgaa
tttatcgtgcaggtatttcagctgatccgtggcggtcgtgaaccggcattgcagcagcgtgcgttgttgccaa-
cgcttcaggcgctggaaaatctgggg
ctgctgccggtagagcaggtgttgcagttgcgtaacagctatctgttcctgcgacgtctggaaaacctgttgc-
aggccattgctgacgagcaaacgcaa
accttaccgtccgatgagctgaatcaggcgcgtctggcgtgggggatgaattacgctggctggccgcagctta-
ggatgcagtgaatgctcacatgca
ggccgtacgcgcggtatttaacgatctgattggcgatgacacgccagatgccgaagatgacgtgcaactctcc-
cggttcagcagtttatggattgatac
gcttgagcctgacgagctggctccgctggtgccgcaacttgacgaaaatgcgcaacggcatgttttacatcag-
attgctgattttcgccgtgacgtggat
aaacgcacgatagggccacgtgggcgtgatcagttggatttgctgatgccgcgtttactggcccaggtctgca-
cctataaaaatgcggatgtgacgtta
cagcgcctgatgcagttgctgctcaatatcgtcacgcgcacgacgtatatcgagctgctggtggaatatcccg-
gtgcgctcaaacagttaatacgtctgt
gcgctgcctcgccgatggtggcgacgcaacttgcgcgtcatcctttattgctcgacgaactgctcgacccgcg-
cacgctttaccagccgattgagccg
ggcgcgtaccgtgatgaactgcggcaatatctgatgcgggtgccaaccgaagacgaagaacaacagcttgaag-
ccgtgcgccagttcaaacaggc
acagcatttacgtattgcggccggggatatttccggtgcgttgccggtgatgaaagtcagtgaccatttaacc-
taccttgcggaggccattctgacgtt
gtggttgcaacaggcgtgggaacaaatggtcgtaaaatacggtcagccaacccatcttcagcaccgtaaaggg-
cgcggttttgccgtggtgggttacg
gaaaactcggtggctgggagctgggttacagctcggatctggatctggtcttcctgctcgattgcgcgccgga-
agtcatgaccgacggcgaacgcag
cattgacgggcgtcagttttatctgcggctggcgcagcgcatcatgcatttattagcacccgtacgtcgtcag-
gcattctttatgaggttgacccgcgtc
tgcggccttccggtgcttccggcatgctggtcagcaccatcgaagcttttgcggattatcaggccaacgaagc-
ctggacatgggagcatcaggcgctg
gttcgcgcgcgtgtggtttatggtgatccgcaactgacgcagcaatttaatgccacgcgtcgcgacattcttt-
gccgccagcgcgatgccgacggcttg
cgtaaggaagtccgtgaaatgcgcgagaaaatgtatgcccatctgggcagcaaaagagccgacgagtttgatc-
tgaaagccgatccgggtggcata
acggatattgaattcatcgcacaatatctggttctgcgtttcgcgcatgatgagccgaagagacccgctggta-
gataacgtgcggattttcgaactgat
ggcgcgacatgacatcatgccggaagaggaagcacgccatctgacgcaggcttacgtgacattgcgcgatgaa-
attcatcatctggcgtgcaggaa
cacagcgggaaagtggccgcagacagctttgccactgagcgcgcccaaatccgcgccagctgggcaaactggc-
ttggctga 94
atgaaaaaacttttatccatgatggggcttggtgcagtggctttgctaccttcgcttgccatggcagcacc-
accagcagcggcaaacggtgctgataac
gcctttatgatgatttgtaccgcgctggtattgttcatgaccgtacccggtgtggcgttgttctacggcggct-
tactgcgttctaaaaacgttttgtccatgct
gactcaggttattgttacctttgctctggtctgcgtcctgtggatcctctacggttacagccttgccttcagt-
gaaggtaacgcgttcttcggtggtttcagca
acgtaataatgaaaggcattggcctggattctgtgactggcaccttctcgcagatgatccacgttgcattcca-
gtgttcatttgcctgcatcactgtagcgc
tgatcgtaggtggtattgctgaacgtgtgcgtttctcagcagttctgattttcactgtgatctggctgacttt-
ctcttatattccgatggctcacatggtatggg
caggcggtttcctggctgctgacggtgcgctggactttgccggtggtaccgttgttcatatcaatgccgcaat-
tgctggcctggtaggggcttatctgctg
ggtaaacgcgccggttttggcaaagaagctttcaaaccacacaacctgccaatggtcttcactggcgcctcaa-
tcctgtatgtgggctggttcggcttca
atgcgggttcagcaagtgccgcaagctctgttgccgcgctggctttcctgaacactgtcattgctactgctgg-
cgcaatcctgtcctggacgctggttga
gtggatggtgcgcggtaagccctcactgctgggcgcaagctccggtgctatcgcaggtctggtggctatcacg-
cctgcatgtggtacggtcggcgta
ggtggtgctctgattatcggtctggtaggcggtatcactggtctgtggggggttgttaccctgaaaaaatggc-
tgcgtgttgatgacacctgtgatgtgtt
cggtgttcatggcgtgtgcggtatcgtaggttgtctgctgacgggtgtattcacgtccagttcacttggcggc-
gtgggctacgcagaaggcgtgaccat
gggccatcaggtttgggtgcagttcttcagcgtgtgcgtaacattggtctggtcaggcgttgttgccttcatc-
ggttacaaagtggctgacatgatcgtag
gtctgcgtgttcctgaagaacaagaacgcgaaggtctggacgttaacagccacggcgaaaacgcttacaacca-
ataa 95
tgaatatcactgactcacaagctacctatgtcgaagaattaactaaaaaactgcaagatgcaggcattcgc-
gttaaagccgacttgagaaatgagaaga
ttggctttaaaattcgcgaacacacgctacgccgtgttccttatatgttagtttgtggcgataaagaggtcga-
agcaggcaaagttgctgttcgtactcgtc
gcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgctggcggaaatccgcagcagaag-
tcatcatcaactggaggaataaa
gtattaaaggcggaaaacgagttcaaccggcgtcctaatcgcattaacaaagagattcgcgcgcaagaagttc-
gcctcaccggcgtcgatggcga
gcagattggtattgtcagtctgaatgaagctcttgaaaaagctgaggaagcgggcgtcgatttagtagaaatc-
agtccgaatgccgagccgccagtttg tcgaatc 96
attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgatagtcgagcggtagc-
acagagagcttgctctcgggtgacga
gcggcggaccggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaata-
ccgcataacgtcgcaagaccaaa
gtgggggaccttcgggcctcatgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctca-
cctaggcgacgatccctagctggt
ctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatat-
tgcacaatgggcgcaagcctga
tgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaaggcggtgagg-
ttaataacctcaccgattgacgtta
cccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaa-
ttactgggcgtaaagcgcacgc
acgcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggctaga-
gtcttgtagaggggggtagaattc
caggtgtagggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactga-
cgctcaggtgcgaaagcgtg
gggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgatttggaggttgtgcccttgag-
gcgtggcttccggagctaacgcgtta
aatcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtgg-
agcatgtggtttaattcgatgca
acgcgaagaaccttacctggtcttgacatccacagaactttccagagatggattggtgccttcgggaactgtg-
agacaggtgctgcatggctgtcgtca gctcgtgttgtgaaatgttggg 97
atgaccatgcgtcaatgcgctatctacggtaaaggcggtatcggtaaatccaccaccacccagaatctcgt-
cgcggccctcgccgagatgggtaaga
aagtgatgatcgtcggctgcgatccgaaagcggattccacccgtctgatcctccacgctaaagcccagaacac-
catcatggagatggcggcggaagt
gggctcggtcgaggatctggagctcgaagacgttctgcaaatcggctatggcgatgtccgttgcgccgaatcc-
ggcggcccggagccaggcgtcg
gctgcgccggacgcggggtgatcaccgccatcaacttcctcgaggaagaaggcgcctatgaagaagatttgga-
tttcgtcttctatgacgtcctcggc
gacgtcgtctgcggcggcttcgctatgccgatccgcgaaaacaaagcccaggagatctacatcgtctgctccg-
gcgagatgatggcgatgtatgccg
ccaacaatatctccaaagggatcgtgaagtacgccaaatccggcaaggtgcgcctcggcggcctgatctgtaa-
ctcgcaaaaccgaccgggaag
acgaactgatcatcgccctggcggagaagcttggcacgcagatgatccacttcgttccccgcgacaacattgt-
gcagcgcgcggagatccgccgga
tgacggtgatcgagtacgacccgacctgtcagcaggcgaatgaatatcgtcaactggcgcagaagatcgtcaa-
taacaccaaaaaagtggtgccga
cgccgtgcaccatggacgagctggaatcgctgctgatggagttccgcatcatggaagaagaagacaccagcat-
cattggtaaaaccgccgctgaag aaaacgcggcctga 98
atggttaggaaaagtagaagtaaaaatacaaatatagaactaactgaacatgaccatttattaataagtca-
aataaaaaagcttaaaacacaaaccacttg
cttttttaataataaaggaggggttgggaagactacattagtagcaaatttaggagcagagctatcaataaac-
tttagtgcaaaagttcttattgtggatgcc
gaccctcaatgtaatctcacgcagtatgtatttagtgatgaagaaactcaggacttatatgggcaagaaaatc-
cagatagtatttatacagtaataagacc
actatcctttggtaaaggatatgaaagtgacctccctataaggcatgtagagaatttcggttttgacataatt-
gtcggtgaccctagacttgctttacaggaa
gaccttttagctggagactggcgagatgccaaaggcggtgggatgcgaggaattaggacaacttttgtatttg-
cagagttaattaagaaagctcgtgag
ctaaattatgattttgttttctttgacatgggaccatcattaggcgcaatcaacagggcagtattactggcaa-
tggaattctttgtcgtcccaatgtcaatcga
tgtattttcactatgggctattaaaaatattggctccacggtttcaatatggaaaaaagaattagacacaggg-
attcggctctcagaggaacctagcgaatt
atcacaattatcacctcaaggaaaactaaagtttctcggttacgtcacccaacaacataaagaacgctctgga-
tacgatacaattcagcttgagaatactg
aggaagaaataaaatcgaaacgtcgggtaaaggcgtatgaagacattggagaggtgtttccttctaaaattac-
tgagcatctttctaaactttatgcatca
aaagatatgaacccacaccttggagatatacgtcatttaggtagtttagctccgaaatcacaatcacaacacg-
ttccgatgatatcagtgtctggtacagg
aaattacaccagacttagaaaaagcgcgcgtgaactttatcgagatattgcaagaagatacttagagaacatt-
cagactgctaatggcgagaaatag 99
atgaagggaaaggaaattctggcgctgctggacgaacccgcctgcgagcacaaccagaagcaaaaatccgg-
ctgcagcgcccctaagcccggcg
ctaccgccggcggttgcgccttcgacggcgcgcagataacgctcctgcccatcgccgacgtcgcgcacctggt-
gcacggccccatcggctgcgcg
ggcagctcgtgggataaccgcggcagcgtcagcgccggcccggccctcaaccggctcggctttaccaccgatc-
ttaacgaacaggttgtgattatg
ggccgcggcgaacgccgcctgttccacgccgtgcgtcacatcgtcgaccgctatcatccggcggcggtcttta-
tctacaacacctgcgtaccggcga
tggagggcgatgacatcgaggcggtctgccaggccgcacagaccgccaccggcgtcccggtcatcgctattga-
cgccgccggtttctacggcagta
aaaatcttggcaaccgaatggcgggcgacgtgatgctcaggcaggtgattggccagcgcgaaccggccccgtg-
gccagacaacacgccctttgcc
ccggcccagcgccacgatatcggcctgattggcgaattcaatatcgccggcgagttctggcaggtccagccgc-
tgctcgacgagctggggatccgc
gtcctcggcagcctctccggcgacggccgctttgccgagatccagaccctgcaccgggcgcaggccaatatgc-
tggtgtgctcgcgcgcgctgatc
aacgtcgcccgggggctggagctgcgctacggcacgccgtggtttgaaggatgcttctacgggatccgcgcca-
cctccgacgccttgcgccagct
ggcgacgctgctgggggatgacgacctgcgccgccgcaccgaggcgctgatcgcccgcgaagagcaggcggcg-
gagcaggctcttgcgccgt
ggcgtgagcagctccgcgggcgcaaagtgctgctctataccggcggcgtgaaatcctggtcggtggtatcggc-
cctgcaggatctcggcatgaccg
tggtggccaccggcacgcgcaaatccaccgaggaggacaaacagcggatccgtgagctgatgggcgacgaggc-
ggtgatgcttgaggagggca
atgcccgcaccctgctcgacgtggtgtaccgctatcaggccgacctgatgatcgccggcggacgcaatatgta-
caccgcctggaaagcccggctgc
cgttctcgatatcaatcaggagcgcgagcacgcctacgccggctatcagggcatcatcaccctcgcccgccag-
ctctgtctgaccctcgccagcccc gtctggccgcaaacgcatacccgcgccccgtggcgctag
100
atgaccaacgcaacaggcgaacgtaaccttgcgctcatccaggaagtcctggaggtgtttcccgaaaccg-
cgcgcaaagagcgcagaaagcacat
gatgatcagcgatccgcagatggagagcgtcggcaagtgcattatctcgaaccgtaaatcgcagcccggggtg-
atgaccgtgcgaggctgcgccta
tgcgggctcgaaaggggtggtgtttgggccaatcaaagacatggcccatatctcgcacggccccatcggctgc-
ggccagtattcccgcgccggacg
gcgcaactactataccggcgtcagcggtgtcgacagcttcggcaccctgaacttcacctctgattttcaggag-
cgcgatattgttttcggcggcgataaa
aagctgaccaaactgatcgaagagatggagctgctgttcccgctgaccaaagggatcaccatccagtcggagt-
gcccggtgggcctgatcggcgat
gacatcagcgccgtagccaacgccagcagcaaggcgctggataaaccggtgatcccggtgcgctgcgaaggct-
ttcgcggcgtatcgcaatcgct
gggccaccatatcgccaacgacgtggtgcgcgactgggtgctgaacaatcgcgaagggcagccgtttgccagc-
accccgtacgatgttgccatcatt
ggcgattacaacatcggcggcgacgcctgggcctcgcgcattctgctggaagagatgggcctgcgcgtagtgg-
cgcagtggtccggcgacggca
ccctggtggagatggagaacaccccattcgttaagcttaacctcgtccactgctaccgttcgatgaactatat-
cgcccgccatatggaggagaaacatc
agatcccatggatggaatataacttcttcggcccgaccaaaatcgccgaatcgctgcgcaagatcgccgatca-
atttgatgacaccattcgcgccaatg
cggaagcggtgatcgcataatatgaggggcagatggcggccatcatcgccaaatatcgcccgcggctggaggg-
gcgcaaagtgctgctgtacatg
ggggggctgcggccgcgccacgtcatcggcgcctatgaggatctcgggatggagatcatcgccgccggctacg-
agtttgcccataacgatgattac
gaccgcaccctgccggacctaaagagggcaccctgctgtttgacgatgccagcagctatgagctgtgaggcct-
tcgtcaaagcgctgaaacctgac
ctcatcggctccgggatcaaagagaaatatatcttccagaaaatgggggtgccgttccgccagatgcactcct-
gggactattccggcccctatcacgg
ctatgacggcttcgccatctttgcccgcgatatggatatgaccctgaacaatccggcgtggaacgaactgact-
gccccgtggctgaagtctgcgtga 101
atggcagatattatccgcagtgaaaaaccgctggcatgagcccgattaaaaccgggcaaccgctcggggc-
gatcctcgccagcctcgggctggc
ccaggccatcccgctggtccacggcgcccagggctgcagcgccttcgccaaagttttctttattcagcatttc-
catgacccggtgccgctgcagtcgac
ggccatggatccgaccgccacgatcatgggggccgacggcaatatcttcaccgcgctcgacaccctctgccag-
cgccacagcccgcaggccatcg
tgctgctcagcaccggtctggcggaagcgcagggcagcgatatcgcccgggtggtgcgccagtttcgcgaggc-
gcatccgcgccataacggcgtg
gcgatcctcaccgtcaataccccggatttttttggctctatggaaaacggctacagcgcggtgatcgagagcg-
tgatcgagcagtgggtcgcgccgac
gccgcgtccggggcagcggccccggcgggtcaacctgctggtcagccacctctgttcgccaggggatatcgaa-
tggctgggccgctgcgtggag
gcctttggcctgcagcccgtgatcctgccggacctctcgcagtcaatggatggccacctcggtgaaggggatt-
ttacgcccctgacccagggcggcg
cctcgctgcgccagattgcccagatgggccagagtctgggcagcttcgccattggcgtgtcgctccagcgggc-
ggcatcgctcctgacccaacgca
gccgaggcgacgtgatcgccctgccgcatctgatgaccctcgaccattgcgatacctttatccatcagctggc-
gaagatgtccggacgccgcgtacc
ggcctggattgagcgccagcgtggccagctgcaggatgcgatgatcgactgccatatgtggcttcagggccag-
cgcatggcgatggcggcggagg
gcgacctgctggcggcgtggtgtgatttcgcccgcagccaggggatgcagcccggcccgctggtcgcccccac-
cagccaccccagcctgcgcca
gctgccggtcgagcaagtcgtgccgggggatcttgaggatctgcagcagctgctgagccaccaacccgccgat-
ctgctggtggctaactctcacgc
ccgcgatctggcggagcagtttgccctgccgctgatccgcgtcggttttcccctcttcgaccggctcggtgag-
tttcgtcgcgtccgccaggggtacgc
cggtatgcgagatacgctgtttgaactggccaatctgctgcgcgaccgccatcaccacaccgccctctaccgc-
tcgccgcttcgccagggcgccgac ccccagccggcttcaggagacgcttatgccgcccattaa
102
atgagccaaacgatcgataaaattcacagctgttatccgctgtttgaacaggatgaataccagaccctgt-
tccagaataaaaagacccttgaagaggcg
cacgacgcgcagcgtgtgcaggaggtttttgcctggaccaccaccgccgagtatgaagcgctgaaatccagcg-
cgaggcgctgaccgtcgaccc
ggccaaagcctgccagccgctcggcgccgtactctgcgcgctggggttcgccggcaccctgccctacgtgcac-
ggctcccagggctgcgtcgcct
attttcgcacctactttaaccgccattttaaagagccggtcgcctgcgtctccgactccatgaccgaggacgc-
ggcggtgttcggcggcaacaacaac
atgaatctgggcctgcagaatgccagcgcgctgtataaacccgagattatcgccgtctccaccacctgtatgg-
ccgaggtgatcggcgacgatctgca
ggcgtttatcgccaacgccaaaaaagagggatttgttgacgaccgcatcgccattccttacgcccataccccc-
agctttatcggcagccatgtcaccgg
ctgggacaatatgttcgaagggttcgcgaagacctttaccgctgactacgccgggcagccgggcaaacagcaa-
aagctcaatctggtgaccggattt
gagacctatctcggcaacttccgcgtgctgaagggatgatggcgcagatggatgtcccgtgcagcctgctctc-
cgacccatcagaggtgctcgaca
cccccgccgacggccattaccggatgtacgccggcggcaccagccagcaggagatcaaaaccgcgccggacgc-
cattgacaccctgctgctgca
gccgtggcagctggtgaaaagcaaaaaggtggttcaggagatgtggaaccagcccgccaccgaggtggccgtt-
ccgctgggcctggccgccacc
gacgcgctgctgatgaccgtcagtcagctgaccggcaaaccgatcgccgacgactgaccaggagcgcggccgg-
ctggtcgacatgatgctggat
tcccacacctggctgcatggcaaaaaattcggcctctacggcgatccggatttcgtgatggggctgacgcgct-
tcctgctggagctgggctgcgagcc
gacggtgatcctcagtcataacgccaataaacgctggcaaaaagcgatgaagaaaatgctcgatgcctcgccg-
tacggtcaggaaagcgaagtgttc
atcaactgcgacctgtggcacttccggtcgctgatgttcacccgtcagccggactttatgatcggtaactcct-
acggcaagtttatccaggcgataccc
tggcaaagggcaaagccttcgaagtgccgctgatccgtctgggctttccgctgttcgaccgccatcatctgca-
ccgccagaccacctggggctatgaa
ggcgcaatgaacatcgtcacgacgctggtgaacgccgtgctggaaaaactggaccacgacaccagccagttgg-
gcaaaaccgattacagcttcgac ctcgttcgttaa 103
atgaccctgaatatgatgctcgataacgccgcgccggaggccatcgccggcgcgctgactcaacaacatc-
cggggctgttttttaccatggtggaaca
ggcctcggtggccatctccctcaccgatgccagcgccaggatcatttacgccaacccggcgttttgccgccag-
accggctattcgctggcgcaattgtt
aaaccagaacccgcgcctgctggccagcagccagacgccgcgcgagatctatcaggagatgtggcataccctg-
ctccagcgtcagccctggcgcg
gtcagctgattaatcagcgtcgggacggcggcctgtacctggtggagattgacatcaccccggtgcttagccc-
gcaaggggaactggagcattatct
ggcgatgcagcgggatatcagcgtcagctacaccctcgaacagcggctgcgcaaccatatgaccctgatggag-
gcggtgctgaataatatccccgc
cgccgtggtagtggtggacgagcaggatcgggtggtgatggacaacctcgcctacaaaaccttctgcgctgac-
tgcggcggccgggagctgctcac
cgagctgcaggtctcccctggccggatgacgcccggcgtggaggcgatcctgccggtggcgctgcgcggggcc-
gcgcgctggctgtcggtaacc
tgctggccgttacccgccgtcagtgaagaggccagccgctactttatcgacagcgcgctggcgcggaccctgg-
tggtgatcgccgactgtacccag
cagcgtcagcagcaggagcaagggcgccttgaccggctgaagcagcaaatgaccgccggcanctgctggcggc-
gatccgcgagtcgctggac
gccgcgctgatccagctgaactgcccgattaatatgctggcggcagcccgtcggctgaacggcgagggaagcg-
ggaatgtggcgctggaggccg
cctggcgtgaaggggaagaggcgatggcgcggctccagcgctgtcgcccatcgctggaactcgaaaaccccgc-
cgtctggccgctgcagccctttt
tcgacgatctgtgcgccctctaccgtacacgcttcgatcccgacgggctgcaggtcgacatggcctcaccgca-
tctgatcggctttggccagcgcacc
ccactgctggcgtgcttaagcctgtggctcgatcgcaccctggccctcgccgccgaactcccctccgtgccgc-
tggcgatgcagctctacgccgagg
agaacgacggaggctgtcgctgtatctgactgacaacgtaccgctgctgcaggtgcgctacgctcactccccc-
gacgcgctganctcgccgggca
aaggcatggagctgcggctgatccagaccctggtggcgcaccatcgcgagccattgagctggcttcccgaccg-
cagggcggcacctgcctgacc ctgcgtttcccgctgtttaacaccctgaccggaggtgaagcatga
104
atgatccctgaatccgacccggacaccaccgtcagacgcttcgacctctctcagcagttcaccgccatga-
gcggataagcgtggtgctgagccggg
ccaccgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgatgcctttatgcagcacgggat-
gatctgcctgtacgacagcgagc
aggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgcagatccgcta-
tcgccccggcgagggactggt
ggggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgttttctcgaccgc-
ctgagcactacgattacgatctg
ccgtttatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctggcggcccagccgatggcgc-
gccaggaagagcggctgccgg
cctgcacccgttttctcgaaaccgtcgccaacctcgtcgcccagaccatccggctgatgatccttccggcctc-
acccgccctgtcgagccgccagccg
ccgaaggtggaacggccgccggcctgctcgtcgtcgcgcggcgtgggccttgacaatatggtcggcaagagcc-
cggcgatgcgccagatcgtgg
aggtgatccgtcaggtttcgcgctgggacaccaccgtgctggtacgcggcgaaagcggcaccgggaaagagct-
gatcgccaacgccatccatcac
cattcgccacgggctggcgccgccttcgtcaaatttaaactgcgcggcgctgccggacaccctgctggaaagc-
gaactgttcggccatgagaaaggc
gcctttaccgagcggtgcgtcagcgtaaaggacgttttgagctggcggatggcggcaccctgttcctcgatga-
gattggtganagcagcgcctcg
ccaggccaagctgctgcgatcctccaggagggggagatggagcgggtcggcggcgatgagaccctgcgggtga-
atgtccgcatcatcgccgcc
accaaccgtcacctggaggaggaggtccggctgggccatttccgcgaggatctctactatcgctgaacgtgat-
gccatcgccctgcccccgagc
gcgagcgtcaggaggacatcgccgagctggcgcacttcctggtgcgcaaaatcggccagcatcaggggcgcac-
gctgcggatcagcgagggcg
cgatccgcctgctgatggagtacagctggccgggtaacgttcgcgaactggagaactgcctcgaacgatcggc-
ggtgatgcggagagtggcctga
tcgatcgcgacgtgatcctcttcactcaccaggatcgtcccgccaaagccctgcctgccctgcgggccagcgg-
aagacagctggctggacaacagcc
tggacgaacgtcagcgactgatcgccgcgctggaaaaagccggctgggtgcaggccaagccggcacggctgct-
ggggatgacgccgcgccagg
tcgcttatcggatccagatcatggatatcaccctgccgcgtctgtag 105
atgatgccgctttctccgcaattacagcagcactggcagacggtcgctgaccgtctgccagcggattttc-
ccattgccgaactgagcccacaggccag
gtccgtcatggcgttcagcgattttgtcgaacagagtgtgatcgcccagccgggctggctgaatgagcttgcg-
gactcctcgccggaggcggaagag
tggcggcattacgaggcctggctgcaggatcgcctgcaggccgtcactgacgaagcggggttgatgcgagagc-
tgcgtctcttccgccgccagatg
atggtccgcatcgcctgggcgcaggcgctgtcgctggtgagcgaagaagagactctgcagcagctgagcgtcc-
tggcggagaccctgattgtcgcc
gcccgcgactggctgtacgccgcctgctgtaaggagtggggaacgccatgcaatgccgagggccagccgcagc-
cgctgctgatcctcgggatgg
gaaaagctgggcggcggcgagctgaacttctcttccgatatcgatctgatctttgcctggcctgagcatggcg-
ccacccgcggcggccgccgcgagct
ggataacgcccaggctttacccgtctggggcagcggctgatcaaggcccttgaccagccgacgcaggacggct-
ttgtctatcgggttgacatgcgcc
tgcggccgtttggcgacagtgggccgctggtactcagttttgcggcgctggaagattattaccaggagcaggg-
tcgggactgggaacgctatgcgat
ggtgaaagcgcggatcatgggcgataacgacggcgtgtacgccagcgagttgcgcgcgatgctccgtcctttc-
gtcttccgccgttatatcgacttca
gcgtgatccagtcgctgcgtaacatgaaaggcatgatcgcccgcgaagtgcggcgtcgcgggctgaaagacaa-
catcaagctcggcgccggcgg
gatccgtgaaattgagtttatcgttcaggtctttcaactgatccgcggtggtcgcgaacctgcactgcagcag-
gcgccctgctgccgacgctggcgg
cgattgatgagctacatctgctgccggaaggcgacgcggcgctgctgcgcgaggcctatctgttcctgcgccg-
gctggaaaacctgctgcaaagcat
caacgatgagcagacccagaccctgccgcaggatgaacttaaccgcgccaggctggcgtgggggatgcatacc-
gaagactgggagacgctgagc
gcgcagctggcgagccagatggccaacgtgcggcgagtgtttaatgaactgatcggcgatgatgaggatcagt-
ccccggatgagcaactggccga
gtactggcgcgagctgtggcaggatgcgctggaagaagatgacgccagcccggcgctggcgcatttaaacgat-
accgaccgccgtagcgtgctgg
cgctgattgccgattttcgtaaagagctggatcggcgcaccatcggcccgcgggccgccaggtgctggatcag-
ctgatgccgcatctgctgagcga
aatctgctcgcgcgccgatgcgccgctgcctctggcggatcacgccgctgttgaccgggatcgtcacccgtac-
cacctatcttgagctgctgagc
gaattccccggcgcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgc-
gccacccgctgctgctggatga
gctcgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacct-
gctgcgcgtgccggaagaggatga
agagcagcagctggaggcgttgcgccagtttaagcaggcgcagcagagcatatcgcggcggcggatatcgctg-
gtaccctgccggtgatgaagg
tcagcgatcatttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggt-
cgctcgctacggccagccgaccc
acctgcacgatcgccagggtcgcggcttcgccgtcgtcggctacggtaagcttggcggctgggagctgggcta-
cagctccgatctcgatctggtgttc
ctccatgactgcccggcggaggtgatgaccgacggcgagcgggagattgacggccgtcagttctacctgcggc-
tggcccagcggatcatgcacct
gttcagcacccgcacctcgccggtattctctacgaagtggacgcccggctgcgtccttctggcgcggcgggga-
tgctggtcaccaccgccgacgc
gtttgctgactatcagcagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctat-
ggcgacccggcgctgcaggcgc
gctttgacgccattcgtcgcgatatcctgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcga-
gatgcgcgagaagatgcgcgcc
caccttggcaacaaacatcccgatcgtttgatatcaaagccgatgccggcgggatcaccgatattgaatttat-
tactcagtatctggtcctacgctatgcc
agtgacaagccgaagctgacccgctggtctgacaacgtgcgtattcttgagctgctggcgcagaagacatcat-
ggacgaggaggaggcgcgcgc
cttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggccctgcaggagcagccgggacacgtg-
gcgccagaggccttcagccggga
gcgtcagcaggtcagcgccagctggcagaagtggctgatggcttaa 106
agcgtcaggtaccggtcatgattcaccgtgcgattctcggttccctggagcgcttcattggcatcctgac-
cgaagagttcgctggcttcttcccaacctg
gattgcaccagtgcaggtagtggtcatgaatattaccgattctcaggctgaatacgttaacgaattgacgcgt-
aaactacaaaatgcgggcattcgtgta
aaagcagacttgagaaatgagaagattggctttaaaatccgcgagcacactttacgtcgtgcccgtatatgtt-
ggtctgtggcgacaaagaagtcgaa
gccggcaaagtggccgtgcgcacccgtcgcgggaaagacctcggcagcatggacgtaagtgaagtgattgaga-
agctgcaacaagagattcgca
gccgcagtcttcaacaactggaggaataaggtattaaaggcggaaaacgagttcaaacggcacgtccgaatcg-
tatcaatggcgagattcgcgccct ggaagttcgc 107
atgaaaatggcaacaatgaaatcgggtctgggggcattagcccttcttccgggactggcaatggccgcgc-
ccgcagtggcggacaaagccgataac
gcgtttatgatgatttgcaccgcgctggttctgtttatgaccatcccggggatcgcgctgttttacggcggcc-
tgatccgcggcaaaaacgtcctttccat
gctgactcaggtgattgtgacctttggcctcgtctgcgtactgtgggtgatttatggctataccctggccttc-
ggcaccggcggcagcttcttcgctagttt
tgactgggtgatgctgaaaaatattgaactgaaagcgctgatgggcaccttctatcagtacatccacgtggca-
tccagggctcgttcgcctgtatcacc
gtcgggctgatcgtgcgggcgctggctgagcgtattcgtttctccgccgtgctgatttttgtggtggtgtgga-
tgacgctctcttatgttccgattgcgcac
atggtctggggcggcggtctgctggcgacccacggcgcgctggacttcgcgggcggcaccgttgtacacatca-
acgccgcggttgccgggctggt
gggtgcgtacatgatgggcaaacgtggggcttcggcaaagaagcgttcaaaccgcacaatctgccgatggtgt-
tcaccggaaccgccatcctctac
gtgggctggttcggcttcaacgccggctccgccagcgcagcgaacgaaattgccgcattggctttcgtcaaca-
ccgtcgtcgccacagcggctgcca
tcctggcgtggacctttggcgaatgggccctgcgcggtaaaccttcactgctgggcgcctgctccggggcgat-
tgccggtctggttggcgtcacacca
gcctgtgggtatatcggtgtcggtgggcgttgattgtgggtatcgcatctggtctggcgggcatctggggcgt-
aacggcgctgaaacgctggctgcg
ggttgatgacccttgcgacgtcttcggcgtccacggcgtctgcggcatcgtcggctgtatcctgaccggtatc-
ttcgcggccacctctctgggcggcgt
gggttatgcagaaggcgtcaccatgggccatcagctgctggtgcaactcgagagtatcgcgattaccatcgtc-
tggtcgggcgttgtcgctttcattgg
ctacaaagtggcggacatgaccgtggggctgcgcgtaccagaagagcaggagcgcgaaggactggacgtcaac-
agccatggcgaaaacgccta caacgcctga 108
cgccgtcctcgcagtaccattgcaaccgactttacagcaagaagtgattctggcacgcatggaacaaatt-
cttgccagtcgggctttatccgatgacga
acgcgcacagcttttatatgagcgcggagtgttgtatgatagtctcggtctgagggcattagcgcgaaatgat-
ttttcacaagcgctggcaatccgaccc
gatatgcctgaagtattcaattacttaggcatttacttaacgcaggcaggcaattttgatgctgcctatgaag-
cgtttgattctgtacttgagcttgatc 109
gctaaagttctcggctaatcgctgataacatttgacgcaatgcgcaataaaagggcatcatttgatgccc-
tttttgcacgctttcataccagaacctggctc
atcagtgattttttttgtcataatcattgctgagacaggctctgaagagggcgtttatacaccaaaccattcg-
agcggtagcgcgacggcaagtcagcgtt
ctcctttgcaatagcagggaagaggcgccagaaccgccagcgttgaagcagtttgaacgcgttcagtgtataa-
tccgaaacttaatttcggtttgga 110
gcccgctgaccgaccagaacttccaccttggactcggctatacccttggcgtgacggcgcgcgataactg-
ggactacatccccattccggtgatctta
ccattggcgtcaataggttacggtccggcgactttccagatgacctatattcccggcacctacaataacggta-
acgtttacttcgcctgggctcgataca
gttttaattcgctaagtcttagcaataaatgagataagcggtgtgtcttgtggaaaaacaaggactaaagcgt-
tacccactaaaaaagatagcgacttttat
cactttttagcaaagttgcactggacaaaaggtaccacaattggtgtactgatactcgacacagcattagtgt-
cgatttttcatataaaggtaattttg 111
ttgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggtagc-
acagagagcttgctctcgggtgacgag
cggcggacgggtgagtaatgtagggaaactgcctgatggagggggataactactggaaacggtagctaatacc-
gcataacgtcgcaagaccaaag
tgggggaccttcgggcctcatgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctcac-
ctaggcgacgatccctagctggtct
gagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattg-
cacaatgggcgcaagcctgat
gcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaaggcgntnaggt-
taataaccttgtcgattgacgttac
ccgcagaagaagcaccggctaactccggccagcagccgcggtaatacggagggtgcaagcgttaatcggaatt-
actgggcgtaaagcgcacgca
ggcggtagtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggctagagt-
cttgtagaggggggtagaattcc
agggtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgac-
gctcaggtgcgaaagcgtgg
ggagcaaacaggattagaccctggtagtccacgctgtaaacgatgtcgatttggaggttgtgcccttgaggcg-
tggcttccggagctagcgcgttaa
atcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtgga-
gcatgtggtttaattcgatgcaa
cgcgaagaaccttacctggtcttgacatccacagaactttccagagatggattggtgccttcgggaactgtga-
gacaggtgctgcatggctgtcgtcag
ctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtnnggcc-
gggaactcaaaggagactgccagtg
ataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaa-
tggcatatacaaagagaagcgac
ctcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagt-
cggaatcgctagtaatcgtagatca
gaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtacaccatgggagtgggttgcaaaagaa-
gtaggtagcttaaccttcgggag
ggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttgg-
atcacctcctt 112
atgaccatgcgtcaatgcgctatctacggtaaaggcggtatcggtaaatccaccaccacccagaatctcg-
tcgcggcctcgccgagatgggtaaga
aagtgatgatcgtcggctgcgatccgaaagcggactccacccgtctgatccttcacgctaaagcccagaacac-
catcatggagatggcggcggaagt
gggctcggtcgaggatctggagctcgaagacgttctgcaaatcggctatggcgatgtccgttgcgccgaatcc-
ggcggcccggagccaggcgtcg
gctgcgccggacgcggggtgatcaccgccatcaacttcctcgaggaagaaggcgcctatgaggaagatttgga-
tttcgtcttctatgacgtcctcggc
gacgtagtctgcggcggcttcgccatgccgatccgcgaaaacaaagcccaggagatctacatcgtctgctccg-
gcgagatgatggcgatgtatgccg
ccaacaatatctccaaggggatcgtgaagtacgcgaaatctggcaaggtgcgcctcggcggcctgatctgtaa-
ctcgcgcaaaaccgaccgggaag
acgaactgatcatcgccctggcggagaagcttggcacgcagatgatccacttcgttccccgcgacaacattgt-
gcagcgcgcggagatccgccgga
tgacggtgatcgagtacgacccgacctgtcagcaggcgaatgaatatcgtcaactggcgcagaagatcgtcaa-
taacaccaaaaaagtggtgccaa
cgccgtgcaccatggacgagctggaatcgctgctgatggagttcggcatcatggaagaagaagacaccagcat-
cattggtaaaaccgccgctgaag aaaacgcggcctga 113
atgaccaacgcaacaggcgaacgtaaccttgcgctcatccaggaagtcctggaggtgtttcccgaaaccg-
cgcgcaaagagcgcagaaagcacat
gatgatcagcgatccgcagatggagagcgtcggcaagtgcattatctcgaaccgtaaatcgcagcccggggtg-
atgaccgtgcgtggctgcgcctat
gcgggcttcgaaaggggtggtgtttgggccaatcaaagacatggcccatatctcgcacggccccatcggctgc-
ggccagtactgcgcgccggacg
gcgcaactactataccggcgtcagcggtgtcgacagcttcggcaccctgaacttcacctctgattttcaggag-
cgcgatattgttttcggcggcgataaa
aagctgaccaaactgatcgaagagatggagctgcttcccgctgctccaaagggatcaccatccaatcggagtg-
cccggtgggcctgatcggcgat
gacatcagcgccgtggccaacgccagcagcaaggcgctggataaaccggtgatcccggtgcgctgcgaaggct-
ttcgcggcgtatcgcaatcgct
gggccaccatatcgccaacgacgtggtgcgcgactgggtgctgaacaatcgcgaagggcagccgtttgccagc-
accccgtatgatgttgccatcatt
ggcgattacaacatcggcggcgacgcctgggcctcgcgcattctgctggaagagatggggctgcgcgtagtgg-
cgcagtggtccggcgacggca
ccctggttgagatggagaacaccccattcgttaagcttaacctcgccactgctaccgacgatgaactatatcg-
cccgccatatggaggagaaacatc
agatcccgtggatggaatataacttcttcggcccgaccaaaatcgccgaatcgctgcgcaagatcgccgatca-
atttgatgacaccattcgcgccaatg
cggaagcggtgatcgccaaatatgaggggcagatggcggccatcatcgccaaatatcgcccgcggctggaggg-
gcgcaaagtgctgctgtacatg
gagggctgcggccgcgccacgtcatcggcgcctatgaggatctcgggatggagatcatcgccgccggctacga-
gtttgcccataacgatgattac
gaccgcaccctgccggacctgaaagagggcaccctgctgtttgacgatgccagcagctatgagctggaggcct-
tcgtcaaagcgctgaaacctgac
ctcatcggctccgggatcaaagagaaatatatcttccagaaaatgggggtgccgttccgccagatgcactcct-
gggactattccggcccctatcacgg
ctatgacggcttcgccatctttgcccgccatatggatatgaccctgaacaatccggcgtggaacgaactgact-
gccccgtggctgaagtctgcgtga 114
atgaagggaaaggaaattaggcgctgctggacgaacccgcctgcgagcacaaccagaagcaaaaatccgg-
ctgcagcgctcctaagcccggcg
caaccgccggcggctgcgccttcgacggcgcgcagataacgctcctgcccatcgccgacgtcgcgcacctggt-
gcacggccccatcggctgcgc
gggcagctcgtgggataaccgcggcagcgtcagcgccggcccggccctcaaccggctcggctttaccaccgat-
cttaacgaacaggatgtgattat
gggccgcggcgaacgccgcctgttccacgccgtccgtcacatcgtcgaccgctatcatccggcggcggtcttt-
atctacaacacctgcgtaccggcg
atggagggggatgacctggaggccgtctgccaggccgcacagaccgccaccggcgtcccggtcatcgccattg-
acgccgccggtttctacggcag
taaaaatcttggcaaccgaatggcgggcgacgtgatgctcaggcaggtgattggccagcgcgaaccggccccg-
tggccagacaacacgccctttg
ccccggcccagcgccacgatatcggcctgattggcgaattcaatatcgccggcgagttctggcaggtccagcc-
gctgctcgacgagctggggatcc
gcgtcctcggcagcctctccggcgacggccgctttgccgagatccagaccctgcaccgggcgcaggccaatat-
gctggtgtgctcgcgcgcgctga
tcaacgtcgcccgggggctggagctgcgctacggcacgccgtggtttgaaggcagcttctacgggatccgcgc-
cacctccgacgccttgcgccagc
tggcggcgctgctgggaatgacgacctgtgccgccgcaccgaggcgctgatcgcccgcgaagagcaggcggcg-
gagcaggcgctggcgccg
tggcgcgagcagctccgtgggcgcaaagtgttgctctacaccggcggcgtgaaatcctggtcggtggtatcag-
ccctgcaggatctcggcatgacc
gtggtggccaccggcacgcggaaatccaccgaggaggacaaacagggatccgtgagctgatgggcgacgaggc-
ggtgatgcttgaggagggc
aatgctcgcaccctgctcgacgtggtgaccgctatcaggccgacctgatgatcgccggcggacgcaatatgta-
caccgcctggaaagcccggctg
ccgtttctcgatatcaatcaggagcgcgagcacgcctacgccggctatcagggcatcatcaccctcgcccgcc-
agactgtctgaccctcgccagtcc cgtctggccgcaaacgcatacccgcgccccgtggcgctag
115
atggcagacattatccgcagtgaaaaaccgctggcggtgagcccgattaaaaccgggcaaccgctcgggg-
cgatcctcgccagcctcgggctggc
ccaggccatcccgctggtccacggcgcccagggctgcagcgccttcgccaaagttttctttattcagcatttc-
catgacccggtgccgctgcagtcgac
ggccatggatccgaccgccacgatcatgggggccgacggcaatatcttcaccgcgctcgacaccctctgccag-
cgccacagcccgcaggccatcg
tgctgctcagcaccggtctggcggaagcgcagggcagcgatatcgcccgggtggtgcgccagtttcgtgaggc-
gcatccgcgccataacggcgtg
gcgatcctcaccgtcaataccccggatttttttggctcgatggaaaacggctacagcgcggtgatcgagagcg-
tgatcgagcagtgggcgcgccga
cgccgcgtccggggcagcggccccggcgggtcaacctgctggtcagccacctctgttcgccaggggatatcga-
atggctgggccgctgcgtggag
gcctttggcctgcagccggtgatcctgccggacctctcgcagtcaatggatggccacctcggtgaaggggatt-
ttacgcccctgacccagggcggcg
cctcgctgcgccagattgcccagatgggccagagtctgggcagcttcgccattggcgtgtcgctccagcgggc-
ggcatcgctcctgacccaacgca
gccgcggcgacgtgatcgccctgccgcatctgatgaccctcgaccattgcgatacctttatccatcagctggc-
gaagatgtccggacgccgcgtacc
ggcctggattgagcgccagcgcggccagctgcaggatgcgatgatcgactgccatatgtggcttcagggccag-
cgcatggcgatggcggcggag
ggcgacctgaggcggcgtggtgtgatttcgcccgcagccaggggatgcagcccggcccgctggtcgcccccac-
cagccaccccagcctgagcc
agctgccggtcgatcaggtcgtgccgggggatcttgaggatctgcagcagctgctgagccaccaacccgccga-
tctgctggtggctaactctcacgc
ccgctgatctggcggagcagtttgccctgccgctgatccgcgtcggttttcccctatcgaccggctcggtgag-
atcgtcgcgtccgccaggcgtacgc
cggtatgcgagatacgctgtttgagctggccaatctgctgcgcgaccgccatcaccacaccgccctctaccgc-
tcgccgcttcgccagggcgccgac cccctgccggcttcaggagacgcttatgccgcccattaa
116
gtgccgctgatccgtctgggctttccgctgttcgaccgccatcatctgcaccgccagaccacctggggct-
atgaaggcgcaatgaacatcgtcacgac
gctggtgaacgccgtgctggaaaaactggaccacgacaccagttgggcaaaaccgattacagcttcgacctcg-
ttcgttaa 117
atgaccctgaatatgatgctcgataacgccgcaccggaggccatcgccggcgcgctgactcaacaacatc-
cggggctgttttttaccatggtggaaca
ggcctcggtggccatatccctcaccgatgccagcgccaggatcatttacgccaacccagcgtttgccgccaga-
ccggctattcgctggcgcaattgt
aaaccagaacccgcgcctgctggccagcagccagacgccgcgcgcgatctatcaggagatgtggcataccctg-
ctccagcgtcagccctggcgcg
gtcagctgattaatcagcgtcgggacggcggcctgtgcctggtggagattgacatcaccccggtgcttagccc-
gcaaggggaactggagcattatct
ggcgatgcagcgggatatcagcgtcagctacaccctcgaacaacggctgcgcaaccatatgaccctgatggag-
gcggtgctgaataatatccccgc
cgccgtggtggtggtggacgagcaggatcgggtggtgatggacaacctcgcctacataaccttctgcgctgac-
tgcggcggccgggagctgctca
ccgagctgcaggtctcccctggccggatgacgcccggcgtggaggcgatcctgccggtagcgctgcgcggggc-
cgcgcgctggctgtcggtaac
ctgctggccgttgcccggcgtcagtgaagaggccagccgctactttatcgacagcgcgctggcgcggaccctg-
gtggtgatcgccgactgtaccca
gcagcgtcagcagcaggagcaaggacgccttgaccggctgaagcagcaaatgaccgccggcaagctgctggcg-
gcgatccgcgagtcgctgga
cgccgcgctgatccagctgaactgcccgattaatatgaggcggcagcccgtcggctgaacggcgagggaagcg-
ggaatgtggcgctggaggcc
gcctggcgtgaagggcaagaggcgatggcgcggctccagcgctgtcgcccatcgctggaactcgaaaaccccg-
ccgtctggccgctgcagccctt
tttcgacgatctgtgcgccctctaccgtacccgcttcgatcccgacgcgctgcaggtcgacatggcctcaccg-
catctgatcggctttggccagcgcac
cccgctgctgccgtgcttaagcctgtggctcgaccgcaccctggccctcgccgccgaattgccctccatgcgc-
tggcgatgcagctctatgccgag
gagaacgacggctggctgtcgctgtacctgactgataacgtaccgctgttgcagggcgctacgcccactcccc-
cgacgcgctgaactcgccgggta
aaggcatggagctgcgcctgatccagaccctggtggcgcaccatcgcggggccattgagctggcttcccgacc-
gcagggcggcacctgcctgacc
ctgcgtttcccgctgtttaacaccctgaccggaggtgaagcatga 118
atgatccctgaatccgacccggacaccaccgtcagacgcttcgacctctacagcagttcaccgccatgca-
gcggataagcgtggtgctgagccggg
ccaccgaggccagcaaaacgctgcaggaggtactcactgtattgcacaacgatgccatatgcagcacgggatg-
atctgcctgtacgacagcgagca
ggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgcagatccgctat-
cgccccggcgagggactggtg
gggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgttttctcgaccgcc-
tgagcctctacgattacgatctgc
cgtttatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctggcggcccagccgatggcgcg-
ccaggaagagcggctgccggc
ctgcacccgttttacgaaaccgtcgccaacacgtcgcccagaccatccggctgatgatccttccggcctcacc-
cgccagtcgagccgccagccgc
cgaaggtggaacggccgccggcctgctcgtcgtcgcgcggcgtgggccttgacaatatggtcggcaagagccc-
ggcgatgcgccagatcgtgga
ggtgatccgtcaggtttcgcgctgggacaccaccgtgctggtgcgcggtgaaagcggcaccgggaaagagctg-
atcgccaacgccatccatcacc
attcgccacgggctggcgccgccttcgtcaaatttaactgcgcggcgctgccggacaccctgctggaaagcga-
actgttcggccatgagaaaggcg
cctttaccggggcggtgcgtcagcgtaaaggacgttttgagctggccgatcgcggcaccctgttcctcgatga-
gattggtgaaagcagcgcctcgttc
caggccaagctgctgcgtatcctccaggagggggagatggagcgggtcggcggcgatgagaccctgcgggtga-
atgtccgcatcatcgccgcca
ccaaccgtcacctggaggaggaggtccggctgggccatttccgcgaggatctctattatcgtctgaacgtgat-
gcccatcgccctgcccccgctgcgc
gagcgtcaggaggacatcgccgagctggcgcacttcctggtgcgcaaaatcggccagcatcaggggcgcacgc-
tgcggatcagcgagggcgcg
atccgcctgctgatggagtacagctggccgggtaacgttcgcgaactggagaactgcctcgaacgatcggcgg-
tgatgtcggagagtggcctgatc
gatcgcgacgtgatcctcttcactcaccaggatcgtcccgccaaagccctgcctgccaggggccagcggaaga-
cagctggctggacaacagcct
ggacgaacgtcagcgactgatcgccgcgctggaaaaagccggctgggtgcaggccaaggcgccacggctgctg-
gggatgacgccgcgccaggt cgcttaccggatccagatcatggatatcaccctgccgcgtctgtag
119
atgatgccgctttctccgcaattacagcagcactggcagacggtcgctgaccgtctgccagcggattttc-
ccattgcagaactgagcccacaggccag
gtcggtcatggcgttcagcgattttgtcgaacagagtgtgatcgcccagccgggctggctgaatgagatgcgg-
actcctcgccggaggcggaagag
tggcggcattacgaggcctggctgcaggatcgcctgcaggccgtcactgacgaagcggggttgatgcgagagc-
tgcgtctatccgccgccagatg
atggtccgcatcgcagggcgcaggcgctgtcgctggtgagcgaagaagagaccctgagcagctgagcgccctg-
gcggagaccctgattgtcgc
cgcccgcgactggctctacgccgcctgctgtaaggagtggggaacgccatgcaatgccgagggccagccgcag-
ccgctgctgatcctcgggatgg
gaaagctgggcggcggcgagctgaacttctatccgatatcgatagatctttgcctggcctgagcatggcgcca-
cccgcggcggccgccgcgagct
ggataacgcccagttctttacccgtctggggcagcggctgatcaaggcccttgaccagccgacgcaggacggc-
tttgtctatcgggttgacatgcgcc
tgcggccgtttggcgacagtgggccgctggtactcagctttgcggcactggaagattattaccaggagcaggg-
tcgggactgggaacgctatgcgat
ggtgtaagcgcggatcatgggcgataacgacggcgtgtacgccagcgagttgcgcgcgatgctccgtcctttc-
gtcttccgccgttatatcgacttca
gcgtgatccagtcgctgcgtaacatgaaaggcatgatogcccgcgaagtgcggcgtcgcgggctgaaagacaa-
catcaagctcggcgccggcgg
gatccgtgaaattgagtttatcgttcaggtctttcagctgatccgcggtggtcgcgaacctgcactgcagcag-
cgcgccctgctgccgacgctggcgg
cgattgatgagctacatctgctgccggaaggcgacgcggcgctgctgcgcgaggcctatctgttcctgcgccg-
gctggaaaacctgctgcaaagcat
caacgatgattcagacccagaccctgccgcaggatgaacttaaccgcgccaggctggcgtgggggatgcatac-
cgaagactgggagacgctgagc
gcgcagctggcgagccagatggccaacgtgcggcgagtgtttaatgaactgatcggcgatgatgaggatcagt-
ccccggatgagcaactggccga
gtactggcgcgagctgtggcaggatgcgctggaagaagatgacgccagcccggcgctggcgcatttaaacgat-
accgaccgccgtagcgtgctgg
cgctgattgccgattttcgtaaagagctggatcggcgcaccatcggcccgcgcggccgccaggtgctggatca-
gctgatgccgcatctgagagcga
aatctgctcgcgtgccgatgcgccgagcctctggcgcggatcacgccgctgttgaccgggatcgtcacccgta-
ccacctatcttgagctgctgagcg
aattccccggcgcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcg-
ccacccgctgctgctggatgag
ctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgc-
tgcgcgtgccggaagaggatcaa
gagcagcagctggaggcgtgcgccagtttaagcaggcgcagagctgcatatcgcggcggcggatatcgctggt-
accctgccggtgatgaaggt
caggatcacttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcg-
ctcgctacggtcagccgaccca
cctgcacgatcgccagggtcgcggatcgccgttgtcggctacggtaagctcggcgcctgggagctggactaca-
gctccgatctcgatctggtgttcc
tccatgactgcccggcggaggtgatgaccgacggcgaggggagattgacggccgtcagttctacctgcggctg-
gcccagcggatcatgcacctgt
tcagcacccgcacctcgtccggtattactacgaagtggacgcccggctgcgtcatctggcgcggcaggttgct-
ggtcaccaccgccgacgcgtt
tgctgactatcagagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatggcg-
acccggcgctgcaggcgcgct
ttgacgccattcgtcgcgatatcctgaccaccccgcgggaggggacgaccctgcagaccgaggttcgcaagat-
gcgcgagaagatgcgcgcccac
cttggcaacaaacatcccgatcgattgatatcaaagccgatgccggcgggatcaccgatattgaatttattac-
tcagtatctggtcctacgctatgccagt
gacaagccgaagctgacccgctggtctgacaacgtgcgtattcttgagctgctggcgcaggacgacatcatgg-
acgaggaggaggcgcgcgcctta
acgcatgcatacaccaccttgcgtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgc-
cagaggccttcagccgggagg tcagcaggtcagcgccagctggcagaagtggctgatggcttaa
120
atgaaaatggcaacaatgaaatcgggtctgggggcattagcccttcttccgggactggcaatggccgcgc-
ccgcagtggcggacaaagccgataac
gcgtttatgatgatttgcaccgcgaggttctgtttatgaccatcccggggatcgcgctgattacggcggcctg-
atccgcggcaaaaacgtcctttccat
gctgactcaggtgattgtgacctttggcctggtntgcgtactgtgggtgatttatggctataccctggccttc-
ggaaccggcggcagcttcttcggtagctt
tgactgggtgatgctgaaaaatattgaactgaaagcgctgatgggcaccttctatcagtacatccacgtggcc-
ttccagggctcgttcgcctgtatcacc
gtcgggctgatcgtgggggcgctggctgagcgtattcgtttctccgccgtgctgattttcgtggtggtgtgga-
tgacgctacttatgttccgattgcgca
catggtctggggcggcggtctgctggcgacccacggcgcgctggacttcgcgggcggcaccgttgtacacatc-
aacgccgcggttgccgggctgg
tgggtgcgtatatgatgggcaaacgtgtgggcttcggcaaagaagcgttcaaaccgcacaatctgccgatggt-
gttcaccggaaccgccatcctctac
gtgggctggttcggcttcaacgccggctccgccagcgcagcgaacgaaattgccgcactggctttcgtcaaca-
ccgtcgtcgccacagcggcagcc
atcctggcctggacctttggcgaatgggctctgcgcggcaaaccttcactgctgggcgcctgctccggggcga-
ttgccggtctggttggcgtcacacc
agcctgtgggtatatcggtgtcggtggggcgttgattgtgggtatcgcatctggtctggcgggcatctggggc-
gtaacggcgctgaaacgctggctgc
gggttgatgacccttgcgacgtcttcggcgtccacggcgtctgcggtcgtcggctgtatcctgaccggtatct-
tcgcggccacctctctgggcggc
gtgggttatgcagaaggcgtcaccatgggccatcagctgctggtgcaactcgagagtatcgcgattaccatcg-
tctggtcgggcgttgtcgattcattg
gctacaaagtggcggacatgaccgtggggctgcgcgtaccagaagagaggagcgcgaaggactggacgtcaac-
agccatggcgaaaacgcct acaacgcctga 121
ctgaagagtttgatcctggctcagattgaacgctagcgggatgccttacacatgcaagtcgaacggcagc-
acggacttcggtctggtggcgagtggc
gaacgggtgagtaatgtatcggaacgtgcctagtagcgggggataactacgcgaaagcgtagctaataccgca-
tacgccctacgggggaaagcag
gggatcgcaagaccttgcactattagagcggccgatatcggattagatagttggtggggtaanggatcaccaa-
ggcgacgatccgtagctggattgag
aggacgaccagccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaattttggac-
aatgggggaaaccctgatcca
gccatcccgcgtgtgcgatgaaggccttcgggatgtaaagcacttttggcaggaaagaaacgtcatgggntaa-
taccccgtgaaactgacggtacctg
cagaataagcaccggctaactacgtgccagcagccgcggtaatacgtagggtgcaagcgttaatcggaattac-
tgggcgtaaagcgtgcgcaggcg
gttcggaaagaaagatgtgaaatcccagagcttaactttggaactgcatttttaactaccgggctagagtgtg-
tcagagggaggtggaattccgcgtgta
gcagtgaaatgcgtagatatgcggaggaacaccgatggcgaaggcagcctcctgggataacactgacgctcat-
gcacgaaagcgtggggagcaa
acaggattagataccctgctagtccacgccctaaacgatgtcaactagctgttggggccttcgggccttagta-
gcgcagataacgcgtgaagttgacc
gcctggggagtacggtcgcaagattaaaactcaaaggaattgacggggacccgcacaagcggtggatgatgtg-
gattaattcgatgcaacgcgaaa
aaccttacctacccttgacatgtctggaattctgaagagattcggaagtgctcgcaagagaaccggaacacag-
gtgctgcatggctgtcgtatgatcgt
gtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcattagttgctacgaaagggcactctaa-
tgagactgccggtgacaaaccggag
gaaggtggggatgacgtcaagtcctcatggcccttatgggtagggcttcacacgtcatacaatggtcgggaca-
gagggtcgccaacccgcgagggg
gagccaatcccagaaacccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgcta-
gtaatcgcggatcagcatgtcgcg
gtgaatacgttcccgggtcttgtacacaccgcccgtcacaccatgggagtgggttttaccagaagtagttagc-
ctaacc 122
ctgaagagtttgatcctcgctcagattgaacgctaggggatgccttacacatgcaagtcgaacggcagca-
cggacttcggtctggtggcgagtggc
gaacgggtgagtaatgtatcggaacgtgcctagtagcgggggataactacgcgaaagcgtagctaataccgca-
tacgccctacgggggaaagcag
gggatcgcaagaccttgcactattagagcggccgatatcggattagatagttggtggggtaaaggctcaccaa-
ggcgacgatccgtagatggtttgag
aggacgaccagccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaattttggac-
aatgggcgaaaccctgatcca
gccatcccgcgtgtgcgatgaaggccttcgggttgtaaagcacttttggcaggaaagaaacgtcatgggttaa-
taccccgtgaaactgacggtacctg
cagaataagcaccggctaactacgtgccagcagccgcggtaatacgtagggtgcaagcgttaatcggaattac-
tgggcgtaaagcgtgcgcaggcg
gttcggaaagaaagatgtgaaatcccagagcttaactttggaactgcatttttaactaccgggctagagtgtg-
tcagagggaggtggaattccgcgtgta
gcagtgaaatgcgtagatatgcggaggaacaccgatggcgaaggcagcctcctgagataacactgacgctcat-
gcacgaaagcgtggggagcaa
acaggattagataccctggtagtccacgccctaaacgatgtcaactagctgttggggcatcgggccttagtag-
cgcagctaacgcgtgaagttgacc
gcctggggagtacggtcgcaagattaaaactcaaaggaattgacggggacccgcacaagcggtggatgatgtg-
gattaattcgatgcaacgcgaaa
aaccttacctacccttgacatgtctggaattcngaagagattnggaagtgctcgcaagagaaccggaacacag-
gtgctgcatggctgtcgtcagctcg
tgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcattagttgatacgattagggcactct-
aatgagactgccggtgacaaaccgga
ggaaggtgggcatgacgtcaagtcctcatggcccttatgggtagggcttcacacgtcatacaatggtcgggac-
agagggtcgccaacccgcgaggg
ggagccaatcccagaaacccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgct-
agtaatcgcggatcagcatgtcgc
ggtgaatacgttcccgggtcttgtacacaccgcccgtcacaccatgggagtgggttttaccagaagtagttag-
cctaaccgnaaggggggcgattacc
acggtaggattcatgactggggtgaagtcgtaacaaggtagccgtatcggaaggtgaggctggatcacctcct-
tt 123
tacggagagtttgatcctggctcaggatgaacgctcgcggcctgcttaacacatgcaagtcgaacggttg-
aacacggagcttgctctctgggatcagtg
gcgaacgggtgagtaacacgtcagcaacctgcccctgactctgggataagcgctggaaacggcgtctaatact-
ggatatgtgacgtggccgcatgga
ctgcgtctggaaagaatttcggttggggatgggatcgcggcctatatgcttgttggtgaggtaatggctcacc-
aaggcgtcgacgggtagccggcctg
agagggtgaccggccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgc-
acaatgggcgcaagcctgatg
cagcaacgccgcgtgagggatgacggccttcgggagtaaacctcttttagcagggaagaaggaaagtgacggt-
acctgcagaaaaagcgccgg
ctaactacgtgccagcagccgcggtaatacgtagggcgcaagcgttatccggaattattgggcggaaagagct-
cgtaggcggtttgtcgcgtctgctgt
gaaatccggaggctcaacctccggcctgcagtgggtacgggcagactagagtgcggtaggggagattggaatt-
cctggtgtagcggtggaatgcgc
agatatcaggaggaacaccgatggcgaaggcagatctctgggccgtaactgacgctgaggagcgaaagggtgg-
ggagcaaacaggcttagatac
cctggtagtccaccccgtaaacgttgggaactagttgtggggtccattccacggattccgtgacgcagataac-
gcattaagttccccgcctggggagta
cggccgcaaggctaaaactcaaaggaattgacggggacccgcacaagcgacggagcatgcggattaattcgat-
gcaacgcgaagaaccttaccaa
ggcttgacatatacgagaacgggccagaaatggtcaactctttggacactcgtaaacaggtggtgcatggttg-
tcgtcagatcgtgtcgtgagatgttg
ggttaagtcccgcaacgagcgcaaccctcgttctatgttgccagcacctaatggtgggaactcatgggatact-
gccggggtcaactcggaggaaggt
ggggatgacgtcaaatcatcatcccccttatgtcttgggcttcacgcatgctacaatggccggtacaaagggc-
tgcaataccgcgaggtggaccgaat
cccaaaaagccggtcccagttcggattgaggtctgcaactcgacctcatgaagtcggagtcgctagtaatcgc-
agatcagcaacgctgcggtgaata
cgttcccgggtcttgtacacaccgcccgtcaagtcatgaaagtcggtaacacctgaagccggtggcctaaccc-
ttgtggagggagccgtcgaaggtg
ggatcggtaattaggactaagtcgtaacaaggtagccgtaccggaaggtgcggctggatcacctccttt
124
attgaagagtttgatcatggctcagattgaacgctggcggcagccctaacacatgcaagtcgaacggtag-
cacagagagcttgctctcgggtgacga
gtggcggacgggtgagtaatgtctgggaaactgcccgatggagggggataactactggaaacggtagctaata-
ccgcataatgtcgcaagaccaaa
gagggggaccttcggccctcttgccatcggatgtgcccagatgggattagattgatggtgaggtaatggatca-
ccaaggcgacgatccctagatggtc
tgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatatt-
gcacaatgggcgcaagcctgat
gcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaaggcgatncggt-
taataaccgtgttgattgacgttac
ccgcagaagaagcaccggctaactccgtgccagcagccccggtaatacggagggtgcaagagataatcggaat-
tactgggcgtaaagcgcacgca
ggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggcttgag-
tcttgtagaggggggtagaattcca
ggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgac-
gctcaggtgcgaaagcgtggg
gagcaaacaggattagataccctggtagtccacgccgtaaacgatgtcgacttggaggttgtgcccttgaggc-
gtggcttccggagctaacgcgttaa
gtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtgga-
gcatgtggtttaattcgatgcaa
cgcgaagaaccttacctggtcttgacatccacggaattnggcagagatgccttagtgccttcgggaaccgtga-
gacaggtgctgcatggctgtcgtca
gctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggc-
cgggaactcaaaggagactgccagt
gataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctaca-
atggcatatacaaagagaagcga
cctcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaag-
tcggaatcgctagtaatcgtggatc
agaatgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaag-
aagtaggtagcttaaccttcggga
gggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttg-
gatcacctcctt 125
atgaccatgcgtcaatgcgccatttatggcaaaggtgggatcggcaaatccaccaccacgcaaaacctcg-
tcgccgctctcgcggaaatgggtaaaa
aagtgatgatcgtcggctgcgacccgaaagggactccacccgtctgatcctgcatgcgaaagcacagaacacc-
attatggagatggccgccgaag
tgggttcagtggaagaccttgaactggaagatgtgctgcaaatcggttacggcggcgtgcgttgtgcagaatc-
cggcggcccggagccaggcgtgg
gttgtgcaggccgcggcgttattaccgccattaacttccttgaagaagaaggcgcctatgtcagcgacctcga-
ctttgtcttctatgacgtcctcggtgac
gtggtctgcggcgggttcgccatgccgattcgtgaaaacaaagcgcaagagatctatatcgtctgctccgggg-
aaatgatggcgatgtatgccgctaa
caacataccaaaggcatcgtgaaatacgctaaatccggcaaggtgcgcctgggcgggctgatttgtaactccc-
gtcagaccgaccgcgaagatgaa
ctgatcatcgcgctggcagaaaaactgggcacccagattgattcactttgtgccacgcgacaacatcgtccag-
cgcgcggaaattcgccgtatgacgg
ttatcgaatatgacccgaaatgcaaccaggccgacgaataccgcgcgctggcgaacaagatcgtcaacaacac-
cctgatggtcgtcccgaccccttg
caccatggatgaactggaagagctgctgatggaattcggcattatggatgtggaagacgccagcatcatcggt-
aaaaccgccgccgaagaaaacgc ggcctga 126
atgaacgataacgatgtccttttctggcgcatgctgccgctatttcagtgtctgccggaactgcaacccg-
cgcagatcctggcctggctgacaggagaa
cgcgacgacgccttaaccccggcgtacctcgataagcttaacgtccgcgaactggaagcgaccttcccgtctg-
aaacggcgatgatgtcgcccgca
cgctggagccgcgttaacgcgtgccttcacggtacgctgcccgcacacctgcaggtaaaaagcaccactcgtc-
aggggcaattacgggtagcattt
gttcacaggatggattgctgatcaatggtcattttggtcaggggcggctgttttttatctacgcctttgatga-
acagggcggatggctacacgcgttacgc
cgtcttccctcggccccgcaaacccaggagccgaatgaagttcgcgcgcagctcctgagtgattgccacctgc-
tgttttgtgaagccattggcggccc
tgcggcggcccggctgattcgtcacaatatccacccgatgaaagtgtcgccagggatgtccattgccgcccag-
tgtgatgccattaccgcactgctga
gcggacgtctgccaccgtggctggcaaaacgtcttgagaaagccaacccgctggagaagggtgttttaa
127
atgaagggaaatgacattctcgcgctgctggatgaacccgcctgcgaacacaatcacaaacagaaatccg-
gctgtagcgcccctaaacccggtgcc
acggcgggcggttgcgcgttcgacggcgcgcaaatcaccctgttgccgctgtcggtagtggcgcacctggtcc-
acggaccgattggctgcacggg
aagctcctgggataaccggggcagtatgagctccggccccagtctcaaccggctcggctttaccaccgacctg-
aacgagcaggatgtcaaatgggg
cgcggcgaacggcggcttttccacgcggtgcgtcatatcgtcaaccgttatcaccctgccgccgtgtttatct-
ataacacctgcgttccggcgatggag
ggtgatgatattgacgccgtctgtcaggcggcggaaaccgccaccggcgtgccagtgattgccgttcatgccg-
ccgggttctatggcagcaaaaacc
ttggcaaccgtctcgcgggtgaagtgatggttaacaaggtcattggacggcgcccgcccgccccctggccgga-
cgatacccccttcgcgccggaac
accgccacgatatcggcctgattggcgaatttaatatcgccggggagttctggcacgttcagccgctgctcga-
tgagctgggtattcgcgtgctgggca
gcctttccggggatggccgttttagtgaaatccagaccctgcaccacgcgcaggtcaatatgctggtctgctc-
aagagcgctgatcaatgttgcccgca
ccctggaacagcgctatggcaccccctggtttgagggcagtttttacggcgtgcgcgctacctccgatgccct-
gcgtcaactggcatccctgcttggcg
acagcgatctgattgcccccaccgaagccgttattgcccgcgaagaagccacggcaaatcagccgctcgcccc-
gtggcgcgaacggctacagggt
cgcaaagtgctgctctataccggtggcgtgaaatcaggtcggtgatctccgcattgcaggatttagggatgac-
cctcgtgccgactggcacccgcaa
atctaccgaagaagataagcagcgtattcgcgaattaatgggcgatgacgcgctaatgctggaagaaggcaac-
gcccgcaccctgctggatgtggtg
taccgctatcaggcggatttgatgatcgctggggggcgtaacatgtataccgcgtacaaagcgggctgccgtt-
tctggatatcaaccaggagcgtga
acacgcctttgcgggttatcgcggcatcgtcaccctcgcccaacagctttgccagactattgaaagccccgtc-
tggccgcaaacacacgcccgcgcg ccgtgccaataa 128
atgagcaatgcaacaggcgaacgtaatctggaaattatccaggaagtgctggagatctttcccgaaaaaa-
cgcgcaaagaacgcagaaagcacatg
atggttaccgacccggagatggaaagcgtcgggaaatgcatcatctctaaccgcaaatcgcagccgggtgtga-
tgactgtccgcggctgacctacg
ccgggtcgaaaggcgtggtttttgggccgattaaagatatggcccacatctcccacggcccgatcggctgtgg-
gcagtactcccgtgccgggcggc
gcaactactacaccggggtcagcggcgttgattccttcgggacgctgaactttacctctgattttcaggagcg-
cgatatcgtcttcggcggcgataaaaa
gctcaccaaactgattgaggagatggaggaactgttcccgctgaccaaaggcatctccattcagtcggagtgc-
ccggtaggtttaatcggtgacgatat
cgaagcggtggcgaatgccagtaaaaaagcgctcaacaagccggtgatcccggtgcgttgcgaaggctttcgc-
ggcgtgtcgcagtcgctcggtca
ccatatcgccaacgacgttatccgcgactgggtgctggataaccgcgaagggaagcccttcgaatctaccccc-
tatgacgtggccatcatcggcgatt
acaacatcgggggggatgcctgggcgtcgcgcattctgcttgaagagatggggttacgcgtggtggcgcagtg-
gtccggtgacggcacgctggtag
agatggtaaacaccccgttcgtcaagctgaacaggtgcactgctaccgctctatgaactacatctctcgccat-
atggaagagaaacacggtatcccgt
ggatggagtacaacttcttcggcccgaccaattatcgccgaatcgagcgtaagatcgccgatcaatttgacga-
caccatccgcgccaatgcggaagc
ggtgatcgccaaatatcaggcgcaaattcgatgcgattatcgccaaataccgcccgcgtctcgaaggccgcaa-
ggtgctgctctatatgggtggcctg
cgtcctcgccacgtgattggcgcgtatgaggatttgggcatggagattgtcgccgccgggtatgaatttgccc-
ataacgacgattacgaccgcaccct
gccggacctcaaagagggcacgctgttgttcgacgatgccagcagttatgaactggaagccttcgtgaaggcg-
attaagccggacctcattggctca
ggcatcaaggaaaaatacattttccagaaaatgggggtaccgtttcgccagatgcactcctgggattactccg-
gcccgtatcacggctatgacggcttt
gccatctttgcccgcaatatggacatgacgctcaacaatcccgcctggggcgagttgaccgcaccctggctga-
aatcagcctga 129
atggcagatatcatccgtaatcagaaaccgctggcggtaagcccggtaaaaagcggccagccgttaggcg-
ccattctggcgagcctcggctttgtgc
acagattccactggtgcacggtgcgcagggatgcagcgcgttcgccaaagtgttttttatccaacattttcat-
gaccctattccgctgcaatccacggcg
atggaccccacctcaacggtcatgggggcggacggcaatatccttgccgcgctcaatacgctgtgccagcgca-
ttcaccccgaaagctatcgtcctgt
tgagtaccggcctgtctgaggcgcagggcagcgatatcagccgcgtggtacgtcagtttcgtgaggattttcc-
ccgccacaaaaatatcgccctcctg
acggtcaacaccccggatttttacggcacgctggagaacggctttagtgcggtggtggaaagcgtcatcgaac-
agtgggtgccggaaagcctcag
catggcctgcgtaaccggcgggtcaacttgttgttaagtcacctgctgacgcccggtgatgttgagttgctgc-
gcagctacgtcgaggcttttggcctgc
aaccggtgatcgtgccggatctttcacagtcgctggatggtcacctggcaagcggtgatttttcgccggtcac-
tcaggggggaacgcccctgcgcatt
atcgaacagatgggacagagcctgtgcacgtttgctattggcgtgtcgctgtcccgtgcggcatcgagctggc-
acagcgtagccgtggcgangtga
tcgtgcttccccatctgatgaccatggaacattgcgaccgttttattcatcaactgaagatcatttccgggcg-
cgaggttcccgcctggattgagcgccag
cgcggacaattgcaggatgcgatgatcgattgtcatatgtggttgcaggatacccggctcgcgctggccgccg-
agggcgatctgctggcgggctggt
gtgatttcgcccgtagccagggcatgctccccggccccgttgtggcgccggtcagccagccgggcctgcgaca-
gcttcccgtggagaaagtggtca
ttggcgatctggaagatatgcaggatttactctgcgctatacctgctgacctgctggtcgccaactcccatgc-
cgcagacctggccgaacaattctccat
cccgctgatccgcgccgggttccctatcttcgacaggcttggcgaatttcgtcgcgtgcgtcagggatacccc-
ggcattcgcgacacgctgtttgagct
ggcgaacctgatgcgcgaacgtcatcaccacctgcccgtctaccgctcccocctgcgccagcaatttgcccag-
gacgctgacggaggccgctatgc aacatgttaa 130
atgagccaaactgctgagaaaattgtcacctgtcatccgctgtttgaacaggacgaataccagacgctgt-
ttcgcaataagcgcggtctggaagaggc
gcacgacccgcagcgcgtgaagaggtttttgaatggaccaccacggcggagtatgaagcgctgaactttaagc-
gtgaagcgttaaccgtcgatccg
gcaaaggcctgccagcctttaggatcggtactctgctcgctgggttttgccaatacgctgccttagtgcacgg-
ttcccagggctgtgtggcctatttccg
cacctattttaaccgtcatttcaaagagccgatcgcttgcgttccgactctatgacagaggatgcggtcttcg-
gcgccggcaacaacaacctaacacc
gggttgcaaaatgccagcgccctgacaaaccggaaattgtcgctgtgctccactacctgtatggcggaggtca-
tcggcgatgacctgcaggcctttatc
gccaacgccaaaaaggacgggtttattgatgccgccattccggtgccctacgcccatacgccaagttttatcg-
gtagccacatcaccggctgggacaa
catgtttgaaggtttcgcccgggcatttaccgccgatcacgtggcgcaaccgggcaaactggcgaagctaaac-
ctggtgaccggttttgaaacctatct
tggcaattaccgcgtgctcaaacgcatgatggcccagatggaggtgccctgtagcctgctgtctgacccgtct-
gagggttagatacgccagccgacg
gccactatcgcatgtatgcgggcggcacaacgcaacaagagatgcgcgacgcccccgatgctatcgacaccca-
gctgctgctgcaaccctggcatctgg
tgaagagtaaaaaagtggtgcaggagtcctggggccagcccgccacagaagtgtccatcccaatgggactgac-
cgggaccgacgaactgctgatg
gcagtcagtcagttaaccggcaaaccggtggccgatgaactgacgctggagcgtgggcgcctggtggatatga-
ttctcgattcacacacctggctgc
acggtaagaaattcggtctctacggcgatccggatttgtgatggggctgacgcgtttcctgctggaactgggc-
tgcgagccgacggttatcctctgtca
taacggtagcaagcgctggcagaaagcgatgaagaaaatgcttgaggcatcgccctacggtcaggagagcgaa-
gtgttcatcaactgcgatctgtg
gcatttccgctcgctgatgtttacccgcaaaccggactttatgatcggcaactcgtacgccaaattcatccag-
cgtgacacgctggcgaaaggcgaaca
gtttgaagttccgctgatccgtcttggcttcccgttgttcgaccgccaccacctgcatcgccagaccacatgg-
ggttatgaaggggcgatgaatatcgtc
accaccctggtcaacgccgtgctggaaaaagtcgaccgcgataccatcaaactgggcaaaacggactacagct-
tccgaccttgtccgctaa 131
atgacctttaatatgatgctggagaccagcgcaccgcagcacattgcgggcaacctctcacttcaacatc-
ccggactgttttccacgatggttgaacag
gctccgatcgcgatttcgctgaccgacccggacgcgaggattctgtacgctaatccggccttttgtcgccaga-
ccggttatagcctggaagagctgctc
aaccagaaccatcgcatactggcaagccancagacgccgcgcagcatttatcaggaactgtggcaaacgctgc-
tgcaacagatgccctggcgcggt
cagctcatcaatcgccgtcgggatggcagcctttatctggctgaggtcgatatcaccccggtcgtcaacaaac-
agggcgaactggaacactacctcgc
catgcaacgtgatatcagcgccagctatgcgctcgaacagcgattgcgcaatcacaccaccatgagcgaggcg-
gtgctgaacaacattcctgccgcc
gtggtggtggtcaacgagcaggaccaggtagtcatggacaacctcgcctacaaaaccttctgtgccgactgcg-
gtggcaaggagctgctcaccgaa
ctggatttctcccggcgcaaaagcgatctctatgccgggcaaatactgcctgtggtgctgcgcggcgccgtgc-
gctggactctgtcacctgaggac
cttgccgggggtgagcgaagaagccagccgctactttattgataccgcgagccccgcaccctggtggtgatca-
ccgactgcacccagcaacaaca
acaggcccgaacagggccgtctcgatcgtctcaaacaggagatgaccaccgggaagctgctggccgcgatccg-
tgaatcgttggatgccgcgctggt
tcagctaaactgccccatcaatatgctggcggcggcgcgacgtctcaacggtgaagataaccataacgtggcg-
ctggatgccgcgtggcgcgaggg
ggaagaggcgctggcccgcctgcaacgctgccgcccttctctcgatctggaagagagcgcgctgtggcctctg-
caaccgctgtttgacgacctgcg
cgccctttaccatacccgctataacaatggcgaaaatctgcacgttgaaatggcctctccgcatctggcgggg-
tttggtcagcgcacgcagatccttgc
ctgctcagtttgtggctcgaccgtacgctggccctcgccgccgcgctaccggacagaacgctgcatacccagc-
tttacgcccgtgaagaagatggct
ggctgtgtccatttggctgacagataatggccgctcatccatgtgcgatacgcccactcccccgatgccctga-
acgcccccggcaaagggatggagct
gcgattgattcaaaccctggttgcccatcatcgcggcgcaatagaactaactacccgccagatggcggtacct-
gcctgaccctgcgattcccgttattt cattcactgacccgaggcccacgatga 132
atgacccagcgacccgagtcgggcaccaccgtctggcgttttgatctctcacagcaatttaccgccatgc-
agcgcatcagcgtggtgttgagtcgcgc
aaccgagataagccagacgctgcaggaggtgctgtgtgttctgcataatgacgcatttatgcaacacggcatg-
ctgtgtctgtatgacaaccagcagg
aaattctgagtattgaagccttgcaggaggcagaccaacatctgatccccggcagctcgcaaattcgctatcg-
ccctggcgaagggctggtaggagc
cgtactgtcccagggacaatctcttgtgctgccgcgtgtcgccgacgatcaacgctttctcgacaggcttggc-
atctatgattacacctgccgtttatcg
ccgtccccttaatggggccaggcgcgcagacgattggcgtgctcgccgcgcagccgatggcgcgtctggagga-
gcggcttccttctgtacgcgct
ttctggaaaccgtcgccaatctggtcgcacagacagtccggctgatgaccccgcctgccgccgccacaccgcg-
cgccgcgattgcccagaccgaa
cgccagcgcaactgtggcactcctcgccccttcggctttgagaatatggtgggcaaaagcccggccatgcagc-
agacaatggacattatccgccagg
tttcgcgctgggataccacggtactggtgcgcggcgaaagcggcaccggtaaagaacttatcgccaatgctat-
tcatcacaactcccctcgcgccgcc
gcgccctttgtgaaatttaactgcgcggcgctaccggatacgctggagagagcgaattgttcggccatgaaaa-
aggggcgttcaccggcgcggttc
gccagcgtaaaggacgtttgaactggccgatggcggcacactgtttcttgatgaaattggcgaaagcagcgcc-
tcgttccaggccaaactgctgcgt
attttgcaggagggtgaaatggagcgcgttggcggcgacgaaaccctgcgcgtcaatgtgcgtatcatcgccg-
ccaccaaccggaatctggaagaa
gaggtgcggatgggcaatttccgcgaggatctctattatcgcctcaacgtaatgcccatctccctgcccccgc-
tgcgtgaacgtcaggaggacattgc
cgagctggcgcactttctggtgcgcaaaatcgcccataaccaggggcgtacgctgcgcatcagtgatggcgcc-
atccgtctgctgatgggttacaact
ggcccggtaacgtgctgtgagctggaaaattgcctggaacgttcggcagtgatgtcagaaaacggcctgatcg-
accgcgatgtggtgctctttaaccac
cgtgagaacacgccaaaactcgctatcgccgccgcgccaaaagaggatagctggcttgatcaaacgctggatg-
aacgtcaacggctgattgccgcg
ctggaaaaagccgggtgggtgcaggccaaagcggcgcgtctgctgggtatgacgccccgtcaggtcgcctatc-
ggatacaaattatggatatcagc atgcccaggatgtga 133
atgatgccgcactctccacagctacagcagcactggcaaactgtactggcccgcttgcctgagtcattca-
gtgaaacaccgcttagtgaacaagcgca
gttagtgcttactttcagtgattttgtgcaggatagccttgccgcgcatcctgactggctggctgagctggaa-
agcgcaccgccacaggcggacgagtg
gaagcagtatgcgcaaacccttcgcgaatcgctggaaaggtgtgggagatgaggcatcattaatgcgtgcgct-
gcgcctgttccgtcgccatatgatgg
tgcgcattgcctgggcgcagtcgctggcgctggtggcagaagatgagacgttgcagcagttgagcgtactggc-
ggagaccctgatcgtcgctgcac
gcgactggctttacgatgcctgctgtcgcgagtggggaacgccgtgcaatcagcagggggaaccgcagccgtt-
gctgatcctgggcatgggcaag
ctgggtggcggggagcttaacttttcgtccgatatcgatctgatttttgcctggccggaaaacggttcaacgc-
gcggtgggcgacgcgaacttgataac
gcccagttttttactcgcttgggacagcgcctgatcaaagtgctcgaccagccgacgcaggatggctttgtct-
atcgcgtggatatgcggctgcgcccg
tttggcgacagcggtccgctggtgctgagttttgccgcgctggaagattattatcaggagcaggggcgcgact-
gggaacgttatgcgatggtgaaagc
ccgcattatgggcgataaggacgatgtttacgctggcgaattacgggccatgctgcggccgttcgtcttccgt-
cgctatatcgatttcagcgttattcagt
ctctgcgtaacatgaaagggatgattgcccgcgaagtgcgccgccgtggtctgaaagataacattaagctggg-
cgcgggcggcatccgtgagattga
gtttatcgttcaggtgttccagttgatacgcggtgggcgcgagccgtcgttgcagtcccgttcactgttaccg-
acgctggacgctatcgataagctgggt
ttgctgccgcctggcgatgcaccggcgttacgccaggcctatttgtatctgcgccgtctggaaaaacctgctg-
caaagcattaacgacgaacaaacgca
gacgctgccgacagatgaactcaatcgcgcgcgtctggcctgggggatgcgggtcgcagactgggaaaccctg-
accgctgagcttgaaaagcaga
tgtctgccgtacgagggatattcaacaccctgattggcgatgacgaagccgaagagcagggggatgcgctctg-
cgggcaatggagtgagttgtggc
aggatgcgtttcaggaagatgacagcacgcctgtgctggcgcacctttctgacgatgatcgccgccgcgtggt-
cgcgatgattgctgattttcgcaaag
agctggataaacgcaccattggcccacgcggccgccaggtgctcgaccatctgatgccgcatctgttgagtga-
tgtctgctcccgtgaggatgcccct
gtaccgttgtctcgcgtgacgccgctgttaacgggaattgtcacgcgtacgacgtatcttgagctgctcagcg-
agtttcctggtgcgcgtaagcatctga
tttcactctgtgccgcctcgccgatggtggccagtaagctggcgcgctatccgttattgctggatgagttgct-
cgatccgaataccctttatcagcccacg
gcgatgaatgcctaccgggatgagctacgtcagtatctgctgcgtgtgccggatgacgatgaagagcagcaac-
tggaggcgttacgccagtttaaac
aggctcaattgttgcgtgtggcggcagcagatctggcaggcacactccccgtgatgaaagtgagcgatcactt-
aacatggcttgccgaagccatcatt
gaagccgtggtacaacaggcgtggagcctgatggtatcgcgttatgggcagccgaaacacttacgcgaccgtg-
aaggccgtgggtttgcagtggtc
ggttacggcaaactgggcggttgggagctgggctatagttccgatctggatttgattttccttcatgactgtc-
cggtggacgtgatgactgacggcgagc
gggaaatcgatggccgccaattttatctgcgccttgcccagcgcgtgatgcacctgttcagtacgcgcacctc-
atccgggatcctgtatgaggtagacg
cgcgcttgcgcccgtccggtgcggcgggaatgctggtgacctcaaccgaatcctttgccgactaccagcgcac-
cgaagcctggacctgggaacatc
aggcgctggttcgcgcccgcgttgtctatggcgatccacaattaaacgcgcaatttgatgccatccgccgcga-
tatcaccatgaccgtgcgtaatggtg
caacgttacaaaccgaggtgcgcgagatgcgcgaaaaaatgcgcgcccacttgagcaataagcacaaggatcg-
ctttgatattaaagccgatgagg
gtggaattaccgatatcgaatttatcacccagtatctggtgctgcgttatgcccatgccaaaccgaaactgac-
gcgctggtcggacaatgtccgcattct
ggaagggctggcgcaaaacggcattatggaagagcaggaagcgcaggcacttaccaccgcctatacaacgttg-
cgtgatgagctgcatcacctgg
cgctacaggagctgccaggacatgttccggaggcatgttttgtcgctgaacgcgcgatggtgcgagcctgctg-
gaacaagtggttggtggagccgtg cgaggacgcgtaa 134
atgaagaaagcactattaaaagcgggtctggcctcgctggcattactgccgtgtctggctatggcagccg-
atccggttgtcgtcgataaagccgacaat
gcctttatgatgatttgcaccgcgctggtgctgtttatgtcaattccgggcatcgccctgttctatggtggtt-
taatccgcggtaaaaacgtcctttctatgct
gacacaggttgcggttacgttcgcactggtgtgcgtgctgtgggtggtttacggctactctctggcctttggc-
actggcggcagcttcttcggtagcttcg
actgggtgatgctgaaaaatattgagctgaaagcgctgatgggcaccatctatcagtacattcacgttgcgtt-
ccagggctcgtttgcctgtattaccgtc
ggcctgattgtcggtgcgctggcagaacgtatccgtttctccgcagtactgattttcgtcgtggtatggctga-
cgctgtcctacgtgccgatcgcacacat
ggtctggggcggcggtctgctggcaacccatggcgccatggattttgcgggcggtacagtcgttcacatcaac-
gcagccgttgcaggcctggtgggt
gcttacctgattggcaaacgtgtcggtttcggtaaagaagcgtttaaaccgcacaacctgccgatggtgttta-
ccggtacggcaatcctctactttggctg
gttcggattcaacgcgggttctgcaagcgcggcgaacgaaattgcgggtctggcttttgttaacaccgtcgtg-
gcaacagcgggtgcaatcctctcctg
ggtcttcggtgagtgggcgctgcgcggcaaaccgtctctgttgggtgcctgttctggtgcgattgctggcctc-
gtgggtatcaccccggcgtgtggtta
cgttggtgtgggtggcgcgctgatcgtgggcatcgttgcaggcctggcgggtctgtggggcgttaccgcgctg-
aaacgctggctgcgtgttgacgac
ccgtgtgatgtcttcggtgttcacggcgtgtgcggtatcgtaggttgtatcatgacaggtatcttcgcagcca-
cttcactgggcggcgtgggttatgccga
aggcgtgaccatgggccatcaggttctggtacaactggaaagtatcgccattactatcgtatggtctggtatc-
gtcgcctttatcggttacaaactggctg
atatgacagtgggtctgcgtgttccggaagatcaggaacgcgaagggctggacgtcaacagccacggcgagaa-
cgcctacaacgcctga 135
ctggggtcactggagcgctttatcggcatcctgaccgaagaatttgccggtttcttcccgacctggctgg-
cccctgttcaggttgtggtgatgaatatcac
tgattctcaagctgaatatgtcaacgaattgacccgtaaattgcaaaatgcgggcattcgtgtaaaagcggac-
ttgagaaacgagaagattggctttaaa
atccgcgagcacactttacgtcgtgtcccttatatgttggtctgtggtgataaagaggtggaagcaggcaaag-
tggccgttcgcacccgccgcggtaa
agacctgggcagcctggacgtaagtgaagtgattgagaagctgcaacaagagattcgcagccgcagtcttcaa-
caactggaggaataaggtattaaa
ggcggaaaacgagttcaaacggcacgtccgaatcgtatcaatggcgagattcgcgcccaggaagacgcttaac-
tggtctggaaggtgagcagctg ggtatt 136
attgaagagtttcatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtag-
cacagagagcttgctctcgggtgacga
gtggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaata-
ccgcataacgtcgcaagaccaaa
gagggggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtgggctaacggctca-
cctaggcgacgatccctagctggt
ctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatat-
tgcacaatgggcgcaagcctga
tgcagccatgccgcgtgtatgaagaaggccttcgggttgtaaagtactttcagcggggaggaaggcganacgg-
ttaataaccgtgttgattgacgtta
cccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgaagcgttaatcggaat-
tactgggcgtaaagcgcacgc
aggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcatccgaaactggcaggcttga-
gtacgtagagggaggtagaattc
caggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactg-
acgctcaggtgcgaaagcgtg
gggagcaaacaggattagataccctggtagtccacgccgtaaacgatgtctatttggaggttgtgcccttgag-
gcgtggcttccggagctaacgcgtta
aatagaccgcctggggagtacggccgcaggttaaaactcaaatgaattgacgggggcccgcacaaccggtgga-
gcatgtggtttaattcgatgca
acgcgaagaaccttacctggtcttgacatccacagaacttgccagagatggcttggtgccttcgggaactgtg-
agacaggtgctgcatggctgtcgtca
gctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggc-
cgggaactcaaaggagactgccagt
gataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctaca-
atggcgcatacaaagaagcga
cctcgcgagagcaagcggacctcataaagtgcgtcgtagtcccgattggagtctgcaactcgactccatgaag-
tcggaatcgctagtaatcgtggatc
agaatgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaag-
aagtaggtagcttaaccttcggga
gggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttg-
gatcacctcctt 137
atgaccatgcgtcaatgcgccatttacggcaaaggtgggatcggtaaatcgaccaccacacagaacctgg-
tcgccgcgctggcggagatgggtaag
aaagtcatgatcgtcggctgcgatccgaaagccgactccacgcgtttgatcctgcatgcgaaagcgcagaaca-
ccattatggagatggccgccgaag
tcggctccgtcgaagacctggaattagaagacgtgctgcaaatcggttacggcggcgtgcgctgcgcggaatc-
cggtggcccggagccaggtgtg
ggttgtgccggtcgtggcgtgatcaccgcgattaacttcctcgaagaagaaggcgcttacgtgccggatctgg-
attttgttttctacgacgtgctgggcg
acgtggtatgcggtggtttcgccatgccgattcgtgaaaacaaagcgcaggagatctacatcgtttgctctgg-
cgaaatgatggcgatgtacgccgcc
aataacatctccaaaggcatcgtgaaatatgccaaatccggtaaagtgcgcctcggcgggctgatttgtaact-
cgcgccagaccgaccgcgaagatg
aactcatcattgcgctggcggaaaaactcggcacgcaaatgatccactttgttccccgcgacaacattgtgca-
gcgtgcggaaatccgccgtatgacg
gttatcgaatatgacccgacctgcaatcaggccaacgaatatcgcagccttgccagcaaaatcgtcaacaaca-
ccaaaatggtggtaccaaccccctg
caccatggatgaactggaagaactgctgatggagttcggcattatggatgtggaagacgccagcatcattggt-
aaaaccgccgccgaagaaaacgc cgtctga 138
atgagcaatgcaacaggcgaacgtaacctggaaatcatcgagcaggtgctggaggttttcccggaaaaga-
cgcgcaaagagcgcagaaaacacat
gatggtgacggacccggagcaggagagcgtcggcaagtgcatcatctctaaccgcaaatcgcagccgggcgtg-
atgaccgtgcgtggctgctcgt
atgccggatcaaaaggggtggtatttgggccaatcaaagatatggcgcatatctcccacggcccgatcggctg-
cgggcagtactcccgcgccgggc
ggcgtaactactataccggcgtcagcggcgtggacagtttcggcacgctcaacttcacctccgatttccagga-
gcgcgacatcgtgtttggcggcgac
aaaaagctcgccaaactgattgaagagctggaagaactgtttccgctgaccaaaggcatttcgattcagtcgg-
aatgcccggtcggcctgattggcga
tgatattgaagccgtggcgaacgccagccgcaaagcgatcaacaaaccggttattccggtgcgttgcgaaggc-
tttcgcggcgtgtcgcaatccctc
ggtcaccatattgccaacgatgtgatccgcgactgggtactggataaccgcgaaggcaaaccgtttgaatcca-
ccccttacgatgtggcgatcatcgg
cgattacaacatcggtggcgacgcctgggcctcgcgcattttgctcgaagagatggggttgcgggtggtcgcg-
cagtggtccggcgacatacgct
ggtgcagatggaaaacacgccgttcgtcaaactgaacctggtgcactgctaccgctcgatgaactacatctcg-
cgccatatggaggagaagcacggt
attccgtggatggaatacaacttctttggcccgacgaaaatcgcggaatcgctgcgcaaaatcgccgacctgt-
tcgacgacaccattcgcgccaacgc
cgaagcggtgatcgcccgataccaggcgcagaacgacgccattatcgccaaatatcgcccacgtctggagggt-
cgcaaagtgttgctctatatgggc
gggctgcgtccgcgccatgtgattggcgcctatgaagatctgggaatggagatcatcgccgccggttatgagt-
ttggtcataacgacgattacgaccg
caccctgccggatctgaaagagggcacgctgctgtttgatgacgccagcagctatgagctggaggcgtttgtc-
aacgcgctgaaaccggatctcatc
ggttccggcatcaaagagaagtacatctttcagaaaatgggcgtgccgtttcgccagatgcactcctgggatt-
actccggcccgtaccacggctatgac
ggcttcgccatcttcgcccgcgatatggatatgacgctcaacaaccccgcctggggtcagttgaccgcgccgt-
ggcttaaatccgcctga 139
atgaaggggaacgacatcctggctctgctcgatgaaccagcctgcgagcataaccataaacagaaaaccg-
gctgtagcgcgccaaacccggcgc
caccgccggaggctgcgccttcgacggcgcacagatcaccctgctgccactttccgatgtggcgcatctggta-
catggcccgattggctgcgccggc
agctcatgggataaccgtggcagcctgagttctggcccgctgattaaccgactcggattcaccactgatttga-
acgaacaggatgtcatcatggggcg
cggcgagcggcggttgtttcacgcggtgcgccatattgtcgagcgctatcacccggcggcggtatttatttac-
aacacctgcgttccggctatggaagg
cgatgacattgacgcggtctgccaggccgccgcgaccgccaccggtgtgcccgtgattgccgtagatgtggcc-
ggtttttacggtagcaaaaacctg
ggtaaccgcctcgcgggcgaggtgatggtgaaaaaagttatcggcgggcgcgaacccgcgccgtggccggaca-
atacaccttttgccccggcgca
ccgccatgacataggcctgattggcgaatttaacatcgccggcgagttctggcatatccagccgctgcttgat-
gagctgggtattcgcgtccttggctcc
ctttccggcgacgggcgctttgccgagatccagacgttgcaccgcgcgcaggtcaatatgctggtgtgctcca-
gggcgctgattaatgtcgccagatc
gcttgaacaacgttatggcacaccctggtttgaaggcagtttttatggcgttcgcgccacctccgatgccctg-
cgccagctggcaacactcaccggcga
tagcgatttaatggcgcgaaccgaacggctgatcgcacgtgaagagcaagccacagaacaggcgctagcaccg-
ctgcgtgaacggttacacggcc
ggaaagtgctgctctataccggtggcgtgaaatcctggtcggtggtttcggcgctgcaggatctcggcatgac-
ggtcgttgctaccggaacgcgcaaa
tccaccgaagaggataaacaacgcatccgtgaactgatgggcgatgacgccatcatgctggatgaaggcaatg-
cccgcgccttgctggatgtggtct
atcgctacaaagccgacatgatgatcgcgggcgggcgcaacatgtacaccgcctataaagcgcgtctgccctt-
tctggatatcaaccaggagcgtga
acacgcgtttgccggttatcgcggcatcatcacgcttgccgaacaactttgtcagacgctggaaagcccggtc-
tggccgcaaacacatgcccgcgcc ccgtggcaataa 140
atgagccagactgctgagaaaatacagaattgccatcccctgtttgaacaggacgcctaccagacactat-
ttgccggtaaacgggcactcgaagagg
ctcactcgccggagcgggtgcaggaagtgtttcaatggaccaccaccccggaatacgaagcgctgaacttcaa-
acgcgaagcgctgactatcgacc
cggcaaaagcctgccagccgctgggggcggtgctagttcgctggggtttgccaacaccctgccgtatgtgcac-
ggttcacagggttgtgtggcctat
ttccgtacgtactttaaccgccacttcaaagaaccggtggcctgcgtgtcggattcgatgacggaagacgcgg-
ccgtgttcggcgggaataacaacct
caacaccgattacaaaacgccagcgcactgtataaaccggtgattatcgccgtctctaccacctgtatggcgg-
aagtgatcggtgatgatttacagg
cgtttatcgccaacgccaaaaaagatggttttctcgatgccgccatccccgtgccctacgcccacacccccag-
ttttatcggtagccatatcaccggctg
ggacaacatgtttgaaggttttgcccgtacctttaccgcaaaccatcagccacagcccggtaaactttcacgc-
ctgaacctggtgaccgggtttgaaac
ctatctcggcaatttccgcgtgagaaacgcatgatggaacaaatggaggtgcaggcgagtgtgctctccgatc-
cgtcggaggtgctggacaccccc
gccaatggccattaccagatgtacgcgggcggtacgacgcagcaagagatgcgcgaggcaccggatgccatcg-
acaccctgctgctgcaaccgtg
gcagctggtgaaaagcaaaaaagtggtgcaggagatgtggaatcagcccgccaccgaggttgccattcccgtc-
gggctggcaggcacagacgaa
ctgttgatggcgattagccagttaaccggcaaagccattcccgattcgctggcgctggagcgcgggcggctgg-
tcgatatgatgctcgactcccacac
ctggttacacggtaaaaaattcggtctgtttggcgatccggattttgtcatgggattgacccgcttcctgctg-
gaactgggctgtgaacctgccgtcatcct
ctgccataacggtaacaaacgctggcaaaaagcgatgaagaaaatgctcgatgcttcaccgtacggccaggag-
agcgaagtgtttatcaactgcgac
ttgtggcatttccgctcgctgatgttcacccgccagccggattttatgattggcaactcgtacgccaagttta-
ttcagcgcgacaccttagccaagggcga
acagtttgaagtcccgctgatccgcctcggttttccgctgttcgaccgtcaccatctgcaccgccagaccacc-
tggggctacgagggcgcgatgagca
ttctcacgacgctggtgaatgcggtactggagaaagtggacaaagagaccatcaagctcggcaaaaccgacta-
cagcttcgatcttatccgttaa 141
atggctgatattgttcgtagtaaaaaaccgctggcggtgagcccgataaaaagcggccagccgctggggg-
cgatcctggcaagcctgggtttcgaac
agtgcataccgctggtacacggcgctcaggggtgcagcgcgttcgcgaaagtgttctttattcaacattttca-
cgacccgatcccgctgcaatcgacgg
cgatggacccgacttccaccattatgggcgccgatgaaaacatttttaccgcgctcaatgttctctgccagcg-
caacgccgcgaaagccatcgtgctgc
tcagcaccgggctgtcagaagcccagggcagcgatatttcacgagtggtgcgccagtttcgtgatgactttcc-
gcggcataaaaacgtggcgctgctc
accgtcaacaccccggatttctacggctcgctggaaaacggctacagcgccgtgctggaaagcatgattgaac-
agtgggtgcccgcgcagcccgcc
gccagcctgcgcaaccgtcgcgtcaacctgctggtcagccatttactgacgccgggcgatatcgaactgttac-
gcagttatgtggaagcattcggtctg
caaccggtgattgtgccggatctatcgcagtcgctggacggacatctggccaacggtgatttttcgcccgtca-
cccaggggggaacaccgctgcgca
tgattgaacagatgggccaaaacctggccacttttgtgattggccactcgctggggcgggcggcggcgttact-
ggcgcagcgcagccgtggcgagg
tgatcgccctgccgcatctgatgacgcttgatgcgtgcgacacctttatccatcgcctgaaaaccctctccgg-
gcgcgacgtgcccgcgtggattgag
cgccagcgcgggcaagtgcaggatgcgatgatcgattgccatatgtggttgcagggcgcggctatcgccatgg-
ccgcagaaggcgatcacctggc
ggcatggtgcgatttcgcccgcagccagggcatgatccccggcccggttgtcgcgccggtcagccagccgggg-
ttgcaaaatctgccggttgaaat
ggtggttcatcggcgatctggaagatatgcaggcttcggctttgcgcgacgcccgccgcgttactggtggcca-
attctcatgccgccgatctcgccacgc
agtttgatatgtcgcttatccgcgccggttttccggtgtatgaccggctgggggaatttcgtcggctgcgcca-
ggggtatagcggcattcgtgacacgc
tgtttgagctggcgaatgtgatgcgcgaacgccattgcccgcttgcaacctaccgctcgccgctgcgtcagcg-
cttcggcgacaacgttacgccagg agatcggtatgccgcatgttaa 142
atgaccctgaatatgatgatggatgccagcgcgcccgaggccatcgccggtgcgctttcgcaacaacatc-
ctgggctgttttttaccatcgttgaagaa
gccccgtcgctatttcactaaccgatgccgaggcacgtattgtctatgccaacccggcattctgccgccagac-
cggctatgagcttgaggagttgttg
cagcaaaatccccgcctgcttgccagtcagcagaccccacgggaaatctaccaggatatgtggcacaccctgt-
tacaacgtcgaccatggcgcggg
caattgatcaaccgccaccgtgacggcagcctttttctggttgagatcgatatcaccccggtgattaacccgt-
ttggcgaactggaacactacctggcca
tgcagcgcgatatcagcgccggttatgcgctggagcagcggttgcgtaatcacatggcgctgaccgaagcggt-
gctgaataacattccggcggcgg
tggtcgtggtcgatgaacgcgatcgtgtggttatggataacctcgcctataaaactttctgtgctgattgcgg-
cggaaaagagctactgagcgaactcca
tttttcagcccgtaaagcggagctggcaaacggccaggtcttaccggtggtgctgcgcggcgcggtgcgctgg-
ttgtcggtcacctgctgggcgctg
ccaggcgtcagcgaagaagccagtcgctactttattgataataccttgacgcgcacgctggtggtcatcaccg-
acgacacccagcagcgccagcag
caagagcaaggacggcttgaccgccttaaacagcagatgaccagcggcaaactgctggcggcgatccgcgaag-
cgcttgacgccgcgctgatcc
agcttaactgccccatcaatatgctggcggcggcgcggcgtttaaacggcagtgataacagcaacgtagcgct-
ggacgccgcgtggcgcgaaggt
gaagaagcgatggcgcggctgaaacggtgccgcccgtcgctggagctggaaagtgccgccgtctggccgctgc-
aacccttttttgacgacttgcgc
gcgctttatcacacccgctacgagcagggtaaaaatttgcaggtcacgctggattcgacgcatctggtgggat-
ttggtcagcgaacccaactgctggc
ctgcctgagtctgtggctcgatcgcacgctggatattgccgtcgggctgcgtgatttcaccgcccaaacgcag-
atttacgcccgggaagaagcgggct
ggctctcgttgtatatcactgacaatgtgccgttgattccgctgcgccatacccattcgccggatgcgcttaa-
cgcaccgggaaaaggtatggagttgc
ggctgatccagacgctggtagcgcatcacaacggcgcgatagaactcacttcacgccccgaagggggaagctg-
cctgaccctacgattcccgctatt tcattcactgaccggaggttcaaaatga 143
atgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttatcccagcagttcaccgcgatga-
gcggataagcgtggttctcagccggg
cgaccgaggttgaacagacactccagcaggtgtgtgcgtattgcacaatgacgcctttttgcagcacggcatg-
atctgtctgtacgacagccagcag
gcgattttgactattgaagcgttgcaggaagccgatcagcagttgatccccggcagctcgcaaattcgctacc-
gtccgggtgaagggctggtcggga
cggtgctttcgcaggggcaatcgttagtgctggcgcgtgtggctgacgatcagcgctttcttgaccgcctggg-
actgtatgattacaacctgccgtttatc
gccgtgccgctgatagggccggatgcgcagacttttggcgtgctgacggcgcaaccgatggcgcgttacgaag-
agcggttacccgcctgcacccg
ctttctggaaacggtcgcgaatctggtggcgcagaccgtgcgtttgatgacgccgccggctgcacgcccttcc-
ccacgcgctgccatcacgccaacc
gccagcccgaaatcgtgcagtacttcacgcgcgttcggcttcgaaaatatggtcggcaacagcccggcaatgc-
gccagaccatggagattatccgtc
aggtttcgcgctgggataccaccgttctggtgcgcggcgagagcggcaccggcaaggaactgattgccaacgc-
catccatcacaattcgccgcgcg
ccagtgcgccatttgtgaaattcaactgtgcggcgctgccggacacattgcttgaaagcgaattatttggtca-
tgaaaaaggcgcctttaccggcgcgg
tacgccagcgtaaaggccgttttgagctggccgatggcggcacgctgtttcttgacgaaattggggaaagcag-
cgcctcgtttcaggctaagctgctg
cgtattttgcaggagggcgaaatggaacgcgtcggtggtgacgagacattgcaagtgaatgtgcgcatcattg-
ccgcgacgaaccgcaaccttgaag
atgaagtacgcctgggacattttcgcgaagatctctattaccgcctgaatgtgatgcccatcgccctgccgcc-
gctgcgcgaacgccaggaccacatc
gccgaactggcacattttctggtgcgtaaaatcgcccacaaccagaaccgcacgctgcgcattagcgagggcg-
ctatccgcctgctgatgagctaca
gctggcccggcaatgtgcgcgaactggaaaactgccttgagcgctctgcggtgatgtoggaaaacggtctgat-
cgatcgggacgtgattttatttaatc
atcgcgaccagccagccaaaccgccggttatcagcgtcacgcccgacgataactggctcgataacacccttga-
cgagcgccagcggctgattgccg
cgctgaaaaaagcgggatgggtacaagccaaagccgcccgcttgctggggatgacgccgcgccaggtcgctta-
tcgtattcagaccatggatatca ccctgccaaggctataa 144
atgccgcaccacgcaggattgtcgcagcactggcaaacggttttttctcgtctgccggaagcgctcaccg-
cgcaaccattgagcgcgcaggcgcagt
cagtgctcacttttagtgattttgttcaggacagcatcatcgtgcatcctgagtggctggcagagcttgaaag-
cgcaccgccgccagcgaacgagtggc
aacactacgcgcaatggctgcaagcggcgctggagggcgtcaccgatgaaacctcgctgatgcgcacgctgcg-
gctgtttcgccgtcgcattatggt
gcgcatcgcctggagtcaggcgctacagttggtggcggaagaggatatcctgcaacagctcagcgtgctggcg-
gaaactctgatcgtcgccgcgcg
cgactggctctatgacgcctgctgccgtgagtggggaacgccgtgcaatccgcaaggcgtcgcgcagccgatg-
ctggtgctcggcatgggcaaact
tggcggcggcgaactcaatttctcatccgatatcgatttgatttttgcctggccggaaaatggcaccacgcgc-
ggcggacgccgtgaactggataacg
cgcagttttttacccgccttggtcaacggctaattaaagtcctcgaccagcccacgcaggatggctttgtcta-
ccgcgtcgatatgcgcttgcgtcccttt
ggcgacagcggcccgctggtgctgagttttgccgcgctggaagattactaccaggagcaggggcgcgactggg-
aacgatacgcgatggtgaaagc
gcgcattatgggggacaacgacggcgaccatgcgcgagagttgcgcgccatgctgcgcccgttcgttttccgc-
cgctatatcgacttcagcgtgatcc
agtctctgcgcaacatgaaaggcatgattgcccgcgaagtgcggcgtcgcggcctgaaggacaacataaaact-
cggcgcgggcggtattcgcgaa
atagagtttatcgtgcaggttttccagttgattcgcggcggtcgcgagcctgcgctgcaatcgcgttcgctgt-
tgccgacgcttgctgccattgatcaact
acatctgctgccagatggtgatgcaccccggctgcgcgaggcgtatttgtggctgcgacggctggaaaacttg-
ctgaaagcattaatgacgaacag
acacagacgctgccggccgatgatttgaatcgcgcgcgcctcgcctggggaatgggcaaagagagctgggaag-
cgctctgcgaaacgctggaag
cgcatatgtcggcggtgggcagattttcaacgatctgattggcgatgatgaaacggattcgccggaagatgcg-
ctttctgagggctggcgcgaattgt
ggcaggatgcgttgcaggaagaggactctacgcccgtgctggcgcatctttccgaggacgatcgccgccgcgt-
ggtggcgctgattgctgattttcg
caaagagctggataaacgcaccattggcccgcgcgggcgacaggtactcgatcacttaatgccgcatctgctc-
agcgatgtatgctcgcgtgacgat
gcgccagtgccgctgtcgcgtctgacgccgctgctcaccggtattattacgcgcaccacttaccttgagctgc-
tgagtgaattccccggtgcgctgaaa
cacctcatttccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcctgctcgatg-
aactgctcgacccgaacacgctcta
tcaaccgacggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaa-
gagcagcaactggaggcgctac
ggcagtttaagcacgcgcagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagt-
gagcgatcacttaacctggctggc
ggaagcgattatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcat-
ctgcacgatcgcgaagggcgc
ggtttcgccgtggtcggttacggcaaacttggcggctgggaattaggttacagctccgatctggatctggtgt-
tcctgcacgactgccccatggatgtga
tgaccgatggcgagcgtgaaatcgatggccgccagttctatttgcgcctcgcgcagcgcgtcatgcacctgtt-
cagcacgcgcacgtcgtccggcatt
ctttatgaagtcgatgcgcgtttgcgcccgtccggcgcggccggaatgctggtgaccactgcggaagcgttcg-
ccgattatcaaaaaaatgaagcctg
gacatgggagcatcaggcgctggcgcgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgac-
gccattcgccgcgatatcctgat
gacctcccgcgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttagt-
aacaagcacaaagaccgtttcga
tctgaaagccgatgaaggcggtatcaccgatattgagtttatcgctcagtatctggtgctgcgctttgcccat-
gagaagccgaaactgacgcgctggtc
ggataatgtgcgcatcctcgaagggctggcgcaaaacggcatcatggatgagcaggaagagcaggcattgacg-
ctggcgtacaccacgttgcgtg
atgagctgcaccacctggcgctgcaagagctgccaggacatgtggcgctctcctgttttgtcgccgagcgtgc-
gcttatcaaaaccagctgggacaa gtggctggtggaaccgtgcgccccggcgtaa 145
atgaaaaacacaacattaaaaacggctcttgctttcgctggcgttgctgccaggcctggcgatggcggct-
cccgctgtggcggataaagccgacaacg
gctttatgatgatttgcaccgcgctggtgctgtttatgaccattccgggcattgcgagttctacggcggtttg-
atccgcggtaaaaacgtgctgtcgatgc
tgacgcaggttgccgtcaccttcgctctggtgtgcatcctgtgggtggtttacggctactctctggcatttgg-
cgagggcaacagcttcttcggcagtttc
aactgggcgatgttgaaaaacatcgaattgaaagccgtgatgggcagcatttatcagtacatccacgtggcgt-
tccagggctcctttgcttgtatcaccg
ttggcctgattgtcggtgcgctggctgagcgtattcgcttctctgcggtgctgatttttgtggtggtatggct-
gacgctttcttatgtgccgattgcgcacat
ggtctggggtggcggtctgctggcaacccacggcgcgctggatttcgcgcgcggtacggttgttcacatcaac-
gccgcgatcgcaggtctggtggg
ggcttacctgattggcaaacgcgtgggctttggcaaagaagcgttcaaaccgcataacctgccgatggtcttc-
accggcaccgcgatcctctatgttgg
ctggtttggcttcaacgccggctctgcaagctcggcgaacgaaatcgctgcgctggctttcgtgaacacggtt-
gttgccactgcggccgctattctggc
gtgggtatttggcgagtgggcaatgcgcggtaagccgtctctgctcggtgcctgttctggtgccatcgcgggt-
ctggttggtatcaccctcggcgtgcg
gttatgtgggtgtcggcggcgcgctgattgtgggtctgattgccggtctggcagggctgtggggcgttactgc-
actgaaacgtatgttggtgttgatga
cccatgcgatgtcttcggtgtgcacggcgtgtgcggcatcgtgggttgtatcctgaccggtatcttcgcgtct-
acgtcgctgggcggtgtcggtttcgct
gaaggggtgaccatgggccatcaggtactggtacagctggaaagcgttgccatcactatcgtgtggtctggcg-
tggtggcctttatcggttacaaactg
gcggatatgacggtaggcctgcgcgtaccggaagagcaagagcgtgaagggctggatgtgaacagccacggcg-
aaaatgcgtataacgcctga 146
ttcttggttctctggagcgctttatcggcatcctgactgaagaatttgcaggcttcttcccaacctggct-
tcacccgtgcaggtagttgtgatgaacatca
ctgattcgcaggctgaatacgttaacgaattgacccgtaaactgcaaaatgcgggcattcgtgtaaaagcaga-
cttgagaaacgagaagattggcttta
aaatccgcgagcacactttacgtcgtgtcccttatatgctggtttgtggtgacaaagaggtcgaagccggcaa-
agttgctgtgcgtacccgtcgcggta
aagacctgggtagcctggacgtaaatgatgttatcgagaagctgcaacaagagattcgcagccgcagtcttca-
acaactggaggaataaggtattaaa
ggcggaaaacgagttcaaacggcgcgtcccaatcgtattaatggcgagattcgcgccacggaagttcgcttaa-
caggtaggaaggcgagcagctt ggtatt 147
cgttctgtaataataaccggacaattcggactgattaaaaaagcgccctcgcggcgctttttttatattc-
tcgactccatttaaaataaaaaatccaatcgga
tttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgttttttaa-
ccattattgaaagtcggtgcttctttgagcga
acgatcaaatttaagtcgattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaacg-
ctacatggagattaactcaatctagagggt attaataatgaatcgtactaaactggtactgggcgc
148
cgcgtcaggttgaacgtaaaaaagtcggtctgcgcaaagcacgtcgtcgtccgcagttctccaaacgtta-
attggtttctgcttcggcagaacgattggc
gaaaaaacccggtgcgaaccgggtttttttatggataaagatcgtgttatccacagcaatccattgattatct-
cttctttttcagcatttccagaatcccctca
ccacaaagcccgcaaaatctggtaaactatcatccaattttctgcccaaatggctgggattgttcattttttg-
tttgccttacaacgagagtgacagtacgc gcgggtagttaactcaacatctgaccggtcgat 149
ttgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgngcggaagc-
acaggagagcttgctctctgggtgacg
agcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaat-
accgcataacgtcgcaagaccaa
agagggggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggtaacggcnc-
acctaggcgacgatccctagctg
gtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaat-
attgcacaatgggcgcaagcct
gatgcagccatgccgcgtgtatgaagaaggccttcgggttgtttaagtactttcagcggggaggaaggtgttg-
nggttaataaccncagcaattgacgt
tacccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggaggntgcaagcgttanncgg-
aatnantgggcgtaaagcgtn
cgcaggcggtntgtnaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggct-
agagtnnngtagaggngggtag
aattccnggtgtagcggtgaaatgcgtagagatcnggangaanaccngtggcgaaggcggcccnctggacaaa-
gactgacgctnaggngcgaa
agcgggggagcaaacaggattagataccctngtagtccacgccgtaaacgatgtcgacttggaggttgtgccc-
ttgaggcgtggcttccggagcta
acgcgttaagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcaca-
agcggtggagcatgtggtttaatt
cgatgcaacgcgaagaaccttacctactcttgacatccagagaacttnncagagatgnnttggtgccttcggg-
aactctgagacaggtgctgcatggc
tgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagc-
ggtncggccgggaactcaaaggagac
tgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacg-
tgctacaatggcgcatacaaagag
aagcgacctcgcgagagcaagcggacctcataaagtgcgtcgtagtccggattggagtctgcaactcgactcc-
atgaagtcggaatcgctagtaatc
gtagatcagaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggtt-
gcaaaagaagtaggtagcttaacct
tcgggagggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctg-
cggttggatcacctcctt 150
atgaccatgcgtcaatgtgccatttacggcaaaggtggtatcggtaaatccactaccacgcaaaacctgg-
tcgccgcgctggcggagatgggcaaga
aagtaatgatcgtcggctgcgacccgaaagcagactccactcgtctgatcctgcatgcgaaagcgcagaacac-
cattatggagatggcggctgaagt
cggctccgtggaagaccttgaactggaagatgtgctgcaaatcggttacggcgacgtacgctgcgcagaatcc-
ggcggcccggaaccaggcgttg
gctgtgctggtcgcggggtaattaccgccatcaacttcctggaagaagaaggcgcctatgttcccgacctcga-
tttcgtcttttacgacgtgttgggcga
cgtggtgtgcggggggttcgccatgccgattcgcgaaaacaaagcgcaggagatctacatcgtctgctccggc-
gaaatgatggcgatgtacgccgc
caacaacatctctaaaggcatcgtgaaatacgccaaatccggcaaaggcgccttggcgggctgatctgtaact-
cccgtcagaccgaccgcgaagat
gagctgatcatagcgctggcggaaaaactcggcacccagatgatccacttcgtgccgcgcgacaacatcgtgc-
aacgcgctgaaatccgccgtatg
acggtgattgagtacgatccgaaatgcaaccaggccaatgaataccgcacgctggcgaacaagatcgtcaact-
tacaccaaaatggtcgtgccaacg
cccatcaccatggacgaactggaagagctgttgatggaattcggcattatggatgtggaagacaccagcatta-
tcggtaaaaccgccgcagaagaaa acgcggtttga 151
atgagcaatgcaacaggcgaacgtaataggagatcatccaggaagtgctggagatctttccggaaaaaac-
gcgcaaagaacgcagaaagcacatg
atggtgagcgacccggagatggaaagcgtcgggaaatgcatcatctccaaccgtaagtcgcagcccggcgtaa-
tgaccgtgcgcggttgctcttac
gccggttctaaaggggtggtattcgggccgatcaaagatatggcccatatttcccacggcccggtcggctgcg-
gtcagtactcccgcgccgggcggc
gtaactactacaccggcgtcagcggtgtggatagcttcggtacgctcaactttacctccgattttcaggagcg-
cgatatcgtgtttggcggcgataaaaa
gctgaccaaactgattgaagagatggagacgctgttcccgctgaccaaagggatctccattcagtccgaatgc-
ccggtcggcctgattggcgacgac
attgaagccgttgccaacgccagccgcaaagccatcaataaaccggtcattccggtgcgctgcgaaggttttc-
gcggcgtttcccagtcactcggtca
ccacattgccaacgacgtgatccgcgactgggtactggataaccgcgaaggcaagccgtttgaggccggtcct-
tatgacgtggcgatcatcgccgat
tacaacatcggcggcgatgcctgggcgtcgcgcattttgctcgaagagatgggcctgcgcgtggtggcgcagt-
ggtccggcgacggcacgctggtt
gagatggagaccacgccgttcgtcaaactcaaccttgtgcactgctaccgctcaatgaactatatctcccgcc-
atatggaggagaaacacggtattccg
tggatggagtacaacttcttcggtccgaccaaagtcgccgaatcgttgcgcaaaatcgccgatatgtttgatg-
acaccattcgcgccaacgccgaagc
ggtgatcgccaaatatcaggcgcagaacgacgccatcatcgccaaataccgtccgcgtctggaaggccgcaaa-
gtgctgctgtatatgggcggttta
cgtcctcgccatgtgattggcgcttatgaagatctgggaatggaaattatcgctgcgggttatgaattcgccc-
acaacgatgactacgaccgcaccctg
ccggatctgaaagaaggcaccttgctgttcgacgatgccagcagttatgaactggaagcctttgtcaaagcgc-
tgaagccggatctgatcggctccgg
cattaaagagaagtacatatccagaaaatgggcgtgccgtttcgccagatgcactcctgggattactccggcc-
cctatcacggttatgacggctttgcc
atcttcgcccgcgatatggatatgacgatcaacaaccccgcgtggggccagttgaccgcgccgtggctgaaat-
ccgcctga 152
atgggacgcggcgagcgccgcctgttccatgccgtgcgccacatcgtcaaccgctaccacccggccgccg-
tctttatctataacacctgcgttcccgc
gatggagggcgacgatatcgaagccgtctgccaggcggcagaaaccgccatccgcgtaccggtgattgccgtt-
gatgtcgccgggttttacggcag
caaaaatctcggcaaccggttggccggtgaagtgatggtgaaaaaaggtgattggcgggcgtgaacccgcgcc-
gtggccggaagataccccttttgc
cccggcgcaccgccacgatatcgggctgattggcgaattcaatattgccggagagttctggcatattcagccg-
ctgctcgatgagctgggtattcgcgt
gctcggcagcctctccggcgacgggcgcttcagtgaaatccagacgctgcaccgggcgcaggtcaatatgctg-
gctcctccagggcgctgatcaa
cgtcgcccgctcgctggagcagcgctacggcacgccgtggtttgaaggcagtttttatggtgttcgcgccacc-
tctgacgccctgcgccaactggcg
gcgctgaccggagaccgcgatctgatgcagcgcaccgattcagctcattgcccgcgaagagcagcaaacagag-
caggcgctggccccgctgcgc
gagcgcctgcgcgggcgcaaagcgctgctctatnccggcggcgtgaaatcctggacggtggtttcggcgcttc-
aggatctgggcatggaagtggtg
gcgaccggcacgcgcaaatccaccgaagaggataaacagcgcatccgcgaactgatgggcgccgacgcgctga-
tgcttgatgaaggtaacgccc
gctcgctgctggacgggtttaccgctacaaggcggacatgatgatcgccgggggacgcaatatgtcaccgcct-
acaaagcgcggctgccgttcct
cgatatcaatcaggagcgcgagcacgcctttgccggctaccgcggcattgtcaccctggccgaacagctctgc-
ctgaccatggaaagcccggtctg gccgcaaacccattcccgcgcaccgtggcaataa 153
atgatggagcaaatggacgtgccgtccagcctgctttccgatccctccgaagtgctggataccccggctg-
acgggcattaccacatgtatgcggggcg
gtacgacccagcaggagatgcgcgaagcgcctgacgctatcgacaccctgctgctgcaaccctggcaactggt-
gtaaaccaaaaaagtggtgcag
gaaagctggaaccagcccgctaccgaggtgcaaatcccaatggggctggccggaaccgacgagctgctgatga-
cggtaagccagttaaccggca
aagccattccggatagcttagcgctggaacgcggtcggctggtggatatgatgctcgactcccacacctggct-
gcacggcaagaaattcggcctgttc
ggtgacccggattttgtcatggggctgacccgcttcctgctggaactgggctgcgaaccgacggtgattctgt-
gccataacggcagcaagcgctggc
agaaagcgatgaagaaaatgcttgaagcctcgccgtacgcgaaagagagcgaagtctttatcaactgcgattt-
gtggcatttccgctcgctgatttac
ccgtcagccggactttatgatcggcaactcctacgccaagtttatccaccgcgatacgctggcgaagggtgag-
cagtttgaagtgccgctgatccgcc
tggggttcccgctgttcgatcgccaccatctgcaccgccagaccacctggggttacgaaggggccatgagtat-
cctcaccacgctggttaatgcggtg
ctggagaaagtcgacagagagaccatcaagctcggcaaaaccgactacagcttcgatcttatccgttaa
154
atgcagcgcgacatcagcaccagctacgcgctggaacaacggctgcgcaatcatatgacgctgaccgaag-
ccgtcttgaataacattccggcggcg
gttgtagtggtggatgaacgcgatcgggtggtgatggataacctcgcctacaaaaccttttgcgccgattgcg-
gcggtaaagaactactcaccgaaatc
aacttttccgcccataaggcggagctggcgcagggcctggtactgccggtagtgctgcgcggcaccgtgcgct-
ggttgtccgttacctgttgggcgct
gccgggcgtcagcgaagaagcaggccgctactttattgatagcgccgtgccgcgcacgctggtggtgatcacc-
gataatactcagcagcagcaaca
acaggagcaggggcgtcttgatcgtctgaagcagcagataaccagcggtaaattgctggcggcgatccgcgaa-
tcgctggacgccgcgctggtac
aactcaattgcccaattaatatgctggccgccgcacgccgcttaaatggcgacgagcatagcaatctggcgct-
ggatgccgcatggcgtgaaggcga
agaagcgatggcgcggttgcagcgctgccgcccgtcgctggaactggaaagcccggcagtctggccgctccag-
ccgttccttgacgatctgcgtgc
cctgtatcacacccgatataaccagggcgaaaacctgcaaattgagctggaatcccccgacctggtgggcttt-
ggccagcgaacacaactgcttgcct
gcctgagcctgtggctcgacagaaccctggatattgccgcggagctacgtgatttcacggtacagactcaact-
ttacgcccgcgaagagagcggctg
gctgtcgttctatttaaacgacaatgtgccgctgattcaggtgcgctacacccattcacccgatgcactnaat-
gcgcccggtaaaggcatggagctgcg
gctgatccagacgctggtcgcccaccatcgaggcgcaatagaactgacctcacgccctcagggaggcacctgt-
ctgatcctgcgtttcccattatttta ctcgctgacaggaggctcactatga 155
atgactcagcgaaccgagtcgggtacaaccgtctggcgctttgacctctcccaacagtttacagccatgc-
agcgtatcagtgtggtgttaagccgcgc
gacggagatcgggcagacgctacaggaagtgctgtgcgtgctgcacaacgatgcctttatgcagcacgggatg-
atctgtccgtacgcgcgggtgcg cgtcttcgcgagcgtatggctttga 156
atgcgcgtggaagactggtcaacgctgaccgaacggctcgatgcccatatggcaggcgtgcgccgaatct-
ttaacgaactgatcggtgatgacgaaa
gtgagtcgcaggacgatgcgctctccgagcactggcgcgagctgtggcaggacgcgcttcaggaagatgacac-
cacgccggtgagacgcactta
accgacgacgcgcgccatcgcgtggtggcgctgatcgctganttccgtcttgagagaacaaacgcgccatcgg-
cccgcgtngcgccaggtgctg
gatcacctgatgccgcacctgagagcgaagtctgctcgcgtgccgatgcgccggtgccgctgtcgcggatgat-
gcccctgagaggggattatca
cccgtactacctaccttgaactcctgagcgagttccctggcgcgcttaagcacctgatttcactctgcgccgc-
gtcgccgatggtggccaacaagctgg
cgcgttacccgctgctgctggatgagctgctcgatccgaataccctttatcaaccgacggcgaccgacgccta-
ccgggacgaactgcgtcagtatctg
ctgcgcgtgccggaagaagacgaagagcaacagctcggaggcgctgcgtcagtttaagcaggcccagatgctg-
cgcgtggcggccgcagatattg
ccggaacgctgccggtgatgaaagtgagcgatcacttaacctggcttgcggaagcgattatcgacgcggtggt-
gcatcaggcctgggtgcagatggt
ggcgcgctatggccagccgaaacatctggctgaccgtgatggtcgcggcttcgctggtggtgggttacggtaa-
gctcggcggttgggagctgggcta
tagctccgatctggatttaatcttcctccacgactgcccggttgatgtgatgaccgacggcgagcgcgagatt-
gacgggcgtcagttctacctgcgcct
ggcgcagcgcatcatgcacctgttcagcacccgcacctcgtcgggcattttgtatgaagtggatgcccgtagc-
gcccgtccggcgcggcgggcatg
ctggtcacctcgacggagtccttcgctgattaccagaagaatgaagcctggacgtgggagcatcaggcgctgg-
tgcgcgcccggtggtgtatggcg
atccgctgctgaaaacgcagtttgacgtgattcgtaaggaagtcatgaccaccgtgcgcgatggcagcacgct-
gcaaacggaagtgcgcgaaatgc
gcgagaaaatgcgcgcgcacttaggcaataaacatcgcgatcgctttgatattaaagccgatgagggcggtat-
taccgatattgagtttattacccagta
tctggtgttgctgcacgcgcatgacaagccgaagctgacgcgctggtcggataacgtgcgcattaggaactgc-
tggcgcaaaacgacattatggac
gagcaggaggcgcaggccttaacccgtgcctatacaacgcttcgcgatgagctccatcatctggcgttgcagg-
agcagcngggncacgtggcgct
ggactgtttcaccgctgaacgcgctcaggtaacggccagctggcagaagtggctggtggaaccgtgcgtaaca-
aatcaagtgtga 157
agatgtgcccagatgggattagctagtaggtggggtaacggcncacctaggcgacgatccctagctggtc-
tgagaggatgaccagccacactggaa
ctgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcag-
ccatgccgcgtgtatgaagaag
gccttcgggttgtaaagtactttcagcggggaggaaggtgttgtggttaataaccncagcaattgacgttacc-
cgcagaagaagcaccggctaactcc
gtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcg-
gtctgtcaagtcggatgtgaaat
ccccgggctcaacctgggaactgcattcgaaactggcaggctagagtcttgtagaggggggtagaattccagg-
tgtagcggtgaaatgcgtagagat
ctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggggagca-
aacaggattagataccctggt
agtccacgccgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaag-
tcgaccgcctggggagtacggccg
caaggttaaacctcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacg-
cgaagaaccttacctactcttgacat
ccagagaacttnncagagatgnnttggtgccttcgggaacctgagacaggtctgcatggctgtcgtcagctcg-
tgttgtgaaatgttgggttaagtc
ccgcaacgagcgcaacccttatcctttgttgccagggtccggccgggaactcaaaggagactgccagtgataa-
actggaggaaggtggggatgac
gtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcgcatacaaagagaagcgacctc-
gcgagagcaagggacctcataa
agtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtagatcag-
aatgctacggtgaatacgttcccggg
ccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggagggc-
gcttaccactttgtgattcatgactg
gggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt 158
atgaccatgcgcaatgtgccatttacggcaaaggtggtatcggtaaatccactaccacgcaaaacctggt-
cgccgcgctggcggagatgggcaaga
aagtaatgatcgtcggctgcgacccgaaagcagactccactcgtctgatcctgcatgcgaaagcgcagaacac-
cattatggagatggcggctgaagt
cggctccgtggaagaccttgaactggaagatgtgctgcaaatcggttacggcgacgtacgctgcgcagaatcc-
ggcggcccggaaccaggcgttg
gagtgctggtcgcggggtaattaccgccatcaacttcctggaagaagaaggcgcctatgttcccgacctcgat-
ttcgtcttttacgacgtgttgggcga
cgtggtgtgcggggggttcgccatgccgattcgcgaaaacaaagcgcaggagatctacatcgtctgctccggc-
gaaatgatggcgatgtacgccgc
caacaacatctctaaaggcatcgtgaaatacgccaaatccggcaaagtgcgccttggcgggctgatctgtaac-
tcccgtcagaccgaccgcgaagat
gagctgatcatagcgctggcggaaaaactcggcacccagatgatccacttcgtgccgcgcgacaacatcgtgc-
aacgcgctgaaatccgccgtatg
acggtgattgagtacgatccgaaatgcaaccaggccaatgaataccgcacgctggcgaacaagatcgtcaaca-
acaccaaaatggtcgtgccaacg
cccatcaccatggacgaactggaagagctgttgatggaattcggcattatggatgtggaagacaccagcatta-
tcggtaaaaccgccgcagaagaaa acgcggtttga 159
atgagcaatgcaacaggcgaacgtaatctggagatcatccaggaagtgctggagatctttccggaaaaaa-
cgcgcaaagaacgcagaaagcacatg
atggtgagcgacccggagatggaaagcgtcgggaaatgcatcatctccaaccgtaagtcgcagcccggcgtaa-
tgaccgtgcgcggttgctcttac
gccggttctaaaggggtggtattcgggccgatcaaagatatggcccatatttcccacggcccggtcggctgcg-
gcagtactcccgcgccgggcggc
gtaactactacaccggcgtcagcggtgtggatagcttcggtacgctcaactttacctccgattttcaggagcg-
cgatatcgtgtttggcggcgataaaaa
gctgaccaaactgattgaagtgatggagacgctgttcccgctgaccaaagggatctccattcagtccgaatgc-
ccggtcggcctgattggcgacgac
attgaagccgttgccaacgccagccgcaaagccatcaataaaccggtcattccggtgcgctgcgaaggttttc-
gcggcgtttcccagtcactcggtca
ccacattgccaacgacgtgatccgcgactgggtactggataaccgcgaaggcaagccgtttgaggccggtcct-
tatgacgtggcgatcatcggcgat
tacaacatcggcggcgatgcctgggcgtcgcgcattttgctcgaagagatgggcctgcgcgtggtggcgcagt-
ggtccggcgacggcacgctggtt
gagatggagaacacgccgttcgtcaaactcaaccttgtgcactgctaccgctcaatgaactatatctcccgcc-
atatggaggagaaacacggtattccg
tggatggagtacaacttcttcggtccgaccaaagtcgccgaatcgttgcgcaaaatcgccgatatgtttgatg-
acaccattcgcgccaacgccgaagc
ggtgatcgccaaatatcaggcgcagaacgacgccatcatcgccaaataccgtccgcgtctggaaggccgcaaa-
gtgctgctgtatatgggcggttta
cgtcctcgccatgtgattggcgcttatgaagatctggggatggaaattatcgctgcgggttatgaattcgccc-
acaacgatgactacgaccgcaccctg
ccggatctgaaagaaggcaccttgctgttcgacgatgccagcagttatgaactggaagcctttgtcaaaggct-
gaagccggatctgatcggctccgg
cattaaagagaagtacatcttccagaaaatgggcgtgccgtttcgccagatgcactcctgggattactccggc-
ccctatcacggttatgacggctttgcc
atcttcgcccgcgatatggatatgacgatcaacaaccccgcgtggggccagttgaccgcgccgtggctgaaat-
ccgcctga 160
atgaaggggaacgagatcctggctttgctcgatgaacctgcctgcgagcacaaccataaacagaaatccg-
gctgcagcgcgccgaaacccggcgc
gacagcgggcggctgcgcctttgacggtgcgcagatcaccctgctgccactctccgatgttgcccacctggta-
cacggccccattggttgtaccggta
gctcatgggataaccgtggcagcttcagttccggcccgacgatcaaccggctgggttaccaccgatctgagcg-
aacaggatgtgatcatgggacg
cggcgagcgccgcctgttccatgccgtgcgccacatcgtcaaccgctaccacccggccgccgtctttatctat-
aacacctgcgttcccgcgatggagg
gcgacgatatcgaagccgtctgccaggcggcagaaaccgccatcggcgtaccggtgattgccgttgatgtcgc-
cgggttttacggcagcaaaaatct
cggcaccggttggccggtgaagtgatggtgaaaaaggtgattggcgggcgtgaacccgcgccgtggccggaag-
ataccccttttgccccggcgc
accgccacgatatcgggctgattggcgaattcaatattgccggagagttctggcatattcagccgctgctcga-
tgagctgggtattcgcgtgctcggca
gcctctccggcgacgggcgcttcagtgaaatccagacgctgcaccgggcgcaggtcaatatgctggtctgctc-
cagggcgctgatcaacgtcgccc
gctcgctggagcagcgctacggcacgccgtggtttgaaggcagtttttatggtgttcgcgccacctctgacgc-
cctgcgccaactggcggcgctgac
cggagaccgcgatctgatgcagcgcaccgaacagctcattgcccgcgaagagcagcaaacagagcaggcgctg-
gccccgctgcgcgagcgcct
gcggggcgcaaagcgctgctctataccggcggcgtgaaatcaggtcggggtttcggcgcttcaggatctgggc-
atggaagtggtggcgaccgg
cacgcgcaaatccaccgaagaggataaacagcgcatccgcgaactgatgggcgccgacgcgctgatgcttgat-
gaaggtaacgcccgctcgctgc
tggacgtggtttaccgctacaaggcggacatgatgatcgccgggggacgcaatatgtacaccgcctacaaagc-
gcggctgccgtcctcgatatcaat
caggagcgcgagcacgcctttgccggctaccgcggcattgtcaccctggccgaacagctctgcctgaccatgg-
aaagcccggtctcgccgcaaac ccattcccgcgcaccgtggcaataa 161
atgagccaaagtgctgagaaaattcaaaactgtcatccgctgtttgaacaggatgcgtaccagatgctga-
taaagataaacggcaactggaagaggc
ccacgatccggcgcgcgtgcaggaggtctttcaatggaccaccaccgccgagtatgaagcgcttaactttcaa-
cgcgaagcgctgactatcgatccg
gccaaagcctgccagcgctgggtgcggtactgtgctcgctgggctttgccaataccctgccctatgttcacga-
ctcccaggggtgcgtggcctatttc
cgcacctattttaaccgtcactttaaagagccgattgcctgtgtttctgactcgatgacggaagatgcggcag-
tattcggcggcaacaacaacctgaaca
ccgggttgcagaacgccaggccctctacaagccggaaatcattgccgtctccaccacctgtatggcggaggtc-
atcggcgacgacctgcaggcgt
ttattgctaacgccaaagaagacggctttatcgacgcggcgatcccggtgccttacgcgcacacgccaagctt-
tatcggcagccatatcaccggctgg
gacaatatgtttgagggcttcgcccgtacctttaccgccgattacagcggacaaccgggcaaattaccgcgta-
tcaatctggtcagcggatttgaaacct
atctcggtaatttccgcgtgctgaaacgcatgatggagcaaatggacgtgccgtgcagcctgctttccgatcc-
ctccgaagtgctggataccccggctg
acgggcattaccacatgtatgcgggcggtacgacccagcaggagatgcgcgaagcgcctgacgctatcgacac-
cctgctgctgcaaccctggcaa
ctggttgaaaaccaaaaaaagtggtgcaggaaagctggaaccagcccgctaccgaggtgataatcccaatggg-
gctggccggaaccgacgagagc
tgatgacggtaagccagttaaccggcaaagccattccggatagcttagcgaggaacgcggtcggctggtggat-
atgatgctcgactcccacacctg
gctgcacggcaagaaattcggcctgttcggtgacccggattttgtcatggggctgacccgcttcctgctggaa-
ctgggctgcgaaccgacggtgattct
gtgccatagcggcagcaagcgaggcagaaagcgatgaagaaaatgcttgaagcctcgccgtacgggaaagaga-
gcgaagtctttatcaaagcga
tttgtggcatttccgctcgctgatgtttacccgtcagccggactttatgatcggcaactcctacgccaagttt-
atccagcgcgatacgctggcgaagggtg
agcagtttgaagtgccgctgatccgcctggggttcccgctgttcgatcgccaccatctgcaccgccagaccac-
ctggggttacgaaggggccatgag
tatcctcaccacgctggttaatgcggtgctggagaaagtcgacagagagaccatcaagctcggcaaaaccgac-
tacagcttcgatcttatccgttaa 162
atgccagaaattatccgtagttaaaaagccgctggccgtcagcccggtaaaaagtggccagccgctgggc-
gcgattctggcgagcatgggctttgaa
cagagcattccgctggttcatggcgctcacgggtgcagcgccttcgcgaaggtcttttttatccagcattttc-
acgatccgatcccgctgcaatcgacgg
caatggacccgacatcgaccattatgggtgccgatgagaacatctttaccgcgctgaatgtgctgtgttcacg-
caacaacccgaaagcgattgttctgct
gagcactggcctttccgaggcgcagggaagcgatatttcgcgcgtggtgcgccagttccgcgatgaatatccg-
cgccataaaggggtggcgctgct
gaccgtcaacacgccggatttttacggcagcctggaaaacggctacagcgcggtgctggagagcatggttgaa-
cagtgggtgccggaaaaaccgc
agccgggcgtgcgcaatcgccgcgtgaacctgctgctcagccatttgcttacgccgggcgacattgagctgct-
gcgaagttatgtcgaggcatttggc
ctgcagccggtgatggtccggatctttcccagtcgctggatggccatctcgccagcggggatttctcgccaat-
tacccagggcggcagcagcctgc
ggctgattgaacagatgggacagagtcttggcacgttcgccattggcgtatccctctcccgcgccgcgcaatt-
gctggcgcagcgcagccatgcgga
agtggtcaccctgccgcatctgatgaccatgagccagtgcgatacgtttattcatcaactgaagcgcctctcc-
gggcgcgatgttccggcgtggatcga
acgccagcgcgggcaactgcaggatgcgatgatcgattgtcatatgtggttgcagggcgcgcctgtcgcgctg-
gccgccgagggcgatctgctcgc
cgcctggtgcgatttcgcctgcgatatgggcatggtgcccggcccggtggtggcgccggtgagccagaaaggg-
ttgcaggatctgccggtcgaaaa
agtcattatcggcgatctggaggatatgcaggatctgttgtgtgaaacgcctgcatcgctgctcgtctctaat-
tctcacgccgctgatttggccgggcagt
tcgacattccgctggtgcgcgccggtttccccctgttcgaccgtctgggcgagtttcgccgcgtgcgccaggg-
ttacgccgggatgcgcgacaccttg
tttgagctggcgaatgccctgcgcgatcgccatcatcatcttgccgcttatcactcgccgctgcgccagcgtt-
tttacgaacccgcatcttcgggaggtg actatgcaacatgttaa 163
atgaccctgaatatgatgatggacgccaccgcgcccgccgagatcgccggagcgctctcacaacagcatc-
ccggattgtttttcaccatggttgaaca
ggcgcccgtcgcgatttcactgaccgatgccgatgcccacattctctacgccaaccccgcgttttgtcgccag-
tcggggtatgaactggaagagttgtt
gcagcaaaacccgcgcctgcttgccagtaagaagacgccgcgtgaaatctaccaggaaatgtggcacaccctg-
ctgcaacaccgtccgtggcgcg
gacaactgatcaaccgtcgccgcgacggcagcctgtttctggtggaaatcgacatcaccccactgtttgatgc-
gttcggcaaactcgaacattacctgg
ccatgagcgcgacatcagcaccagctacgcgctggaacaacggctgcgcaatcatatgacgctgaccgaagcc-
gtcttgaataacattccggcgg
cggttgtagtggtggatgaacgcgatcgggtggtgatggataacctcgcctacaaaaccttttgcgccgattg-
cggcggtaaagaactactcaccgaa
atcaacttttccgcccataaggcggagctggcgcagggcctggtactgccggtagtgctgcgggcaccgtgcg-
ctggttgtccgttacctgttgggc
gctgccgggcgtcagcgaagaagaggccgctactttattgatagcgccgtgccgcgcacgctggtggtgatca-
ccgataatactcagcagcagca
acaacaggagcaggggcgtcttgatcgtctgaagcagagataaccagcggtaaattgctggcggcgatccgcg-
aatcgctggacgccgcgctgg
tacaactcaattgcccaattaatatgctggccgccgcacgccgcttaaatggcgacgagcatagcaatctggc-
gctggatgccgcatggcgtgaagg
cgaagaagcgatggcgcggttgcagcgctgccgcccgtcgctggaactggaaagcccggcagtctggccgctc-
cagccgttccttgacgatctgc
gtgccctgtatcacacccgatataaccagggcgaaaacctgcaaattgagctggaatcccccgacctggtggg-
ctttggccagcgaacacaactgctt
gcctgcctgagcctgtggctcgacagaaccctggatattgccgcggagctacgtgatttcacggtacagactc-
aactttacgcccgcgaagagagcg
gctggctgtcgttctatttaaacgacaatgtgccgctgattcaggtgcgctacacccattcacccgatgcact-
caatgcgcccggtaaaggcatggagc
tgcggctgatccagacgctggtcgcccaccatcgaggcgcaatagaactgacctcacgccctcagggaggcac-
ctgtctgatcctgcgtttcccatta ttttactcgctgacaggaggctcactatga 164
atgactcagcgaaccgagtcggggacaaccgtctggcgctttgacctctcccaacagtttacagccatgc-
agcgtatcagtgtggtgttaaaccgcgc
gacggagatcgggcagacgctacaggaagtgctgtgcgtgctgcacaacgatgcctttatgcagcacgggatg-
atctgtctgtacgacagtaagcaa
gcgatcctttccattgaagccttgcatgaggccgatcagagttaattcccggcagttcacagattcgctaccg-
tccgggcgaagggctggtaggcac
ggtgctttcacagggacagtcgctggtactgccctgtgtctccgacgatcggcgttttctcgatcgcctggga-
ttgtatgattacagcttgccgtttatcgc
cgtgccgctgatggggccaaactcgcagcctatcggcgtgctggccgcccagcctatggcgcgttacgaggag-
ggctgcccgcctgcacgcgttt
tcttgaaaccgtcgccaatctggtggcgcaaaccgttcgcctgatgacaccgcccagcgtcgcgtctccaccc-
cgtgctgctgccgcgcagattgcca
gccagcgcgggtgcgcgtcttcgcgagcgtatggctttgaaaacatggtcggtaaaagcgcggctatgcgtca-
gacgctggaaattattcgccaggt
atcacgctgggacaccaccgtgctggtgcgtggcgaaagcggaaccggtaaagagttgatagccaacgctatc-
caccacaattcaccgcgcgccg
ccgcgccgtttgtcaaattcaactgcgcggcgctgcccgatacgctgaggagagtgaactcttcggtcatgaa-
aaaggcgcgtttaccggcgcggtg
cgccagcgcaaaggccgtttcgaactggcggatggcggtacgctgtttcttgatgagatcggcgaaagtagcg-
cctcgtttcaggcgaaattgctgcg
tatcttgcaggaaggcgaaatggaacgcgtcggcggcgacgaaacgctgcgggtgaatgtacggatcattgcc-
gccaccaaccgcaatctggaag
aggaagtgcggctgggtaattttcgcgaagatctctactatcgccttaatgtgatgccgatctccctgccccc-
gctccgcgagcgtcaggaggacatcg
tcgagctggcgcattttctggtgcgcaaaatcgcgcaaaaccagaaccgcacgctgcgcatcagcgatggcgc-
gatccgtttgttgatgagctatagc
tggcctggaaacgtgcgtgagctggaaaactgccttgagcgatcggcggtgatgtcggaaaacgggctgatcg-
atcgcgacgtgattttgtttcacca
cagggaaaatctgcaaaaacgccacagaccagtgcgccgcgcgaagagagctggctcgatcagaacctcgatg-
aggacaaagattgatcgcc
gcgctggagaaagccggttgggtacaggcaaaagccgcgcgcctgagggaatgaccccgcgccaggtggccta-
tcgtattcagacgatggacat tgccatgccgagattgtag 165
atgccgctttcttcgcaggttacagcagcagtggcagaccgtttgcgaacgtctgcctgagtcattaccg-
gcgtcatcgttaagcgagcaggcaaagag
cgtgctcgtcttcagtgattttgtgcaggaaagtatcaccgccaacccgaactggctggcggaacttgagaac-
gcaccaccgcaggcagaagagtgg
cggcactatgctggctggctgcaaactgtactcgaagacgttacggatgaggccacgctgatgcgcgtcctgc-
gccagttccgtcgtcggctgatggt
ggcattgcctgggctcaggcgctggaactggtgagcgaagagagtacgctgcagcagttaagcgagctggcgc-
aaacgttgattgtcgccgcgc
gagactggctctatgcctgctgtaaagagtggggcacgccgtgcagcgaggaaggggttcctcagccgctgtt-
gattctgggcatgggaaagc
tgggcggctgcgagctgaacttctcctctgatatcgacctgatttttgcctggccggagaacggctccacgcg-
cggaggccgccgcgagctgaacaa
cgcgcagttctttacccgtctcggccagcgcctgattaaagcgctggatcagcccacgcaggacggttttgtt-
taccgcgtggacatgcgcctgcgtcc
gtttggcgacagcgggccgctggtgctgagctttgcggcgctggaagattattaccaggagcaaggtcgcgac-
tgggagcgttacgcgatggtcaa
agcgcggatcatgggcaacagcgacgacgcttatgccaacgagctgcgcgccatgctgcgtccgttcgtgttc-
cgtcgctatatcgacttcagcgtca
tccagtccctgcgaaatatgaaagggatgattgcccgcgaggtgcgccgccgtgggctgaaagacaatatcaa-
gctcggtgcgggcggcatccgc
gaaatcgaatttatcgtccaggtcttccagcttattcgcggcggacgcgagccgtcgctgcagtcccgttcct-
tattaccgacgctgagcgccattgcgc
agctgcatctcctgccggacggcgacgcgcaaaccctgcgcgaggcctatcttttcctgcgtcgtctggaaaa-
cctgctgcaaagcattaatgacgaa
cagacccaaaccctgccgggcgacgaccttaaccgggcgcgtctggcctggggaatgcgcgtggaagactggt-
caacgctgaccgaacggctcg
atgcccatatggcaggcgtgcgccgaatctttaacgaactgatcggtgatgacgaaagtgagtcgcaggacga-
tgcgctctccgagcactggcgcg
agctgtggcaggacgcgcttcaggaagatgacaccacgccggtgctgacgcacttaaccgacgacgcgcgcca-
tcgcgtggtggcgctgatcgct
gatttccgtcttgagctgaacaaacgcgccatcggcccgcgtggtcgccaggtgctggatcacctgatgccgc-
acctgctgagcgaagtctgctcgc
gtgccgatgcgccggtgccgctgtcgcggatgatgcccctgctgaggggattatcacccgtactacctacctt-
gaactcctgagcgagttccctggc
gcgcttaagcacctgatttcactctgcgccgcgtcgccgatggtggccaacaagctggcgcgttacccgctgc-
tgctggatgagctgctcgatccgaa
taccctttatcaaccgacggcgaccgacgcctaccgggacgaactgcgtcagtatctgctgcgcgtgccggaa-
gaagacgaagagcaacagctgg
aggcgctgcgtcagtttaagcaggcccagatgctgcgcgtggcggccgcagatattgccggaacgctgccggt-
gatgaaagtgagcgatcacttaa
cctggcttgcggaagcgattatcgacgcggtggtgcatcaggcctgggtgcagatggtggcgcgctatggcca-
gccgaaacatctggctgaccgtg
atggtcgcggcttcgcggtggtgggttacggtaagctcggcggttgggagctgggctatagctccgatctgga-
tttaatcttcctccacgactgcccgg
ttgatgtgatgaccgacggcgagcgcgagattgacgggcgtcagttctacctgcgcctggcgcagcgcatcat-
gcacctgttcagcacccgcacctc
gtcgggcattgtatgaagtggatgcccgtctgcgcccgtccggcgcggcgggcatgctggtcacctcgacgga-
gtccttcgctgattaccagaaga
atgaagcctggacgtgggtgcatcaggcgctggtgcgcgcccgtgtggtgtatggcgatccgctgctgaaacg-
cagtttgacgtgattcgtaagga
agtcatgaccaccgtgcgcgatggcagcacgctgaaacggaaggcgcgaaatgcgcgagaaaatgcgcgcgca-
cttaggcaataaacatcgc
gatcgctttgatattaaagccgatgagggcggtattaccgatattgagtttattacccagtatctggtgttgc-
tgcacgcgcatgacaagccgaagctgac
gcgctggtcggataacgtgcgcattctggaactgctggcgcaaaacgacattatggacgagcaggaggcgcag-
gccttaacccgtgcctatacaac
gcttcgcgatgagctccatcatctggcgttgcaggagcagccgggacacgtggcgctggactgtttcaccgct-
gaacgcgctcaggtaacggccag
ctggcagaagtggctggtggaaccgtgcgtaacaaatcaagtgtga 166
atgaagatagcaacacttaaaacgggtctgggttcgctggcactgctgccgggcctggcgctggctgctg-
cacctgcggtggcagacaaagccgat
aacgcctttatgatgatcagcaccgcgctggtgctgttcatgtccattccgggcattgcgctgttctatggcg-
gcctgatccgtggcaaaaacgttctctc
catgctgacgcaggttgccgtaacgttcgcgctggtctgcgtactgtgggtggtttacggttactcgctggct-
ttcggcacgggcaacgcgttctttggta
acttcgactgggtgatgctgaaaaatattgaactgaccgcgctgatgggcagtttctaccagtatattcacgt-
tgctttccagggctcgttcgcctgcatta
ccgtcgggctgattgtaggcgcgcttgccgagcgtattcgtttctctgcggtcctgatcttcgtggtggtctg-
gctgacgctctcctatgtgccgattgcg
cacatggtctggggtggcggctgctggcgacgcatggcgcgctggacttcgcgggcggtaccgttgtgcacat-
taacgccgcatagcgggtctg
gttggcgcatacctgattggcaaacgcgtgggcttcggtaaagaagcgttcaaaccgcacaacctgccgatgg-
tcttcaccggtaccgcgatcctcta
ctttggctggtttggtttcaacgccggctcagcaagtgccgcgaacgaaatcgccgcgctggccttcgtgaat-
accgttgtggccacggcaggtgcaa
tcctctcctgggtctttggcgagtgcgctgtgcgcggtaaaccttctctgctgggtgcctgttcgggggcgat-
tgctggtctggtcggtatcaccccagc
atgtggttatgtcggtgtgggtggcgcgctgctggtcggcctggtgtcaggtctggcgggtctgtggggcgtg-
acggcgctgaaacgtattctgcgcg
ttgatgacccttgcgatgtgtttggcgtgcacggcgtgtgcggcatcgtcggctgtatcatgaccggtatctt-
tgcagcgaaatcgctgggtggcgtgg
gctacgcagaaggcgtcaccatggcccatcaggtgctggtgcagctggaaagtattgctgtcaccgtggtgtg-
gtctgccgttgtcgctttcattggcta
caaactggcggacatgacggttggtctgcgcgtgccggaagagcaggaacgcgaaggtctggacgtcaacagc-
cacggcgagaatgcgtataac gcatga 167
gccgagagaggggcccgcgtcggattaggtagttggtgaggtaatggctcaccaagccttcgatccgtag-
ctggtctgagaggatgatcagccaca
ctgggactgagacacggcccagactcctacgggaggcagcagtggggaatattggacaatgggcgcaagcctg-
atccagcaatgccgcgtgagtg
atgaaggccttagggttgtaaagctctttcgcacgcgacgatgatgacggtagcgtgagaagaagccccggct-
aacttcgtgccagcagccgcggta
atacgnagggagcnagcgttnntcggaattactgggcgtaaagngcgcgtaggcggcntgttnagtcagaagt-
gaaagccccgggctcaacctgg
gaatagcttttgatactggcaggcttgagttccggagaggatggtggaattcccngtgtagnggtgaaatncg-
tagatattgggangaacaccngtgg
cgaaggcggcnatctggacgganactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccct-
ngtagtccacgccgtaaacga
tgaatgctagacgtcggggtgcatgacttcggtgtcgccgctaacgcattaagcattccgcctggggagtacg-
gccgcaaggttaaaactcaaagg
aattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgcagaaccttaccaaccc-
ttgacatgtccactttgggctcgag
agatngggtccttcagttcggctgggtggaacacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatg-
ttgggttaagtcccgcaacgagcgc
aacccctaccgtcagttgccatcattcagttgggcactctggtggaaccgccggtgacaagccggaggaaggc-
ggggatgacgtcaagtcctcatg
gcccttatgggttgggctacacacgtgctacaatggcggtgacagtgggaagcgaagtcgcgagatggagcaa-
atccccaaaagccgtctcagttc
ggatcgcactctgcaactcgagtgcgtgaagttggcaatcgctagtaatcgcggatcagcacgccgcggtgaa-
tacgttcccgggccttgtacacacc
gcccgtcacaccatgggagttggttttacccgaaggtggtgcgctaaccgcaaggaggcagccaaccacggta-
aggtcagcgactggggtgaagt
cgtaacaaggtagccgtagggcaacctgcggctggatcacctccttt 168
atggccaaagcgcctctgcgtcagatcgccttttacggcaagggcggtatcggcaagtccaccacctctc-
agaacacgctggccgcgctggtcgag
ctggatcagaggatcctgatcgtcggctgcgacccgaaggccgactcgacccgcctgatcctgcacgcaaagg-
cccaggacaccgtcctgcatctg
gccgccgaggccggctcggtcgaggatctggagctcgaggacgttctcaagatcggctacaagaacatcaagt-
gcgtcgagtccggcggtccgga
gccgggggtcggctgcgccggccgcggcgtcatcacctcgatcaacttcctggaagagaacggcgcctacgac-
gacgtggactatgtgtcctacga
cgtgctgggcgacgtggtctgcggcggcttctccatgccgatccgcgagaacaaggcccaggaaatctacatc-
gtcatgccggcgagatgatggc
gctgtacgccgccaacaacatcgccaagggcatcctgaagtacgcgcacagcggcggcgtccgtctcggcggc-
ctgatagcaacgagcgccag
accgacaaggaatgggatctggccgacgcgctggccaaccgcctgggctccaagctgatccacttcgtgccgc-
gcgacaacatcgtccagcacgc
cgagagcgccgcatgacgctcatcgagtacgccccggacagcaagcaggccggcgaataccgcgcgctcgcca-
acaagatccatgcgaactcc
ggccagggttgcatcccgaccccgatcaccatggaagagctggaagagatgctgatggacttcggcatcatga-
agaccgaggagcagcagctcgc cgagctcgccgccaaggaagcggcgaaggccggcgcctga 169
atgagcctgtccgagaacaccacggtcgacgtcaagaacctcgtcaacgaagtcctcgaagcctatcccg-
aaaaatcccgcaagcgccgcgccaa
gcacctgaacgtgctggaggccgaggccaaggaagggcgtcaagtcgaacgtcaagtccatccccggcgtcat-
gaccatccgcggctgcgcct
atgccggctccaagggcgtggtgtggggtccgatcaaggacatgatccacatctcccacggtccggtcggctg-
cggctactactcaggtccggccg
ccgcaactactacatcggcgacaccggtgtggacagctggggcacgatgcacttcacctccgacttccaggag-
aaggacatcgtcttcggcggcga
caagaagctgcacaaggtcatcgaggaaatcaacgagctgttcccgctggtgaacggcatctcgatccagtcg-
gaatgcccgatcggcctgatcgg
cgacgacatcgaggctgtcgcccgcgccaagtcggcggaaatcggcaagccggtcatccccgtgcgctgcgaa-
ggcttccgcggcgtgtcccagt
cgctgggccaccacatcgccaacgacgccatccgagactgggtgttcgagaagacggaacccaaggccggctt-
cgtctccaccccctatgacgtca
ccatcatcggcgactacaacatcggcggcgacgcaggtcgtcccgcatcctgctggaggagatcggcctgcgc-
gtgatcgcccagtggtcgggc
gacggcacgctcgccgaactggagaacacgccgaaggccaaggtcaacctgatccactgctaccgctcgatga-
actacatcgcgcgccacatgga
agagaagttcaacattccttggatggaatacaacttcttcggcccgagccagatcgccgaatccctgcgcaag-
atcgccgctctcttcgacgacaagat
caaggagaacgccgagaaggtcatcgcccgctaccagccgatggtcgatgcggtcatcgccaagtacaagccg-
cggctcgaaggcaagaaggtc
atgatctacgtcggcggcctgcgtccccgccacgtcgtcgatgcctaccatgacctcggcatggagatcaccg-
gcaccggctacgagttcgcccaca
acgacgactatcagcgcacgcagcactacgtgaaggaaggcacgctgatctacgacgacgtcaccgcgttcga-
actggagaagttcgtcgaggcg
atgcgtcccgacctcgtcgcgtcgggcatcaaggaaaagtacgtgttccagaagatgggcctgccgttccgcc-
agatgcacagctgggactactccg
gcccgtaccacggctatgacggcttcgcgatcttcgcccgcgacatggacctggccatcaacaaccccgtctg-
gggcgtgatgaaggccccgttctg a 170
atgctccaggacaagatccaggatgtcttcaacgaaccgggctgcgcgaccaaccaagccaaatcggcca-
aggagaagaagaagggctgcacca
agtcgctgaaaccgggggcggcagccggcggctgagcctatgacggggcgatgatcgtgctccagccgatcgc-
cgacgccgcccatctggtccat
ggccccatcgcctgcctcggaaacagttgggacaaccgcggctccaaatcctccggctcgcagctctaccgca-
ccggcttcaccaccgatctgtcgg
aactggacgtcatcggcggcggcgagaagaagctctaccgcgccatcaaggagatcgttcagcaatacgaccc-
gccggccgtcttcgtctatcaga
cctgcgtgcccgccatgaccggcgacgacatcgccgcattgcaagttcgccacgcagaagctgagcaagccgg-
tgatcacggtggactcgcc
gggcttcgtcgggtcgaagaatctcggcaacaagctggccggcgaagccctgctggagcatgtcatcggcacg-
gtcgaaccggactacaccaccc
cgaccgacgtctgcatcatcggcgaatacaaccttgccggcgagctgtggctggtcaagccgctgctggacga-
gatcggcatccgcctcctgtcctg
catttccggcgacggccgctaccgggaggtggcgcaggcccaccgcgcccgcgtcaccatgatggtgtgcagc-
caggcgctggtgaatgtcggg
cgcaagatggaggagcgctacggcatcctctatttcgaggggtccttctacggcgtgtccgacatgtcggaca-
ccctgcgcaccatgacccgcatgc
tggtggagcgggcgccgacaagggcctgatcgaccgggcggagggcgtgatcgcgcgggaggaaagccgggtc-
tggcgccggctggaaccc
tacaagccgcgcttcgacggcaagcgcgtccttctcttcaccggcggcgtcaagagctggtcgatggtcagcg-
cgctggagggtgcggggctgacc
atcctcggcacctccaccaagaaatcgaccagggaggacaaggagcgcatcaagaagatgaagggcgaagagt-
tccaccagtgggacgatttgaa
gccgcgcgacatctacaggatgctggccgacgatcaggccgacatcatgatgtccggcggccgctcgcagttc-
atctcgctgaaggccaaggttcc
ctggctcgacatcaaccaggagcgccaccacgcctatgccggctatgacggcatcgtcaatctctgcgaggag-
atcgacaaaacgctgtcgaatcc
gatctggcgtcaggtgcgtcagccggcaccgtgggagtccggcgcgtcctccacccttctggcttcctcgatg-
gcggcggagtga 171
atgtcccacatccagcgcttcccctccgccgccaaggccgcctccaccaacccgctgaagatgagccagc-
cgctgggtgcggctctggcctatctcg
gcgtcgaccgctgcctgccgctgttccatggctcgcagggctgcaccgccttcgggctggtcctgctggtgcg-
ccatttccgcgaggcgatcccgct
ccagaccacggcgatggatcaggtcgccaccatcctcggcggctacgacaatctggagcaggcgatccgcacc-
atcgtcgagcgcaaccagccc
gccatgatcgccgtcgccaccaccggcgtcaccgagaccaagggcgaggatatggccggacagtacacgctgt-
tccgccagcgcaaccccgactt
ggccgacacggccctggtcttcgccaacacccccgacttcgccggcggcttcgaggacggcttcgccgccgcg-
gtcaccgcgatggtcgagcggt
tggtcgaaccgtcgccggtgcgcatcccgacccaggtcaacgtgctggccggctgccatctgtcccccggcga-
cgtggaggaactgcgcgacatc
atcgaaggcttcggcctgtcgccgatcttcctgaccgacctgtcgctgtcgatggcgggccgccagccggccg-
acttcaccgccacctcgctgggcg
gcgtgaccgtcgatcagatccgcgccatgggcgcttcggccctcaccatcgtggtcggtgagcatatgcgggt-
ggccggtaacgcgctggagctga
agaccgacgtgcccagccatttatcaaccgcctgaccgggctggaggcgacggacaagctggtccggctgctg-
atggagttgtcgggcaagccg
gcgcccgcccggctgcggcgccagcgcgaaagcctggtcgatgccatgctcgacgggcatttcttctacagcc-
gcaagcgcatcgccgtcgcgct
ggagcccgacctgctctatgccgtcaccggcttcctcgccgacatgggggccgaggtgatcgccgcggtgtcc-
ccgacgcagagcccggtgctgg
agcggttgaaggccgccaccatcatggtcggcgatcattccgacgtggagacgctggcccgcgacgccgacct-
gatcgctccaactcgcacggg
cggcagggagccgcgcggatcggcgtggctctgcaccgcatgggcctgccgctgttcgaccggctgggggccg-
gcctgcgcgtccaggtcggct
accgaggcacgcgggaactgctgtgcgacatcggcaacctgttcctcgcccgcgagatggaccacgagcacgg-
gcacgagagccacgaccacg
gggaatcccacggctgccgaggcggatcatgcggatgcaacgccgtctga 172
atgaccgacaagctttcgcagagcgccgacaaggtcctcgaccactacaccctcttccggcagcccgaat-
acgcggcgatgttcgagaagaagaag
accgagttcgagtacggccattcggacgaggaagtcgcccgcgtgtccgaatggaccaagtccgaggactaca-
aggcgaagaacttcgcccgtga
agcggtcgtcatcaacccgaccaaggcctgccagccgatcggcgcaatgttcgccgcccagggcttcgaaggc-
accctgcccttcgtccacggctc
ccagggctgcgtcgcctattaccgcacccacctgacccgtcacttcaaggagccgaacagcgcggtctcctcg-
tcgagacggaggacgcggcggt
gttcggcggcctgaacaacatgatcgacggcctggcgaacgcctatgcgctctacaagccgaagatgatcgag-
gtgatgaccacctgcatggccga
agtcatcggcgacgatttgcagggcttcatcgccaatgcgaagaccaaggacaggtcccggccgacttcccgg-
tcccctacgcccacaccccggc
cttcgtcggcagccacatcgtcggctacgacaacatgatcaaggggatcctgaccaacttctggggtacgtcg-
gagaatttcgacacacccaagacc
gagcagatcaacctgatcccgggattcgacggcttcgccgtcggcaacaaccgcgaactgaagcgcatcgccg-
gcgaattcggcgtgaagctgca
aatcctgtccgacgtgtccgacaatttcgacacgccgatgaatggcgagtaccgcatgtatgacggcggcacc-
accatcgaggagaccaaggaggc
cctgcacgccaaggccaccatctccatgcaggagtacaacacgacccagaccctgcaattctgcaaggagaag-
ggtcaggaagtcgccaagttcaa
ctacccgatgggcgtcaccggcaccgacgagctgctgctgaagctcgccgaactgtcgggcaagccggtcccg-
gccagcctgaagctggagcgc
ggccgtctggtcgacgccatcgccgacagccacacccacatgcacggcaagcgcttcgccgtctatggcgacc-
cggacttctgcctgggcatgtcc
aagttcctgctggagctgggtgcggagccggtgcacatcctgtcgacgtcgggctccaagaaggggagaagca-
ggtccagaaggtgctggacgg
ctcgcccttcggcgcctcgggcaaggcccatggcggcaaggatctgtggcacctgcgttcgctgatcttcacc-
gacaaggtggactacatcatcggc
aacagctacggcaagtatctggagcgcgacaccaaggttccgctgatccgcctgacctacccgatcttcgacc-
gccaccaccaccaccgctacccg
acctggggctaccagggcgcgctgaacgtgctggtacggatcctaaccggatcttcgaggacatcgacgccaa-
caccaacatcgtcggccagac cgactactcgttcgacctgatccgctga 173
atgttgacctctgatattgttggcaaattgcgctgcatcgcagcagaccccaaagcgggcatcgcaaggg-
gcctcgacaccgggacgacgaagatc
ggtcccgtttgggagggtgacgtgggcgacaccgtggatttcgaagcgctgcgccagcgggcggtccactccc-
tgttcgaacatctggaatccatgt
gcgtcagcgccgtcgccgtcgaccacaccggccgcatcgcctggatggacgagaagtacaaggctctgctggg-
cgttcccgacgacccgcgcgg
ccggcaggtggaggacgtcatccccaacagccagctgcgccgggtgatcgacagcggccagccgcagccgatg-
gacatcatggagttcgacgac
cggtccttcgtggtgacgcgcatgccgctgttcggcaccgacggttcgatcatcggcgccatcggcttcgtgc-
tgttcgaccgcgccgaatatctccg
cccgctggtccgcaaatacgagaagatgcaggaggagctggcccgcacccagcaggagctggcgcatgagcgc-
cgcgccaaatactccttctcg
cagttcctgggcgccagcgaatcgatccgcgagatcaagcggctggggcgccgcgccgcccagatggattcga-
ccgtcctgctgctgggcgaaac
cgggaccggcaaggagctgctggcccaggccatccattccgccagcccgcaggcgtccaagcccttcgtcggc-
gtcaatgtcgccgccattccgg
aaaccctgctggaggcggagttcttcggcgtcgoccccggcgccttcaccggcgccgaccgccgccaccgcga-
cggcaagttccagctcgccaac
ggcggcaccctgttcctcgacgagatcggcgacatgccgctgccggtgcaggccaagcttctgcgcgtgctgc-
aggagcgggagatcgagccgct
cggctccaacaaggtggtgcgggtcgatgtccgcatcatcgccgccaccagccgtgacctgcacgccctggtg-
cgtgagaagcagttccgcgccg
acctctattaccggctgaatgtggtgccgatcaccctgccgccgctgcgcgaccggccggaggacatcgagag-
catcgccgaccgcatcctggaac
agctggcgatccagcagggcacgccgcogcgcgagctgctggaatcggcggtgcaggtgctgcgcgactatga-
ctggcccggcaatgtgcgcga
gctttacaacacgctggaacgggtggtggcgctgaccgatgcgccgatcctgaccgcgccgcacatccgcagc-
gtgctgcccggccagcatccgg
ccggcgcgtcggccctgccgctggcggccggcgcgcggccgttgcaggaggtgctgacgccgccgagcgccac-
gccatcgccgcggcgcttg
aggaggcgaacggcgtcaaggcgcgggcggcgaagctgctgggcatttcgcgcgcgtcgctgtacgaacgcat-
ggtgacgctggggttggggg cgacgcagtag 174
atgccgagtcccatcgcgttctcaagccccttgccgaagcctttcgacagcgcgcaggggcgctggggat-
ggaggctggcgccagcaggccgc
cgcggcggagccggagacccgcgcctgggcggaagccttcgccgattcggagaccggccgggcgctgatcggg-
gcggtgtgcggcaacagcc
cgtatctcggccacagcctgacgcgggagttgcccttcgtcgcccgtacagtgcaggacggcttcgacgacac-
cttcgccgcgctgatcgccgctct
ccatgccgagcatggcgaggagaaatcgatggaccggctgatggccggcctgcgggtggcgaagcggcgggcg-
gcgagctgatcgcgctggc
cgacatcgccgggcgtggccgctgttccgcgtcaccggcgccctgtcggagctggcggagacgggggtgcagc-
tggccgcgaatttcctgctgc
gccgcgccagggaggccgggacgctgacgctgccggatccgcagcgaccgtgggtcggttcgggcctgatcgt-
tttgggcatgggtaagcttggc
gggcgcgaactcaactattccagcgacatcgacctgatcgtcctgtatgacgacgctgttgtgcagacgcccc-
agccggacaacctcgcgcgaacct
tcatcaggctcgcacgcgatcttgtccgcattatggatgaacggaccaaggacggctacgtcttccgcaccga-
ccttcggcttaggcccgatcccggc
gccacgccgctggcggtttccgtctccgcagccgaaatttattacggcagcgtcggtcagaactgggaacgcg-
cggcgatgatcaaggcccgtccc
atcgccggcgatctggaggcgggcgcgcctcctttgtccgcttcctggagcccttcgtctggcgccgcaacct-
ggatttcgccgccatccaggacatcca
ttcgatcaaacgccagatcaacgcccacaagggccaccgcgaggtgacggtcaacggccacgacatcaaggtc-
ggccgcggcggcatccgcga
gatcgagacttcgcccagacccagcagctgatcttcggcgggcgcgacccgcgcgtgcgaatcgctccgaccc-
tgatggcgaacgaggcgctgc
gcgacgtcggccgcgtgccgccgcagacggtggaagagcttgccggggcctatcatttcctgcgccgtgtcga-
acatcgcatccagatgatcgacg
accagcagacccatcgtattcccgccgacgatgccggggtggcgcatttggccaccttcctcggctatgacga-
ccccgccgccttccgggcggaact
gctggcgacgctggggcaggtggaggaccgctattccgagctgttcgaggaggcgccgtcgctttccggcccc-
ggcaatctggtcttcaccggca
ccgaccccgatccgggcacgatggagacgctgaagggcatgggcttcgccgatccggccgcgtcatcagcgtg-
gtgtcggctggcatcgcgg
ccgctaccgcgccacccggtcgggccgggcgcgggagctgctgacggagctgacgccggccctgctgagtgcg-
ctgaccaagaccccggcccc
cgattcggcgctgatgaacttcgacgatttcctcggcaagctgccggccggcgtcggtctgttctcgctgttc-
gtcgccaatccctggctcctggagctg
gtggcggagatcatgggcatcgcgccgcagatggcgcagacgctgtcgcgcaacccgtcgctgctcgacgccg-
tgctgtcgccggacttcttcgac
ccgctgccgggcaaggaggacgggctggccgacgaccacgcccgcgtgatggcgccggcccgcgatttcgagg-
atgcgctgaccctgtcgcgg
cgctggaccaacgaccagcgcttccgcgccggggtgcatatcctgcgcggcatcaccgatggcgaccgctgcg-
gcgccttcctggccgatctggc
cgacatcgtcgtccccgaccttgcccgccgggtggaggaggagttcgcccagcgccacggccatattcccggc-
ggcgcctgggtggtggtggcga
tgggcaagtcggcagccggcagctgaccatcacgtccgacatcgacctgatcgtcatctacgatgtggcgccc-
ggccaagggggcgggggcgg
tccccgcttgtcggatggtgccaagccgctgtcgcccaacgagtattacatcaagtgactcagcgtctgacca-
acgccattaccgcgccgatgcgc
gacggccggctctacgaggtcgacatgcggctgcgcccgtcgggcaacgccgggccgctcgccacctcgctgg-
acgctttcctgaaatatcaggc
gaccgatgcctggacctgggagcatatggccctgacccgcgcccgggtgatcggcggtgatgcggagctggcc-
gggcgggtgtcggcagcgatc
cgctcggtgctgacggcgccgcgcgatgccgaccggctgctgtgggacgtggccgacatgcggcggcggatcg-
agaaggagttcgggacgacc
aatgtctggaacgtcaaatacgcccgcggcggcctgatcgacatcgagttcatcgcccagtacctgaactgcg-
ccatggtcacgagcggccggac
atcctgcacatcggcaccgccaaggcgctgggctgcgccgcccggacgggcgcgctggcgccggaggtggcgg-
aggatctggagacgacgct
gcggctgtggcggcgggtgcagggctttctgcggttgaccaccgccggggtgctcgatcccaatcaggtgtcg-
cccagcctgctggccgggctggt
ccgcgccgcctttcctgctgactttcagggcgagcgtgagcctcgactgttgacttccccgaactggaccaca-
aaatccgtgccgtcgccgcccgc
gcccatggtcatttcaagaccctggtcgaggaaccggcgggccgtctggccccacccgccaccacgcctccag-
cctga 175
atgaaccgtctgttccaatggccgcaccgatgatggcggttgctctgggcgcggtcggcatgccggccgc-
agcccttgcccaggatccggcggctg
ccgccgctgccgcggctgcggctgcggagccgccgctgctgccgcaccggcggctccggcgctgaatggcggc-
gacaccgcctggatgctcat
ctccaccgcgctggtgctgatgatgaccatccccggcctggcgctgttctacggcggcatggtccgcaagatg-
aacgtgctgtcgacggtgatgcag
agcttcgccatcacctgcctgatcagcgtcctgtggtacgtcatcggctacagcctggccttcaccggcaccg-
gtgcctatgtcggcggtctcgaccg
gctgttcctcaacgggctcgacttcacgaaggccttcgtgctgggcgaggcgaccgggtcgggcgtcccgacg-
accatccccgagccggtcttcat
gatgttccagatgacctttgcgatcatcaccccggccctgatcaccggcgccttcgccgaccgatgaagttct-
cctccctgctggtcttcaccgcgctg
tggtcgatcgtggtctatgcgccgatcgcccactgggtctggtacccgtcgggcttcctgttcggcctagcgt-
gaggacttcgccggcggcacggt
cgtgcacatcaaccgccggcgtcgccggcctggtcgccgcgctggtgatcggcaagcgcaagggctacccgaa-
ggaagccttcatgccgcacaac
ctggtgctgtcgctgatcggcgcctcgctgctgtgggtcggctggttcggcttcaacgccggttcggccctga-
ccgccggtccgcgtgccggcatgg
cgctggccgccacgcacatcgccaccgccggtgccgccatgggctggctgttcgcggagtggatcgtcaaggg-
caagccgtcgatcctcggcatc
atctccggcgccgtcgccggcctggtcgggtgaccccggccgccggcttcgtcgacccgacgggcgccatcgt-
catcggcatcgtcgcccgcgt
ggtctgcttctggtcggccaccagcctcaagcacatgctgggctatgacgacagcctggacgccttcggcgtg-
cacggcgtcggcggcctgatcgg
cgccatcctgaccggcgtcttcgccaagatgtcggtgtccaacagcgaaggcggcttcgcctccgtcctgcag-
gccgacccgaaggccacgctgg
gcctgctggaaggcaacgccgccgccgtctggatccaggtccagggcgtcctctacaccatggtctggtgcgc-
catcgccaccttcgtcctgctgaa
gatcgtcgatgtggtcatgggcctgcgcgtcgaagaggatgtggagcgcgacggtctcgacctcgccctgcat-
ggcgagagcatccactaa 176
atggatgcggcaaagacgggtggcgacgtccttttcgtgctgatgggcgcggtgatggtgctggcgatgc-
attgcggcttcgccctgctggaggtcg
ggacggtccggcgcaagaatcaggtcaacgcgctggtgaagatcctgtcggacttcgccatgtcgaccatcgc-
ctattttttcgtcggttatgccgtgg
cctacggcatcgacttcttcgccgacgcccacacgctggtcggcaagggaagcggcgggttcgcggcctatgg-
ctacgatctggtgaagttcttcttc
ctggcgaccttcgccgccgcggtgccggccatcgtctcgggcggcatcgccgagcgtgctaggttctggccgc-
aggccgccgccacgctggcgct
gatcgcgctgttctatccattgctggaaggcacggtctggggcacccgcttcggcctgcaaagctggatggcc-
gcgaccttcggccagcctttccacg
acttcgccggatctgtggtggtgcatgccttcggcggctgggtggcgctgggtgccgtgctgaacctcggcaa-
ccgccgcggccgctaccgtccga
acggctcgctgatcgccattccgccgtcgaacatccccttcctggcgctgggcgcctgggtgctgtgcgtggg-
gtggttcggcttcaacgtgatgagc
gcccaggtgctggatggcgtgacgggtctggtggcgctgaactcgctgatggcgatggtcggcggcatcgtca-
cctcgctggtgatcagccgcacc
gatcccggcttcgtccacaacggcgcgctggccggtctggtggcggtctgcgccgggtccgacgtgatgcacc-
cgctgggcgcgctggtcaccgg
cggcatcgccgggctgctgttcgtctgggccttcaacaaatgccagatcgactggaagatcgacgacgtgctg-
ggcgtctggccgctgcacggctg
tgcggcctgaccggcggcctgctggccggcgtcttcgggcaggaggcactgggcggccttggcggcgtgtcga-
tcctcagccagatcgtcggcac
ggcaagcggcgccagcttcggattcgctacgggtctggcggtctaccgcctgctgcgcgtcaccgtccgcatc-
cgcctcgatcccgaccaggagta
caagggcgccgacttgtcgttgcaccatatcaccgcgtacccggaagaggacgcgccgaccctgtaa
177
atgaccctgaatatgatgatggattcttggttctctggagcgctttatcggcatcctgactgaagatttg-
caggcttcttcccaacctggcttgcacccgt
gcaggtagttgtgatgaacatcactgattcgcaggctgaatacgttaacgaattgacccgtaaactgcaaaat-
gcgggcattcgtgtaaaagcagactt
gagaaacgagaagattggctttaaaatccgcgagcacactttacgtcgtgtcccttatatgctggtttgtggt-
gacaaagaggtcgaagccggcaaagt
tgctgtgcgtacccgtcgcggtaaagacctgggtagcctggacgtaaatgatgatcgagaagctgcaacaaga-
gattcgcagccgcagtcttcaac
aactggaggaataaggtattaaaggcggaaaacgagttcaaacggcgcgtcccaacgtattaatggcgagatt-
cgcgccacggaagttcgcttaac
aggtctggaaggcgagcagcttggtattgcgatagaactcacttcacgccccgaagggggaagctgcctgacc-
ctacgattcccgctatttcattcact gaccggaggttcaaaatga 178
accggataagagagaaaagtgcgacgtcggtccggttgatattgaccggcgcatccgccagctcgcccag-
tttttggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccganaaaaataccaatatttgccataacacacgctcctgttgaaaaagagat-
cccgcggggaaaatgcggtgaacat
gtcagctattgcgaagagtgtgccagttttgctcacgggcaaaagctgcaccagaatgggattaatgcaccag-
cctggcgctttttttcgcggcacgtc
ccctcgctaatgcccgtctggcgcggctttgacgctgataaggcgctgaataccgatctggatcaaggtttgt-
cgggttatcgtccaaaaggtgcactc
ttgcatggttataagtgcctgacatggtgtccgggcgaacgtcgccaggtggcacaaattgtcagaactacga-
cacgactaaccgaccgcaggagt
gtgcgatgaccctgaatatgatgatggattatggttctctggagcgctttatcggcatcctgactgaagaatt-
tgcaggcttcttcccaacctggcttgca
cccgtgcaggtagttgtgatgaacatcactgattcgcaggctgaatacgttacgaattgacccgtaaactgca-
aaatgcgggcattcgtgtaaaagca
gacttgagaaacgagaagattggctttaaaatccgcgagcacactttacgtcgtgtcccttatatgctggttt-
gtggtgacaaagaggtcgaagccggc
aaagttgctgtgcgtacccgtcgcggtaaagacctgggtagcctggacgtaaatgatgttatcgagaagctgc-
aacaagagattcgcagccgcagtct
tcaacaactggaggaataaggtattaaaggcggaaaacgagttcaaacggcgcgtcccaatcgtattaatggc-
gagattcgcgccacggaagttcgc
ttaacaggtctggaaggcgagcagcttggtattgcgatagaactcacttcacgccccgaagggggaagctgcc-
tgaccctacgattcccgctatttcat
tcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttatccca-
gcagttcaccgcgatgcagcggata
agcgtggttctcagcgggcgaccgaggttgaacagacactccagcaggtgctgtgcgtattgcacaatgacgc-
ctttttgcagcacggcatgatctg
tctgtacgacagccagcaggcgattttgactattgaagcgttgcaggaagccgatcagcagttgatccccggc-
agctcgcaaattcgctaccgtccgg
gtgaagggaggtcgggacggtgctttcgcaggggcaatcgttagtgctggcgcgtgtggctgacgatcagcgc-
tttcttgaccgcctgggactgtat
gattacaacctgccgtttatcgccgtgccgctgatagggccggatgcgcagacttttggcgtgctgacggcgc-
aaccgatggcgcgttacgaagagc ggttacccgcctgcacccgctttctggaaacggtc 179
tccctgtgcgccgcgtcgccgatggtgaccagcaactggcgcgctacccgatcctgctcgagaactgctc-
gacccgaacacgctctatcaaccga
cggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaagagcagca-
actggaggcgctacggcagttta
agcaggcgcagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagtgagcgatca-
cttaacctggctggcggaagcga
ttatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcatctgcacga-
tcgcgaagggcgcggtttcgccg
tggtcggttacggcaaacttacggctgggaattaggttacagctccgatctggatctggtgttcctgcacgac-
tcccccatggatgtgatgaccgatg
gcgagcgtgaaatcgatggcccccagttctatttgcgcctcgcgcagcgcgtgatgcacctgttcagcacgcg-
cacgtcgtccggcattctttatgaag
tcgatgcgcgtttgcgcccgtccgggcggccggaatgctggtgaccactgcggaagcgttcgccgattatcaa-
aaaaatgaagcctggacatggg
agcatcaggcgctggcgcgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcg-
ccgcgatatcctgatgacctcccg
cgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttggtaacaagcac-
aaagaccgtttcgatctgaaagc
cgatgaaggcggatcaccgatattgagtttatcgctcagtatctggtgctgcgctttgcccatgagaagccga-
aactgacgcgctggtcggataatgg
cgcatcctcgaagggctggcgcaaaacggcatcatggatgagcaggaagcgcaggcattgacgctggcgtaca-
ccacgttgcgtgatgagctgca
ccacctggcgctgcaagagctgccaggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttatcaaa-
accagctgggacaagtggctggtg gaaccgtgcgccccggcgtaa 180
taaagcgagcgctcacttacgtgatctgttgacgcagtccgaagcgaccattacttcagccgtttcagca-
gatacggcggtgtggagtgcgcaatcag
ccctggcgaaactggtgctcaccgagtggttagtgacgcagggctggcgaaccttccttgatgaaaaagcgca-
ggctaagtttgccgactcctttaaa
cgctttgctgacgttcatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccagccgctgggcgaca-
gctatcgcgaccagttaccgcggc
tggcgcgtgatatcgacagcgcgttattgctggccggacattacgatcgcgcgcgcgccgtggagtggctgga-
aaactggcaggggcttcagcacg
ctattgaaacgcgccagagagttgaaatcgaacatttccgtaataccgccattacccaggagccgttctggtt-
gcacagggaaaacgttaacgaaag
gatatttcgcatgtccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcagctcg-
atgaactgctcgacccgaacacg
ctctatcaaccgacggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagagg-
atgaagagcagcaactcgaggc
gctacggcagtttaagcaggcgcagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatg-
aaagtgagcgatcacttaacctgg
ctggcggaagcgattatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccga-
cgcatctgcacgatcgcgaagg
gcgcggtttcgccgtggtcggttacgccaaacttggcggctgggaattaggttacagctccgatctggatctg-
gtgttcctgcacgactgccccatgga
tgtgatgaccgatggcgagcgtgaaatcgatggccgccagttctatttgcgcctcgcgcagcgcgtgatgcac-
ctgttcagcacgcgcacgtcgtccg
gcattctttatgaagtcgatgcgcgtttgcgcccgtccggcgcggccggaatgctggtgaccactgcggaagc-
gttcgccgattatcaaaaaaatgaa
gcctggacatgggagcatcaggcgctggcgcgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaat-
ttgacgccattcgccgcgatatc
ctgatgacctcccgcgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatc-
ttggtaacaagcacaaagaccgt
ttcgatctgaaagccgatgaaggcggtatcaccgatattgagtttatcgctcagatctggtgctgcgctttgc-
ccatgagaagccgaaactgacgcgct
ggtcggataatgtgcgcatcctcgaagggctggcgcaaaacggcatcatggatgagcaggaagcgcaggcatt-
gacgctggcgtacaccacgttg
cgtgatgagctgcaccacctggcgctgcaagagctgccaggacatgtggcgactctcctgttttgtcgccgag-
cgtgcgcttatcaaaaccagctggga
caagtggctggtggaaccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcagga-
gacaggaatgaaagttacgctgcca
gagttcaatcaagccggtgtcatggtggtgggtgatgtgatgctggatcgctactggtacggcccaaccagcc-
gcatttctccggaagcgccagttcc
ggttgttaaagtcgatactattgaagagcgaccgggcggtgcggcaaacgtggcgatgaacattgcctcgctg-
ggcgcaacggcgcgtctggttggc
ctgactggcattgatgatgcggcgcgcgcgctgagcaaagcgctggcggatgttaatgttaaatgtgacttcg-
tttctgttccgactcaccccaccatca
ctaagctgcgcgtgctgtcgcgtaaccagcaactgattcgc 181
atgaccctgaatatgatgatggatgccagccgttctgtaataataaccggacaattcggactgattaaaa-
aagcgccctcgcggcgctttttttatattctc
gactccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatg-
gaagctcgctgttttaacacgcgtttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattct-
caacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactca-
cttcacgccccgaagggagaagctgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcacaatga 182
accggataagagagaaaagtgtcgacgtcggtccggttgatattgaccggcgcatccgccagctcgccca-
gtttttggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagat-
cccgcggggaaaatgcggtgaacat
gtcagctattgcgaagactgtgccagttttgctcacgggcaaaagctgcaccagaatgggtattaatgcacca-
gcctggcgctttttttcgcggcacgtc
ccctcgctaatgcccgtctggcgcggctttgacgctgataaggcgctgaataccgatctggatcaaggttttg-
tcgggttatcgtccaaaaggtgcactc
tttgcatggttataagtgcctgacatggtgtccgggcgaacgtcgccaggtgccacaaattgtcagaactacg-
acacgactaaccgaccgcaggagt
gtgcgatgaccctgaatatgatgatggatgccagccgttctgtaataatcaccggacaattcggactgattaa-
aaaagcgccctcgcggcgctttttttat
attctcgactccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaat-
ccgatggaagctcgctgttttaacacgcgtt
ttttaaccttttattgaaagtcgggcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaat-
attctcaacctaaaaaagtttgtgtaatactt
gtaacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgca-
actcacttcacgccccgaagggggaa
gctgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcg-
ggtaataccgtctggcgcttcgattta
tcccagcagttcaccgcgatgcagcggataagcgtggttctcagccggccgaccgaggttgaacagacactcc-
agcaggtgctgtgcgtattgcaca
atgacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgactattgaagcgtt-
gcaggaagccgatcagcagttgatccc
cggcagctcgcaaattcgctaccgtccgggtgaagggctggtcgggacggtgctttcgcaggggcaatcgtta-
gtgctggcgcgtgtggctgacgat
cagcgctttcttgaccgcctgggactgtatgattacaacctgccgtttatcgccgtgccgctgatagggccgg-
atgcgcagacttttggcgtgctgacg
gcgcaaccgatggcgcgttacgaagagcggttacccgcctgcacccgctttctggaaacggtc 183
tccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcctgctcgatgaactgc-
tcgacccgaacacgctctatcaaccga
cggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaagagcagca-
actggaggcgctacggcagttta
agcaggcgcagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagtgagcgatca-
cttaacctggctggcggaagcga
ttatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcatctgcacga-
tcgcgaagggcgcggtttcgccg
tggtcggttacggcaaacttggcggctgggaattaggttacagctccgatctggatctggtgttcctgcacga-
ctgccccatggatgtgatgaccgatg
gcgagcgtgaaatcgatggcccccagttctatttgcgcctcgcgcagcgcgtgatgcacctgttcagcacgcg-
cacgtcgtccggcattctttatgaag
tcgatgcgcgtttgcgcccgtccggcgggccggaatgctggtgaccactgggaagcgttcgccgattatcaaa-
aaaatgaagcctggacatggg
agcatcaggcgctggcgcgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcg-
ccgcgatatcctgatgacctcccg
cgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttggtaacaagcac-
aaagaccgtttcgatctgaaagc
cgatgaaggcggtatcaccgatattgagtttatcgctcagtatctgggctgcgctttgcccatgagaagccga-
aactgacgcgctggtcggataatgtg
cgcatcctcgaagggctggcgcaaaacggcatcatggatgagcagcaagcgcaggcattgacgctggcgtaca-
ccacgttgcgtgatgagctgca
ccacctggcgctgcaagagctgccaggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttatcaaa-
accagctgggacaagtggctggtg gaaccgtgcgccccggcgtaa 184
taaagcgagcgctcacttacgtgatctgttgacgcagtccgaagcgaccattacttcagccgtttcagca-
gatacggcggtgtggagtgcgcaatcag
ccctggcgaaactggtgctcaccgagtggttagtgacgcagggctggcgaaccttccttgatgaaaaagcgca-
ggctaagtttgccgactcctttaaa
cgctttgctgacgttcatctgtcactcagcgccgccgagctgtaaaaagcctttgcccagccgctgggcgaca-
gctattgcgaccagttaccgcggc
tggcgcgtgatatcgacagcgcgttattgctggccggacattacgatcgcgcgcgcgccgtggagtggctgga-
aaactggcaggggcttcagcacg
ctattgaaacgcgccagagagttgaaatcgaacatttccgtaataccgccattacccaggagccgttctggtt-
gcacagcggaaaacgttacgaaag
gatatttcgcatgtccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcctgctc-
gatgaactgctcgacccgaacacg
ctctatcsaccgacggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagagg-
atgaagagcagcaactggaggc
gctacggcagtttaagcaggcccagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatg-
aaagtgagcgatcacttaacctgg
ctggcggaagcgattatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccga-
cgcatctgcacgatcgcgaagg
gcgcggtttcgccgtggtcggttacggcaaacttggcggctgggaattaggttacagctccgataggatctgg-
tgttcctgcacgactgccccatgga
tgtgatgaccgatggccagcctgaaatcgatggccgccagttctatttgcgcctcgcgcagcgcgtgatgcac-
ctgttcagcacgcgcacgtcgtccg
gcattctttatgaagtcgatgcgcgtttgcgcccgtccggcgcggccggaatgctggtgaccactgcggaagc-
gttcgccgattatcaaaaaaatgaa
gcctggacatgggagcatcagccgtagcgcgtgcgcgcgtggtgtaccgcgatccgcaactgaccgccgaatt-
tgacgccattcgccgcagatatc
ctgatgacctcccgcgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatc-
ttggtaacaagcacaaagaccgt
ttcgatctgaaagccgatgaaggcggtatcaccgatattgagtttatcgctcagtatctggtgctgcgctttg-
cccatgagaagccgaaactgacgcgct
ggtcggataatgtgcgcatcctcgaagggctggcgcaaaacggcatggatggatgagcaggaagcgcaggcat-
tgacgctggcgtacaccacgttg
cgtgatgagctgcaccacctggcgctgcaagagctgccaggacatgtggcgctctcctgttttgtcgccgagc-
gtgcgatatcaaaaccagctggga
caagtggctggtggaaccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcagga-
gacaggaatgaaagttacgctgcca
gagttcaatcaagccggtgtcatggtggtgggtgatgtgatgctggatcgctactatacggcccaaccagocg-
catttctccggaagcgccagcc
ggttgttaaagtcgatactattgaagagcgaccgggcggtgcggcaaacgtggcgatgaacattgcctcgctg-
ggcgcaacggcgcgtctggttggc
ctgactggcattgatgatgcggcgcgcgcgctgagcaaagcgctggcggagttaatgttaaatgtgacttcgt-
ttctgttccgactcaccccaccatca
ctaagctgcgcgtgctgtcgcgtaaccagcaactgattcgc 185
atgaccctgaatatgatgatggatgccagccgttctgaataataaccggacaattcggactgattaaaaa-
agcgccctcgcggcgctttttttatattctc
gactccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatg-
gaagacgctgattttaacacgcgttttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttattgtggattcccatcaaaaaaatattc-
tcaacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactca-
cttcacgccccgaagggggaagctgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga 186
accggataagagagaaaagtgtcgacgtcggtccggttgatattgaccggcgcatccgccagctcgccca-
gtttttggtggatctgtttggcgattttgc
gggtcttgccggtgtcgctgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagat-
cccgcggggaaaatgcggtgaacat
gtcagctattgcgaagagtgtgccagttttgctcacgggcaaaagctgcaccagaatgggtattaatgcacca-
gcctggcgctttttttcgcggcacgtc
ccctcgctaatgcccgtctggcgcggctttgacgctgataaggcgctgaataccgatctggatcaaggttttg-
tcgggttatcgtccaaaaggtgcactc
tttgcatggttataagtgcctgacatggtgtccgggcgaacgtcgccaggtggcacaaattgtcagaactacg-
acacgactaaccgaccgcaggagt
gtgcgatgaccctgaatatgatgatggatgccagccgttctgtaataataaccggacaattcggactgattaa-
aaaagcgccctcgcggcgctttttat
attctcgactccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaat-
ccgatggaagctcgctgtttaacacgcgtt
ttttaaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcagaaaaa-
tattctcaacctaaaaaagtttgtgtaatactt
gtaacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgca-
actcacttcacgccccgaagggggaa
gctgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcg-
ggtaataccgtctggcgcttcgattta
tcccagcagtcaccgcgatgcagcggataagcgtggttctcagccgggcgaccgaggttgaacagacactcca-
gcaggtgctgtgcgtattgcaca
atgacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgactattgaagcgtt-
gcaggaagccgatcagcagttgatccc
cggcagctcgcaaattcgctaccgtccgggtgaagggctggtcgggacggtgctttcgcaggggcaatcgtta-
gtgaggcgcgtgtggctgacgat
cagcgctttcttgaccgcctgggactgtatgattacaacctgccgtttatcgccgtgccgctgatagggccgg-
atgcgcagacttttggcgtgctgacg
gcgcaaccgatggcgcgttacgaagagcggttacccgcctgcacccgctttctggaaacggtc 187
tccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcctgctcgatgaactgc-
tcgacccgaacacgctctatcaaccga
cggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaagagcagca-
actggaggcgctacggcagttta
agcaggcgcagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagtgagcgatca-
cttaacctggctggcggaagcga
ttatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcatctgcacga-
tcgcgaagggcgcggtttcgccg
tggtcggttacggcaaacttggcggctgggaattaggttacagctccgatctggatctggtgttcctgcacga-
ctgccccatggatggatgaccgatg
gcgagcgtgaaatcgatggcccccagttctatttgcgcctcgcgcagcgcgtgatgcacctgttcagcacgcg-
cacgtcgtccggcattctttatgaag
tcgatgcgcgtttgcgcccgtccggcgcggccggaatgctggtgaccactgcggaagcgttcgccgattatca-
aaaaaatgaagcctggacatggg
agcatcaggcgctggcgcgtcgcggtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgcc-
gcgatatcctgatgacctcccg
cgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttggtaacaagcac-
aaagaccgtttcgatctgaaagc
cgatgaaggcggtatcaccgatattgagtttatcgctcagtatctggtgctgcgctttgcccatgagaagccg-
aaactgacgcgctggtcggataatgtg
cgcatcctcgaagggctggcgcaaaacggcatcatggatgagcaggaagcgcaggcattgacgctggcgtaca-
ccacgttgcgtgatgagctgca
ccacctggcgctgcaagagagccaggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttatcaaaa-
ccagctgggacaagtggctggtg gaaccgtgcgccccggcgtaa 188
taaagcgagcgctcacttacgtgatctgttgacgcagtccgaagcgaccattacttcagccgtttcagca-
gatacggcggtgtggagtgcgcaatcag
ccctggcgaaactggtgctcaccgagtggttagtgacgcagggctggcgaaccttccttgatgaanagcgcag-
gctaagtttgccgactcctttaaa
cgctttgctgacgttcatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccagccgctgggcgaca-
gctatcgcgaccagttaccgcggc
tggcgcgtgatatcgacagcgcgttattgctggccggacattacgatcgcgcgcgcgccgtggagtggctgga-
aaactggcaggggcttcagcacg
ctattgaaacgcgccagagagttgaaatcgaacatttccgtaataccgccattacccaggagccgttctggtt-
gcacagcggaaaacgttaacgaaag
gatatttcgcatgtccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcctgctc-
gatgaactgctcgacccgaacacg
ctctatcaaccgacggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagagg-
atgaagagcagcaactggaggc
gctacggcagtttaagcaggcccagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatg-
aaagtgagcgatcacttaacctgg
ctggcggaagcgattatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccga-
cgcatctgcacgatcgcgaagg
gcgcggtttcgccgtggtcggttacggaaacttggcggctgggaattaggttacagctccgatctggatctgg-
tgttcctgcacgactgccccatgga
tgtgatgaccgatggcgagcgtgaaatcgatggccgccagttctatttgcgcctcgcgcagcgcgtgatgcac-
ctgttcagcacgcgcacgtcgtccg
gcattctttatgaagtcgatgcgcgtttgcgcccgtccggcgcgccggaatgctggtgaccactgcggaagcg-
ttcgccgattatcaaaaaaatgaa
gcctggacatgggagcatcaggctggcgcgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaattt-
gacgccattcgccgcgatatc
ctgatgacctcccgcgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatc-
ttggtaacaagcacaaagaccgt
ttcgatctgaaagccgatgaaggcggtatcaccgatattgagtttatcgctcagtatctggtgctgcgctttg-
cccatgagaagccgaaactgacgcgct
ggtcggataagtgcgcatcctcgaagggctggcgcaaaacggcatcatggatgagcacgaagcgcaggcattg-
acgctggcgacaccacgttg
cgtgatgagagcaccacctggcgctgcaagagctgccaggacatgtggcgactcctgttttgtcgccgagcgg-
cgcttatcaaaaccagctggga
caagtggctggtggaaccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcagga-
gacaggaatgaaagttacgctgcca
gagttcaatcaagccggtgtcatggtggtgggtgatgtgatgctggatcgctactggtacggcccaaccagcc-
gcatttctccggaagcgccagttcc
ggttgttaaagtcgatactattgaagagcgaccgggcggtgcggcaaacgtggcgatgaacattgcctcgctg-
ggcgcaacggcgcgtctggttggc
ctgactggcattgatgatgcggcgcgcgcgctgagcaaagcgctggcggatgttaatgttaaatgtgacttcg-
tttctgttccgactcaccccaccatca
ctaagctgcgcgtgctgtcgcgtaaccagcaactgattcgc 189
atgaccctgaatatgatgatggatgccagccgcgtcaggttgaacgtaaaaaagtcggtctgcgcaaagc-
acgtcgtcgtccgcagttctccaaacgtt
aattggtttctgcttcggcagaacgattggcgaaaaaacccggtgcgaaccgggtttttttatggataaagat-
cgtgttatccacagcaatccattgattat
ctcttctttttcagcatttccagaatcccctcaccacaaagcccgcaaaatctggtaaactatcatccaattt-
tctgcccaaatggctgggattgttcattttt
gtttgcatacaacgagagtgacagtacgcgcgggtagttaactcaacatctgaccggtcgataactcacttca-
cgccccgaagggggaagctgcctg
accctacgattcccgctatttcattcactgaccggaggttcaaaatga 190
accggataagagagaaaagtgtcgacgtcggtccggttgatattgaccggcgcatccgccagctcgccca-
gtttttggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagat-
cccgcggggaaaatgcgggaacat
gtcagctattgcgaagagtgtgccagttttgctcacgggcaaaagctgcaccagaatgggtattaatgcacca-
gcctggcgctttttttcgcggcacgtc
ccctcgctaatgcccgtctggcgcggctttgacgctgataaggcgctgaataccgatctggatcaaggttttg-
tcgggttatcgtccaaaaggtgcactc
tttgcatggttataagtgcctgacatggtgtccgggcgaacgtcgccaggtggcacaaattgtcagaactacg-
acacgactaaccgaccgcaggagt
gtgcgatgaccctgaatatgatgatggatgccagccgcgtcaggttgaacgtaaaaaagtcggtctgcgcaaa-
gcacgtcgtcgtccgcagttctcca
aacgttaattggtttctgcttcggcagaacgattggcgaaaaaacccggtgcgaaccgggtttttttatggat-
aaagatcgtgttatccacagcaatccatt
gattatctcttctttttcagcatttccagaatcccctcaccacaaagcccgcaaaatctggtaaactatcatc-
caattttctgcccaaatggctgggattgttc
attttttgtttgccttacaacgagagtgacagtacgcgcgggtagttaactcaacatctgaccggcgataact-
cacttcacgccccgaagggggaagct
gcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggt-
aataccgtctggcgcttcgatttatcc
cagcagttcaccgcgatgcagcggataagcgtggttctcagccgggcgaccgaggttgaacagacactccagc-
aggtgctgtgcgtattgcacaat
gacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgactattgaagcgttgc-
aggaagccgatcagcagttgatcccc
ggcagctcgcaaattcgctaccgtccgggtgaagggctggtcgggacggtgctttcgcaggggcaatcgttag-
tgctggcgcgtgtggctgacgatc
agcgctttcttgaccgcctgggactgtatgattacaacctgccgtttatcgccgtgccgctgatagggccgga-
tgcgcagacttttggcgtgctgacgg
cgcaaccgatggcgcgttacgaagagcggttacccgcctgcacccgctttctggaaacggt
191
atggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacc-
cgctgctgctggatgagctgctggat
cccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgc-
cggaagaggatgaagagcagca
gctggaggcgttgcgccagtttaagcaggcgcagcagctgcatatcgcggcggcggatatcgctggtaccctg-
ccggtgatgaaggtcagcgatca
cttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctac-
ggccagccgacccacctgcacg
atcgccagggtcgcggcttcgccgtcgtcggctacggtaagcttggcggctgggagctgggctacagctccga-
tctcgatctggtgttcctccatgac
tgcccggcggaggtgatgaccgacggcgagcgggagattgacgtccgtcagttctacctgcggctggcccagc-
ggatcatgcacctgttgcagcac
ccgcacctcgtccggtattctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctgtgg-
tcaccaccgccgacgcgtttgctgact
atcagcagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatggcgacccggc-
gctgcaggcgcgctttgacgc
cattcgtcgcgatatcctgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgag-
aagatgcgcgcccaccttggca
acaaacatcccgatcgttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagta-
tctggtcctacgctatgccagtgacaag
ccgaagctgacccgctggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgagg-
aggaggcgcgcgccttaacgcat
gcgtacaccaccttgcgtgatgcgctccatcacctggccctgcaggaccagccgggacacgtggcgccagagg-
ccttcagccgggagcgtcagca ggtcagcgccagctggcagaagtggctgatggcttaa 192
cgtaaggcgaccacccagctccgcgcgttgctgaacgacgctgaagccgttctgctggccgcggacaccg-
ccgacgaggcgttattccgcaccga
ggtcgtcggcgccaaactggccctgactgaatggctggtccagcgcggctggcgtccgttcctcaacgaggca-
ggagagaaaaaatagccggat
cgttcaaacggtttgccgatattaacctctcgcgggtggcggccgagctgcgcagcgccgtgcagcatctggc-
ggttgaagatgccgccgaccagtt
gccgaagctgcccgcgacatcgacagcgtccagctgctggcgggcgcctatggcgacgccgtcgcgccgtggc-
tggagaactggcaggagctt
caccgtgcaatagcacatgacgatcgcagcgtctttgaatatttccgtcgccaggcgctggctgccgagccgt-
tctggctgcatagtggaaaacgata
atttcaggccagggagcccttatggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgcca-
gccagctggcgcgccacccgctg
ctgctggatgagctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgc-
gccagtacctgctgcgcgtgccg
gaagaggatgaagagcagcagctggaggcgttgcgccagtttaagcaggcgcagcagctgcatatcgcggcgg-
cggatatcgctggtaccctgcc
ggtgatgaaggtcagcgatcacttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgg-
gggcagatggtcgctcgctacgg
ccagccgacccacctgcacgatcgccagggtcgcggcttcgccgtcgtcggctacggtaagcttggcggctgg-
gagctgggctacagctccgatct
cgatctggtgttcaccatgactgcccggcggaggtgatgaccgacggcgagcgggagattgacggccgcagtt-
ctacctgcggctggcccagcg
gatcatgcacctgttcagcacccgcacctcgtccggtattctctacgaagtggacgcccggctgcgtccttct-
ggcgcggcggggatgctggtcacca
ccgccgacgcgtttgctgactatcagcagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccg-
cgtggtctatggcgacccggcg
ctgcaggcgcgctttgacgccattcgtcgcgatatcctgaccaccccccaaggggatgaccctgcagaccgag-
gttcgcgagatgcgcgagaa
gatgcgcgcccaccttggcaacaaacatcccgatcgttttgatatcaaagccgatgccggcgggatcaccgat-
attgaatttattactcagtatctggtcc
tacgctatgccagtgacaagccgaagctgacccgctggtctgacagcgtgcgtattcttgagctgctggcgca-
gaqacgacatcatgcacgaggagga
ggcgcgcgccttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggccctgcaggagcagccg-
ggacacgtggcgccagaggcct
tcagccgggagcgtcagcaggtcagcgccagctggcagaagtggctgatggcttaactataaaatcgggtgtg-
ctatatcgcgcgcaaagtttgcgt
ctcgcaggagagagtcatgaaagtaacgctgccggagtttgaacgtgcaggagtgttggtggtgggtgatgtg-
atgctggaccgctactggtacggc
cccaccagtcgtatttcccggaagccccggtgccggtggtgaaggtggaaaatatcgaagaacgtcctggcgg-
cgcggcaaacgtagcgatgaa
catcgcctccctgggggcaacgtcgcgcctggtgggattgaccgggattgatgacgctgcccgcgctgagcca-
ggcgctggccaatgtgaatgt
gaagtgcgacttcgtctccgtcccgactcacccgaccatcaccaagctgcgggtgctgtcgcgcaatcagcag-
ctgatccgcctcgactttgaagag
ggcttctccggcgtggatccgcagccgatgcatgagcgcattcagcaggcgctgggagccattggcgcactgg
193
atgaccctgaatatgatgctagaagcgtcaggtaccggtcatgattcaccgtgcgattctcggttccctg-
gagcgcttcattggcatcctgaccgaagag
ttcgctggcttcttcccaacctggattgcaccagtgcaggtagtggtcatgaatattaccgattctcaggctg-
aatacgttaacgaattgacgcgtaaacta
caaaatgcgggcattcgtgtaaaagcagacttgagaaatgagaagattggctttaaaatccgcgagcacactt-
tacgtcgtgtcccgtatatgttggtct
gtggcgacaaagaagtcgaagccggcaaagtggccgtgcgcacccgtcgcgggaaagacctcggcagcatgga-
cgtaagtgaagtgattgagaa
gctgcaacaagagattcgcagccgcagtcttcaacaactggaggaataaggtattaaaggcggaaaacgagtt-
caaacggcacgtccgaatcgtatc
aatggcgagattcgcgccctggaagttcgcgccattgagctggcttcccgaccgcagggcggcacagcctgac-
cctgcgtttcccgctgtttaacac cctgaccggaggtgaagcatga 194
ggccgtcgcccagcgtcggcgtccccaacagcagggcgggtaggccagcaggtccgccagcgtggcgcgg-
ttaatattgaccggggcggcgg
cggcctcccccagctgcttgtggatcattttcgcgatcttgcgggtttttaccggtatcggtaccaaagaaaa-
tgccaatgttcgccatagtacgctcctgt
cggnatggtgttgaaaaaaggaatgacgacagaggtattgcgaaggctgtgccaggttgccctgcaccgcgac-
ggcccatccctgccccatcagga
tcgcttcgcatcacgatgccgcgcgccaaaggcgcacccggcggggcgaaaggtaaaaatccgtgaattttcc-
ccctgtcggatcaatgtttcgcgtg
gtcgttccgataagggcgcacactttgcatggttatccgggttcggcttaccccgccgagttttgcgcacggt-
gtcggacaatttgtcataactgcgaca
caggagtttgcgatgaccctgaatatgatgctagaagcgcaggtaccggtcatgattcaccgtgcgattctcg-
gttccctggagcgcttcattggcatc
ctgaccgaagagttcgctggcttcttcccaacctggattgcaccagtgcaggtagtggtcatgaatattaccg-
attctcaggctgaatacgttaacgaatt
gacgcgtaaactacaaaatgcgggcattcgtgtaaaagcagacttgagaaatcagaagattggctttaaaatc-
cgcgagcacactttacgtcgtgtccc
gtatatgttggtctgtgccgacaaagaagtcgaagccggcnaagtggccgtgcgcacccgtcgcgggaaagac-
ctcggcagcatggacgtaagtg
aagtgattgagaagctgcaaccagagattcgcagccgcagtcttcaacaactggaggaataaggtattaaagg-
cggaaaacgagttcaaacggcac
gtccgaatcgtatcaatggcgagattcgcgccctggaagttcgcgccattgagctggcttcccgaccgcaggg-
cggcacctgcctgaccctgcgtttc
ccgctgtttaacaccctgaccggaggtgaagcatgatccctgaatccgacccggacaccaccgtcagacgctt-
cgacctctctcagcagttcaccgcc
atgcagcggataaggtggtgagagccgggccaccgaggccagcaaaacgctgcaggaggtgctcagcgtatta-
cacaacgatgcctttatgcag
cacgggatgatctgcctctacgacagcgagcaggagatcctcagtatcgaagcgctgcagcaaaccggccagc-
agcccctccccggcagcacgc
agatccgctatcgccccggcgagggactggtggggaccgtgctggcccaggggcagtcgctggtgctgccccg-
ggtcgccgacgatcagcgtttt
ctcgaccgcctgagcctctacgattacgatctgccgtttatcgccgtaccgttgatggggcccaacgcccggc-
caataggggtgctggcggcccagc
cgatggcgcgccaggaagagcggctgccggcctgcacccgttttctcgaaaccgtc 195
atggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacc-
cgctgctgaggatgagctgctggat
cccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgc-
cggaagaggatgaagagcagca
gctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgatcacttaacctggctt-
gccgaagcgatcctcgacgcggtg
gtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacgatcgccagggtcgcg-
gcttcgccgtcgtcggctacg
gtaagcttggcggctgggagctgggctacagctccgatctcgatctggtgttcctccatgactgcccggcgga-
ggtgatgaccgacggcgagcggg
agattgacggccgtcagttctacctgcggctggcccagcggatcatgcacctgttcagcacccgcacctcgtc-
cggtattctctacgaagtggacgcc
cggctgcgtccttaggcgcggcggggatgctggtcaccaccgccgacgcgtttgctgactatcagcagaacga-
agcctggacgtgggaacatcag
gcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgctttgacgccattcgtcgcgata-
tcctgaccaccccgcgggaggg
gatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggcaacaaacatcccgat-
cgttttgatatcaaagccgatgcc
ggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgccagtgacaagccgaagctga-
cccgctggtctgacancgtgcgtattct
tgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcatgcgtacaccaccttg-
cgtgatgcgctccatcacctggc
cctgcaggagcagccgggacacgtggcgccagaggccttcagccgggagcgtcagcaggtcagcgccagctgg-
cagaagtggctgatggcttaa 196
cgtaaggcgaccacccagctccgcgcgttgctgaacgacgctgaagccgttctgctaccgcggacaccgc-
cgacgaggcgttattccgcaccga
ggtcgtcggcgccaaactggccctgactgaatggctggtccagcgcggctggcgtccgttcctcaacgaggca-
ggagagaaaaaaatagccggat
cgttcaaacggtttgccgatattaacctctcgcgggtggcggccgagctgcgcagcgccgtgcagcatctggc-
ggttgaagatgccgccgaccagtt
gccgaagctgtcccgcgacatcgacagcgtccagctgctggcgggcgcctatggcgacgccgtcgcgccgtgg-
ctggagaactggcaggagctt
caccgtgcaatagcacatgaccatcgcagcgtctttgaatatttccgtcgccaggcgctggctgccgagccgt-
tctggctgcatagtggaaaacgata
atttcaggccagggagcccttatggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgcca-
gccagctggcgcgccacccgctg
ctgctggatgagctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagagcg-
ccagtacctgctgcgcgtgccg
gaagaggatgaagagcaggctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagc-
gatcacttaacctggcttgcc
gaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacc-
tgcacgatcgccagggtcgcg
gcttcgccgtcgtcggctacggtaagcttggcggctgggagctgggctacagctccgatctcgatctggtgtt-
cctccatgactgcccggcggaggtg
atgaccgtcggcgagcgggagattgacggccgtcagttctacctgcggctggcccagcggatcatgcacctgt-
tcagcacccgcacctcgtccggt
attctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctggtcaccaccgccgacgcgt-
ttgagactatcagcagaacgaag
cctggacgtgggattcatcaggcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgct-
ttgacgccattcgtcgcgatatcc
tgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccacct-
tggcaacaaacatcccgatcg
ttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctat-
gccagtgacaagccgaagctgacccgct
ggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgcctt-
aacgcatgcgtacaccaccttgc
gtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggccccagaggccttcagccgggagcg-
tcagcaggtcagcgccagctg
gcagaagtggctgatggcttaactataaaatcgggtgtgctattatcgcgcgcaaagtttgcgtctcgcagga-
gagagtcatgaaagtaacgctgccg
gagtttgaacgtgcaggagtgttggtggtgggtgatgtgatgctggaccgctactggtacggccccaccagtc-
gtatttccccggaagcccoggtgcc
ggtggtgaaggtggtaaatatcgaagaacgtcctggcggcgcggcaaacgtagcgatgaacatcgcctccctg-
ggggcaacgtcgcgcctggtgg
gattgaccgggattgatgacgctgcccgcgcgctgagccaggcgctggccaatgtgaatgtgaagtgcgactt-
cgtctccgtcccgactcacccgac
catcaccaagctgcgggtgctgtcgcgcaatcagcagctgatccgcctcgactttgaagagggcttctccggc-
gtggatccgcagccgatgcatgag cgcattcagcaggcgctgggagccattggcgcactgg 197
atgaccctgaatataagctcgacgccgtcctcgcagtaccattgcaaccgactttacagcaagaagtgat-
tctggcacgcatggaacaaattcttgcca
gtcgggctttatccgatgacgaacgcgcacagcttttatatgagcgcggagtgttgtatgatagtctcggtct-
gagggcattagcgcgaaatgatttttca
caagcgctggcaatccgacccgatatgcctgaagtattcaattacttaggcatttacttaacgcaggcaggca-
attttgatgctgcctatgaagcgtttgat
tctgtacttgagcttgatcgccattgagctggcttcccgaccgcagggcggcacctgcctgaccctgcgtttc-
ccgctgtttaacaccctgaccggaggt gaagcatga 198
cccaacagcagggccgggtaggccagcaggtccgccagcgtggcgcggttaatattgaccggggcggcgg-
cggcctcccccagctgcttgtgga
tcattttcgcgatcttgcgggttttaccggtatcggtaccaaagaaaatgccaatgttcgccatagtacgctc-
ctgtcggaatggtgttgaaaaaaggaat
gacgacagaggtattgcgaaggctgtgccaggttgccctgcaccgcgacggcccatccctgccccatcaggat-
cgcttcgcatcacgatgccgcgc
gccaaaggcgcacccggcggggcgaaaggtaaaaatccgtgaattttccccctgtcggatcaatgatcgcgtg-
gtcgttccgataagggcgcacac
tttgcatggttatccgggttcggcttaccccgccgcgttttgcgcacggtgtcggacaatttgtcataactgc-
gacacaggagtttgcgatgaccctgaat
atgatgctcgacgccgtcctcgcagtaccattgcaaccgactttacagcaagaagtgattctggcacgcatgg-
aacaaattcttgccagtcgggctttat
ccgatgacgaacgcgcacagcattatatgagcgcggagtgttgtatgatagtctcggtctgagggcattagcg-
cgaaatgatttttcacaagcgctggc
aatccgacccgatatgcctgaagtattcaattacttaggcatttacttaacgcaggaggcaattttgatgctg-
cctatgaagcgtttgattctgtacttgag
cttgatcgccattgagaggcttcccgaccgcagggcggcacctgcctgaccctgcgtttcccgctgtttaaca-
ccctgaccggaggtgaagcatgat
ccctgaatccgacccggacaccaccgtcagacgcttcgacctctctcagcagttcaccgccatgcagcggata-
agcgtggtgctgagccgggccac
cgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgatgcctttatgcagcacgggatgatc-
tgcctgtacgacagcgagcagga
gatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgcagatccgctatcgc-
cccggcgagggactggtgggg
accgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgttttctcgaccgcctga-
gcctctacgattacgatctgccgtt
tatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctggcggcccagccgatggcgcgccag-
gaagagcggctgccggcctgc acccgttttctcgaaaccgtc 199
atggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacc-
cgctgctgctggatgagctgctggat
cccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctccgcgtgc-
cggaagaggatgaagagcagca
gctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgatcacttaacctggctt-
gccgaagcgatcctcgacgcggtg
gtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacgatcgccagggtcgcg-
gcttcgccgtcgtcggctacg
gtaagcttggcggctgggagctgggctacagctccgatctcgatctggtgttcctccatgactgcccggcgga-
ggtgatgaccgacggcgagcggg
agattgacggccgtcagttctacctgcggctggcccagcggatcatgcacctgttcagcacccgcacctcgtc-
cggtattctctacgaagtggacgcc
cggctgcgtccttctggcgcggcggggatgctggtcaccaccgccgacgcgtttgctgactatcagcagaacg-
aagcctggacgtgggaacatcag
gcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgctttgacgccattcgtcgcgata-
tcctgaccaccccgcgggaggg
gatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggcaacaaacatcccgat-
cgttttgatatcaaagccgatgcc
ggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgccagtgacaagccgaagctga-
cccgctggtctgacaacgtgcgtattct
tgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcatgcgtacaccaccttg-
cgtgatgcgctccatcacctggc
cctgcaggagcagccgggacacgtggcgccagaggccttcagccgggagcgtcagcaggtcagcgccagctgg-
cagaagtggctgatggcttaa 200
cgtaaggcgaccacccagctccgcgcgttgctgaacgacgctgaagccgttctgctggccgcggacaccg-
ccgacgaggcgttattccgcaccga
ggtcgtcggcgccaaactggccctgactgaatggctggtccagcgcggctggcgtccgttcctcaacgaggca-
ggagagaaaaaaatagccggat
cgttcaaacggtttgccgatattaacctctcgcgggtggcggccgagctgcgcagcgccgtgcagcatctggc-
ggttgaagatgccgccgaccagtt
gccgaagctgtcccgcgacatcgacagcgtccagctgctggcgggcgcctatggcgacgccgtcgcgccgtgg-
ctggagaactggcaggagctt
caccgtgcaatagcacatgacgatcgcagcgtctttgaatatttccgtcgccaggcgctggctgccgagccgt-
tctggctgcatagtgggaaaacgata
atttcaggccagggagcccttatggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgcca-
gccagctggcgcgccacccgctg
ctgctggatgagctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgc-
gccagtacctgctgcgcgtgccg
gaagaggatgaagagcagcagctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtca-
gcgacacttaacctggcttgcc
gaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacc-
tgcacgatcgccagggtcgcg
gcttcgccgtcgtcggctacggtaagcttggcggctgagagctgggctacagctccgatctcgatctggtgtt-
cctccatgactgcccggcggaggtg
atgaccgacggcgagcgggagattgacggccgtcagttctacctccggctggcccagcggatcatgcacctgt-
tcagcacccgcacctcgtccggt
attctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctggtcaccaccgccgacgcgt-
ttgctgactatcagcagaacgaag
cctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgctt-
tgacgccattcgtcgcgatatcc
tgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccacct-
tggcaacaaacatcccgatcg
ttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctat-
gccagtgacaagccgaagctgacccgct
ggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgcctt-
aacgcatgcgtacaccaccttgc
gtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagaggccttcagccgggagcg-
tcagcaggtcagcgccagctg
gcagaagtggctgatggcttaactataaaatcgggtgtgctattatcgcgcgcaaagtttgcgtctcgcagga-
gagagtcatgaaactaacgctgccg
gagtttgaacgtgcaggagtgttggtggtgggtgatgtgatgctggaccgctactggtacggccccaccagtc-
gtatttccccggaagccccggtgcc
ggtggtgaaggtggaaaatatcgaagtacgtcctggcggcgcggcaaacgtagcgatgaacatcgcctccctg-
ggggcaacgtcgcgcctggtgg
gattgaccgggattgatgacgctgcccgcgcgctgagccaggcgctggccaatgtgaatgtgaagtgcgactt-
cgtctccgtcccgactcacccgac
catcaccaagctgcgggtgctgtcgcgcaatcagcagctgatccgcctcgactttgaagagggcttctccggc-
gtggatccgcagccgatgcatgag cgcattcagcaggcgctgggagccattggcgcactgg 201
atgaccctgaatatgatgctcgagctaaagttctcggctaatcgctgattacatttgacgcaatgcgcaa-
taaaagggcatcatttgatgccctttttgcac
gctttcataccagaacctggctcatcagtgattttttttgtcataatcattgctgagacaggctctgaagagg-
gcgtttatacaccaaaccattcgagcggt
agcgcgacggcaagtcagcgttctcctttgcaatagcagggaagaggcgccagaaccgccagcgttgaagcag-
tttgaacgcgttcagtgtataatc
cgaaacttaatttcggtttggagccattgagctggcttcccgaccgcagggcggcacctgcctgaccctgcgt-
ttcccgctgtttaacaccctgaccgg aggtgaagcatga 202
ggccgtcgcccagcgtcggcgtccccaacagcagggccgggtaggccagcaggtccgccagcgtggcgcg-
gttaatattgaccggggcggcgg
cggcctcccccagctgcttgtggatcattttcgcgatcttgcgggttttaccggtatcggtaccaaagaaaat-
gccaatgttcgccatagtacgctcctgt
cggaatggtgttgaaaaaaggaatgacgacagtggtattgcgaaggctgtgccaggttgccctgcaccgcgac-
ggcccatccctgccccatcagga
tcgcttcgcatcacgatgccgcgcgccaaaggcgcacccggcggggcgaaaggtaaaaatccgtgaattttcc-
ccctgtcggatcaatgtttcgcgtg
gtcgttccgataagggcgcacactttgcatggttatccgggttcggcttaccccgccgcgttttgcgcacggt-
gtcggacaatttgtcataactgcgaca
caggagtttgcgatgaccctgaatatgatgctcgagctaaagttctcggctaatcgctgataacatttgacgc-
aatgcgcaataaaagggcatcatttgat
gcccttttgcacgctttcataccagaacctggctcatcagtgattttttttgtcataatcattgctgagacag-
gctctgaagagggcgtttatacaccaaac
cattcgagcggtagcgcgacggcaagtcagcgttctcctttgcaatagcagggaagaggcgccagaaccgcca-
gcgttgaagcagtttgaacgcgt
tcagtgtataatccgaaacttaatttcggtttggagccattgagctggcttcccgaccgcagggcggcacctg-
cctgaccctgcgtttcccgctgtttaac
accctgaccggaggtgtagcatgatccctgaatccgacccagacaccaccgtcagacgcttcgacctctctca-
gcagttcaccgccatgcagcggat
aagcgtggtgctgagccgggccaccgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgat-
gcctttatgcagcacgggatgat
ctgcctgtacgacagcgagcaggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctcccc-
ggcagcacgcagatccgctatc
gccccggcgagggactggtggggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacga-
tcagcgttttctccaccgcctg
agcctctacgattacgatctgccgtttatcgccgtaccgttgatggggcccaacgcccggccaataggggtgc-
tggcggcccagccgatggcgcgc
caggaagagcggctgccggcctgcacccgttttctcgaaaccgtc 203
atgaccctgaatatgatgctcgagcccgctgaccgaccagaacttccaccttggactcggctataccctt-
ggcgtgacggcgcgcgataactgggact
acatccccattccggtgatcttaccattggcgtcaataggttacggtccggcgactttccagatgacctatat-
tcccggcacctacaataacggtaacgttt
acttcgcctgggctcgtatacagttttaattcgctaagtcttagcaataaatgagataagcggtgtgtcttgt-
ggaaaaacaaggactaaagcgttaccca
ctaaaaaagatagcgacttttatcactttttagcaaagttgcactggacaaaaggtaccacaattggtgtact-
gatactcgacacagcattagtgtcgatttt
tcatataaaggtaattttggccattgagctggcttcccgaccgcagggcggcacctgcctgaccctgcgtttc-
ccgctgtttaacaccctgaccggaggt gaagcatga 204
ggccgtcgcccagcgtcggcgtccccaacagcagggccgggtaggccagcaggtccgccagcgtggcgcg-
cttaatattgaccggggcggcgg
cggcctcccccagctgcttgtggatcattttcgcgatcttgcgggttttaccggtatcggtaccaaagaaaat-
gccaatgttcgccatagtacgctcctgt
cggaatggtgttgaaaaaaggaatgacgacagaggattgcgaaggctgtgccaggttgccctgcaccgcgacg-
gcccatccctgccccatcagga
tcgcttcgcatcacgatgccgcgcgccaaaggcgcacccggcggggcgaaaggtaaaaatccgtgaattttcc-
ccctgtcggatcaatgtttcgcgtg
gtcgttccgataagggcgcacactttgcatggttatccgggttcggcttaccccgccgcgttttgcgcacggt-
gtcggacaatttgtcataactgcgaca
caggagtttgcgatgaccctgaatatgatgctcgagcccgctgaccgaccagaacttccaccttggactcggc-
tatacccttggcgtgacggcgcgcg
ataactgggactacatccccattccggtgatcttaccattggcgtcaataggttacggtccggcgactttcca-
gatgacctatattcccggcacctacaat
aacggtaacgtttacttcgcctgggctcgtatacagttttaattcgctaagtcttagcaataaatgagataag-
cggtgtgtcttgtggaaaaacaaggacta
aagcgttacccactaaaaaagatagcgacttttatcactttttagcaaagttgcactggacaaaaggtaccac-
aattggtgtactgatactcgacacagca
ttagtgtcgatttttcatataaaggtaattttggccattgagctggcttcccgaccgcagggcggcacctgcc-
tgaccctgcgtttcccgctgtttaacacc
ctgaccggaggtgaagcatgatccctgaatccgacccggacaccaccgtcagacgatcgacctctctcagcag-
ttcaccgccatgcagcggataag
cgtggtgctgagccgggccaccgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgatgcc-
tttatgcagcacgggatgatctg
cctgtacgacagcgagcaggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggc-
agcacgcagatccgctatcgcc
ccggcgagggactggtggggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatca-
gcgttttctcgaccgcctgagc
ctctacgattacgatctgccgtttatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctgg-
cggcccagccgatggcgcgccag gaagagcggctgccggcctgcacccgttttctcgaaaccgtc
205
atggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacc-
cgctgctgctggatgagctgctggat
cccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgc-
cggaagaggatgaagagcagca
gctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgatcacttaacctggctt-
gccgaagcgatcctcgacgcggtg
gtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacgatcgccagggtcgcg-
ccttcgccgtcgtcggctacg
gtaagcttggcggctgggagagggctacagctccgatctcgatctggtgttcctccatgactgcccggcggag-
gtgatgaccgacggcgagggg
agattgacggccgtcagttctacctgcggctggcccagcggatcatgcacctgttcagcacccgcacctcgtc-
cggtattctctacgaagtggacgcc
cggctgcgtccttctggcgcggcggggatgctggtcaccaccgccgacgcgtttgctgactatcagcagaacg-
aagcctggacgtgggaacatcag
gcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgctttgacgccattcgtcgcgata-
tcctgaccaccccgcgggaggg
gatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggcaacaaacatcccgat-
cgttttgatatcaaagccgatgcc
ggcgggatcaccgatattgaatttattactcagatctggtcctacgctatgccagtgacaagccgaagctgac-
ccgctggtctgacaacgtgcgtattct
tgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcatgcgtacaccaccttg-
cgtgatgcgctccatcacctggc
cctgcaggagcagccgcgacacgtggcgccagaggccttcagccgggagcgtcagcaggtcagcgccagctgg-
cagaagtggctgatggcttaa 206
cgtaaggcgaccacccagctccgcgcgttgctgaacgacgctgaagccgttctgctggccgcggacaccg-
ccgacgaggcgttattccgcaccga
ggtcgtcggcgccaaactggccctgactgaatggctggtccagcgcggctggcgtccgttcctcaacgaggca-
ggagagaaaaaaatagccggat
cgttcaacggtttgccgatattaacctctcgcaggtggcggccgagctgcgcagcgccgtgcagcatctggcg-
gttgaagatgccgccgaccagtt
gccgaagctgtcccgcgacatcgacagcgtccagctgctggcgggcgcctatggcgacgccgtcgcgccgtgg-
ctggagaactggcaggagctt
caccgtgcaatagcacatgacgatcgcagcgtctttgaatatttccgtcgccaggcgctggctgccgagccgt-
tctggctgcatagggaaaacgata
atttcaggccagggagcccttatggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgcca-
gccagctggcgcgccacccgctg
ctgctggatgagctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgc-
gccagacctgctgcgcgtgccg
gaagaggatgaagagcagcagctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtca-
gcgatcacttaacctggcttgcc
gaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacc-
tgcacgatcgccagggtcgcg
gcttcgccgtcgtcggctacggtaagcttggcggctgggagctgggctacagctccgatctcgatctggtgtt-
cctccatgactgcccggcggaggtg
atgaccgacggcgaggggagattgacggccgcagttctacctgcggctggcccagcggatcatgcacctgttc-
agcacccgcacctcgccggt
attctctacgaagtggacgcccggctgcgtccttctggcgcggcgcggatgctggtcaccaccgccgacgcgt-
ttgctgactatcagcagaacgaag
cctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctattgcgacccggcgctgcaggcgcgctt-
tgacgccattcgtcgcgatatcc
tgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccacct-
tggcaacaaacatcccgatcg
ttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctat-
gccagtgacaagccgaagctgacccgct
ggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgcctt-
aacgcatgcgtacaccaccttgc
gtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagaggccttcagccgagagcg-
tcagcaggtcagcgccagctg
gcagaagtggctgatggcttaactataaaatcgggtgtgctattatcgcgcgcaaagtttgcgtctcgcagga-
gagagtcatgaaagtaacgctgccg
gagtttgaacgtgcaggagttggtggtgggtgatgtgatgctggaccgctactggtacggccccaccagtcgt-
atttccccggaagccccggtgcc
ggtggtgaaggtggaaaatatcgaagaacgtcctggcggcgcggcaaacgtagcgatgaacatcgcctccctg-
ggggcaacgtcgcgcctggtgg
gattgaccgggattgatgacgctgcccgcgcgctgagccaggcgctggccaatgtgaatgtgaagtgcgactt-
cgtctccgtcccgactcacccgac
catcaccaagctgcgggtgctgtcgcgcaatcagcagctgatccgcctcgactttgaagagggcttctccggc-
gtggatccgcagccgatgcatgag cgcattcagcaggcgctgggagccattggcgcactgg 207
atgaccctgaatatgatgctagaagcgtcaggtaccggtcatgattcaccgtgcgattctcggttccctg-
gagcgcttcattggcatcctgaccgaagag
ttcgctggcttcttcccaacctggattgcaccagtgcaggtagtggtcatgaatattaccgattctcaggctg-
aatacgttaccgaangacgcgtaaacta
caaaatgcgggcattcgtgtaaaagcagacttgagaaatgagaagattggctttaaaatccgcgagcacactt-
tacgtcgtgtcccgtatatgtggtct
gtggcgacaaagaagtcgaagccggcaaagtggccgtgcgcacccgtcgcgggaaagacctcggcagcatgga-
cgtaagtgaagtgattgagaa
gctgcaacaagagattcgcagccgcagtcttcaacaactggaggaataaggtattaaaggcggaaaacgagtt-
caaacggcacgtccgaatcgtatc
aatgccgagattcgcgccctcgaagttcgcgccattgagctggcttcccgaccgcagggcgccacctgcctga-
ccctgcgtttcccgctgtttaacac cctgaccggaggtgaagcatga 208
ggccgtcgcccagcgtcggcgtccccaacagcagggccgggtaggccagcaggccgccagcgtggcgcgg-
ttaatattgaccggggcggcgg
cggcctcccccagagcttgtggatcattttcgcgatcttgcgggttttaccggtatcgaaccaattgaaaatg-
ccaatgttcgccatagtacgctcctgt
cggaatggtgttgaaaaaaggaatgacgacagaggtattgcgaaggctggccaggttgccctgcaccgcgacg-
gcccatccctgccccatcagga
tcgcttcgcatcacgatgccgcgcgccaaaggcgcacccggcggggcgaaaggtaaaaatccgtgaattttcc-
ccctgtcggatcaatgtttcgcgtg
gtcgttccgataagggcgcacactttgcatggttatccgggttcggcttaccccgccgcgttttgcgcacggt-
gtcggacaatttgtcataactgcgaca
caggagtttgcgatgaccctgaatatgatgctagaagcgtcaggtaccggtcatgattcaccgtgcgattctc-
ggttccctggagcgatcattggcatc
ctgaccgaagagttcgctggcttcttcccaacctggattgcaccagtgcaggtagtggtcatgaatattaccg-
attctcaggctgaatacgttaacgaatt
gacgcgtaaactacaaaatgcggcattcgtgtaaaagcagacttgagaaatgagaagattggctttaaaatcc-
gcgagcacactttacgtcgtgtccc
gtatatgttggtctgtggcgacaaagaagtcgaagccggcaaagtggccgtgcgcacccgtcgcgggaaagac-
ctcggcagcatggacgtaagtg
aagtgattgagaagctgcaacaagagattcgcagccgcagtcttcaacaactggaggaataaggtattaaagg-
cggaaancgagttcaaacggcac
gtccgaatcgtatcaatggcgagattcgcgccctggaagttcgcgccattgagctggcttcccgaccgcaggg-
cggcacctgcctgaccctgcgtttc
ccgctgtttaacaccctgaccggaggtgaagcatgatccctgaatccgacccggacaccaccgtcagacgctt-
cgacctctctcagcagttcaccgcc
atgcagcggataagcgtggtgctgagccgggccaccgaggccagcaaaacgctgcaggtggtgctcagcgtat-
tacacaacgatgcctttatgcag
cacgggatgatctgcctgtacgacagcgagcaggagatcctcagtatcgaagcgctgcagcaaaccggccagc-
agcccctccccggcagcacgc
agatccgctatcgccccggcgagggactggtggggaccgtgctggcccaggggcagtcgctggtgctgccccg-
ggtcgccgacgatcagcgtttt
ctcgaccgcctgagcctctacgattacgatctgccgtttatcgccgtaccgttgatggggcccaacgcccggc-
caataggggtgctggcggcccagc
cgatggcgcgccaggaagagcggctgccggcctgcacccgttttctcgaaaccgtc 209
atggcgctgaagcacctgatcacgactgcgcggcgtcgccgatggtcgccagccagctcgcgcgccaccc-
gctgctgctggatgagctgctggat
cccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgc-
cggaagaggatgaagagcagca
gctggaggcgttgcgccagtttaagcaggcgcagcagctgcatatcgcggcggcggatatcgctggtaccctg-
ccggtgatgaaggtcagcgatca
cttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctac-
ggccagccgacccacctgcacg
atcgccagggtcgcggcttcgccgtcgtcggctacggtaagcttacggctgggagctgggctacagctccgat-
ctcgatctggtgttcctccatgac
tgcccggcggaggtgatgaccgacggcgagcggcagattgacggccgtcagttctacctgcggctggcccagc-
ggatcatgcacctgttcagcac
ccgcacctcgtccggtattctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctggtc-
accaccgccgacgcgtttgctgact
atcagcagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccgcggtctatggcgacccggcgc-
tgcaggcgcgctttgacgc
cattcgtcgcgatatcctgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgag-
aagatgcgcgcccaccttggca
acaaacatcccgatcgttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagta-
tctggtcctacgctatgccagtgacang
ccgaagctgacccgctggtagacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgagga-
ggaggcgcgcgccttaacgcat
gcgtacaccaccttgcgtcatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagagg-
ccttcagccgggagcgtcagca ggtcagcgccagctggcagaagtggctgatggcttaa 210
cgtaaggcgaccacccagctccgcgcgttgctgaacgacgctgaagccgttctgctggccgcggacaccg-
ccgacgaggcgttattccgcaccga
ggtcgtcggcgccaaactggccctgactgaatggctggtccagcgcggctggcgtccgttcctcaacgaggca-
ggagagaaaaaaatagccggat
cgttcaaacggtttgccgatattaacctctcgcgggtggcggccgagctgcgcagcgccgtgcagcatctggc-
ggttgaagatgcgccgaccagtt
gccgaagctgtcccgcgacatcgacagcgtccagctgctggcgggcgcctatggcgacgccgtcgcgccgtgg-
ctggagaactggcaggagctt
caccgtgcaatagcacatgacgatcgcagcgtctttgaatatttccgcgccaggcgctggctgccgagccgtt-
ctggcttcatagtggaaaacgata
atttcaggccagggagcccttatggcgctgaagcacctgatcacgctagcgcggcgtcgccgatggtcgccag-
ccagctggcgcgccacccgctg
ctgctggatgagctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgc-
gccagtacctgctgcgcgtgccg
gaagaggatgaagagcagcagctggaggcgttgcgccagtttagcaggcgcagcagctgcatatcgcggcggc-
ggatatcgctggtaccctgcc
ggtgatgaaggtcagcgatcacttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgg-
gggcagatggtcgctcgctacgg
ccagccgacccacctgcacgatcgccagcgtcgcggcttcgccgtcgtcggctacggtaagcttggcggctgg-
gagctgggctacagctccgatct
cgatctggtgttcctccatgtctgcccggcggaggtgatgaccgacggcgagcgggagattgacggccgtcag-
ttctacctgcggctggcccagcg
gatcatgcacctgttcagcacccgcacctcgtccggtattctctacgaagtggacgcccggctgcgtccttct-
ggcgcggcggggatgctggtcacca
ccgccgacgcgtttgctgactatcagcagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccg-
cgtggtctatgccgacccggcg
ctgcaggcgcgctttgacgccattcgtcgcgatatcctgaccaccccgcgggaggggatgaccctgcagaccg-
aggttcgcgagatgcgcgagaa
gatgcgcgcccaccttggcaacaaacatcccgatcgttttgatatcaaagccgatgccggcgggatcaccgat-
attgaatttattactcagtatctggtcc
tacgctatgccagtgacaagccgaagctgacccgctggtctgacaacgtgcgtattcttgagctgctggcgca-
gaacgacatcatggacgaggagga
ggcgcgcgccttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggccctgcaggagcagccg-
ggacacgtggcgccagaggcct
tcagccgggagcgtcagcaggtcagcgccagctggcagaagtggctgatcgcttaactataaaatcgggtgtg-
ctattatcgcgcgcaaagtttgcgt
ctcgcaggagagagtcatgaaagtaacgctgccggagttgaacgtgcaggagtgttggtggtggggatgtgat-
gctggaccgctactggtacggc
cccaccagtcgtatttccccggaagccccggtgccggtggtgaaggtggaaaatatcgaagaacgtcctggcg-
gcgcggcaaacgtagcgatgaa
catcgcctccctgggggcaacgtcgcgcctggtgggattgaccgggattgatgacgctgcccgcgcgctgagc-
caggcgctggccaatggaatgt
gaagtgcgacttcgtctccgtcccgactcacccgaccatcaccaagctgcgggtgctgtcgcgcaatcagcag-
ctgatccgcctcgactttgaagag
ggcttctccggcgtggatccgcagccgatgcatgagcgcattcagcaggcgctgggagccattggcgcactgg
211
atggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacaacttgcacgtcatcct-
ttattgctcgatgaactgctcgacccgc
gcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatagatgcgggtgccaaca-
gaagacgaagaacagcagcttg
aagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccggt-
gatgaaagtcagtgaccatttaacc
taccttgccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggcagc-
ccgcgcatcttcagcaccgtgagg
ggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggatct-
ggtcttcctgctcgattgcgcgccgg
aggtgatgacggacggcgaacgcagcatcgacggacgtcagttttatatcggctggcgcagcgcattatgcac-
ttattcagcacccggacatcgtca
ggcattctttacgaggttgatccgcgtagcgaccttccggcgcatccggcatgctggtcagtaccattgaagc-
gtttgcagattatcaggccaatgaag
cctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatt-
taacgccacgcgtcgcgacattct
ttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatctg-
gggagtaaaaaagcccacgagtt
tgatctgaaagccgatccgggtggcatcacggatattgaattcattgcaccatacctggttctgcgtttcgcg-
cntgatgagccgaagctgacgcgctg
gtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaagcgcgccatctg-
acgcaggcttatgtgacgctgcgcg
atgaaattcatcatctggcgttgcaggaacacagcgggaagtggccgcggacagctttgctactgagcgcgcg-
cagatccgtgccagctgggcaa agtggctcggctga 212
cggtactggaacagaaatcggcggatgcgtaggagatttgttatgacacggcctgtctgaagtgcaagta-
gtgcttacttcctggctggcaacctcag
gctggacgccgtttattgatgataaatctgcgaagaaactggacgcttccttcaaacgttttgctgacatcat-
gctcggtcgtaccgcagcggatctgaaa
gaagcctttgcgcagccactgacggaagaaggttatcgcgatcagctggcgcgcctgaaacgccagatcatta-
ccttccatttgcttgccggtgcttac
cctgaaaaagacgtcgatgcgtatattgccggctgggtggacctgcaacaggccatcgttcagcagcaacacg-
cctgggaggattcggcccgttctc
acgcggtgatgatggatgattctggttaaacgggcaacctcgtaactgactgactagcctgggcaaactgccc-
gggcttttttttgcaaggaatctgat
ttcatggcgctcaaacagttaatccgctgtggccgcctcgccgatggtcgcgacacaacttgcacgtcatcct-
ttattgctcgatgaactgctcgaccc
gcgcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgggtgcca-
acagaagacgaagaacagcagct
tgaagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccg-
gtgatgaaagtcagtgaccatttaa
cctaccttgccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggca-
gcccgcgcatcttcagcaccgtga
ggggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggat-
ctggtcttcctgctcgattgcgcgcc
ggaggtgatgacggacggcgaacgcagcatcgacggacgtcagttttatcttcggctggcgcagcgcattatg-
cacttattcagcacccggacatcgt
caggcattctttacgaggttgatccgcgctgcgaccttccggcgcatccggcatgaggtcagtaccattgaag-
cgtttgcagattatcaggccaatga
agcctggacgtgggagcatcaggcgaggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaat-
ttaacgccacgcgtcgcgacat
tctttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccat-
ctggggagtaaaaaagcccacga
gtttgatctgaaagccgatccgggtggcatcacggatattgaattcattgcacaatacctggttctgcgtttc-
gcgcatgatgagccgaagctgacgcgc
tggtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgcccgaagaggaagcgcgccatc-
tgacgcaggcttatgtgacgctgcg
cgatgaaattcatcatctggcgttgcaggaacacagcgggaaagtggccgcggacagctttgctactgagcgc-
gcgcagatccgtgccagctgggc
aaagtggctcggctgagggtttttattcggctaacaggcgcttgtgatattatccggcgcattgtatttaccc-
gatttgatttatctgttttggagtcttgggat
gaaagtgactttgcctgattttcaccgcgcaggtgtgctggttgtcggtgacgtaatgttagaccgttactgg-
tatggcccgaccaatcgtatttctccgga
agctccggtgccggtggtgaaggtcagtaccattgaagagcggcctggcggtgagctaacgtggcgatgaaca-
tttcatctctgggcgcctcttcct
gtctgatcggcctgaccggcgtagacgacgctgcgtgccctcagtgagcgtctggcagaagtgaaagaaactg-
cgatttcgtcgcactatccaca
catcctaccatcaccaaactgcgaattttgtcccgtaaccagcaactgatccgcctcgactttgaggaaggtt-
ttgaaggcgttgatctcgagccgatgct gaccaaaataga 213
atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatc-
cttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgg-
gttttgcacttgagacacttttgggcg
agaaccaccgtctgctggaattttttttcacaaaggtagcgttattgaatcgcacattttaaactgttggccg-
ctgtggaaggaatattggtgaaaggtg
cggttttaaggcctttttctttgactctctgtcgttacaaagttaatatgcgcgccctccgtctctgaagctc-
tcggtgaacattgttgcgaggcaggatgcg
agctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgt-
tttcccgtccggggaatggcatggag
ctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggca-
ccttgctgacgttacgcctgccggtac agcaggttatcaccggaggcttaaaatga 214
tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtg-
cgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtg-
gctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgacgaaaaatgcacataaattgcctg-
cgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgat-
gcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggtt-
ccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaaca-
tccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgcc-
agacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggaattttttttcacaaagcgtagcgttattgaatcgcacattttaaactgt-
tggccgctgtggaagcgaatattggtga
aaggtgcggttttaaggcctttttctttgactctctgtcgttacaaagttaatatgcgcgccctccgtctctg-
aagctctcggtgaacattgttgcgaggcag
gatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaag-
cggcgttttcccgtccggggaatggc
atggagctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatg-
gcggcaccttgctgacgttacgcctgc
cggtacagcaggttatcaccggaggcttaaaatgacccagttacctaccgcgggcccggttatccggcgcttt-
gatatgtctgcccagtttacggcgctt
tatcgcatcagcgtggcgctgagtcaggaaagcaacaccgggcgcgcactggcggcgatcctcgaagtgcttc-
acgatcatgcatttatgcaatacg
gcatggtgtgtctgtttgataaagaacgcaatgcactctttgtggaatccctgcatggcatcgacggcgaaag-
gaaaaaagagacccgccatgtccgtt
accgcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggcgctggtgttaccgcgcatttcaga-
cgatcagcgttttctcgaccgcct
gaatatttacgattacagcctgccgttgattggcgtgccgatccccggtgcggataatcagccatcgggcgtg-
ctggtggcacagccgatggcgttgc
acgaagaccggctgactgccagtacgcggtttttagaaatggtc 215
atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatc-
cttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgg-
gttttgcacttgagacacttttgggcg
agaaccaccgtctgctggaattttttttcacaaagcgtagcgttattgaatcgcacattttgatgttggccgc-
tgtggaagcgaatattggtgaaaggtg
cggttttaaggcctttttctttgactctctgtcgttacaaagttaatatgcgcgccctccgtctctgaagctc-
tcggtgaacattgttgcgaggcaggatgcg
agctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggttgcggcgt-
tttcccgtccggggaatggcatggag
ctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggca-
ccttgctgacgttacgcctgccggtac agcaggttatcaccggaggcttaaaatga 216
tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtg-
cgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtg-
gctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaattgcct-
gcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgat-
gcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggtt-
ccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaaca-
tccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgcc-
agacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggaattttttttcacaaagcgtagcgttattgaatcgcacattttaaactgt-
tggccgctgtggaagcgaatattggtga
aaggtgcggttttaaggcctttttctttgactctctgtcgttacaaagttaatatgcgcgccctccgtctctg-
aagctctcggtgagcattgttgcgaggcag
gatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaag-
cggcgttttcccgtccggggaatggc
atggagctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatg-
gcggcaccttgctgacgttacgcctgc
cggtacagcaggttatcaccggaggcttaaaatgacccagttacctaccgcgggcccggttatcccgcgcttt-
gatatgtctgcccagtttacggcgctt
tatcgcatcagcgtggcgctgagtcaggaaagcaacaccgggcgcgcactggcggcgatcctcgaagtgcttc-
acgatcatgcatttatgcaatacg
gcatggtgtgtctgtttgataaagaacgcaatgcactctttgtggaatccctgcatggcatcgacggcgaaag-
gaaaaaagagacccgccatgtccgtt
accgcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggcgctggtgttaccgcgcatttcaga-
cgatcagcgttttctcgaccgcct
gaatatttacgattacagcctgccgttgtttggcgtgccgatccccggtgcggataatcagccatcgggcgtg-
ctggtggcacagccgatggcgttgc
acgaagaccggctgactgccagtacgcggtttttagaaatggtc 217
atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatc-
cttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgg-
gttttgcacttgagacacttttgggcg
agaaccaccgtctgctggttaaaaacgtgaccacgagcattaataaacgccacgaaatgtggcgtttatttat-
tcaaaaagtatcttctttcataaaaagtg
ctaaatgcagtagcagcaaaattgggataagtcccatggaatacggctgttttcgctgcaatttttaactttt-
tcgtaaaaaaagatgtttctttgagcgaac
gatcaaaatataggttaaccggcaaaaaattattctcattagaaaatagtttgtgtaatacttgtaacgctac-
atggagattaacttaatctagagggtttta
taccgtctctgaagctacggtgaacattgttgcgaggcaggatgcgagctggttgtgttttgacattaccgat-
aatgtgccgcgtgaacgggtgcgttat
gcccgcccggaagcggcgttttcccgtccggggaatggcatggagagcgccttatccagacgctgatcgccca-
tcatcgcggttctttagatctctcg
gtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggcttaaaat-
ga 218
gtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtgc-
gaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtg-
gctgcaacaactgatcaacgcct
gagcctgttctccttatgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaattgcctg-
cgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgat-
gcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggtt-
ccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaaca-
tccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgcc-
agacgggttttgcacttgagacactttt
gggcgagaaccaccgtagctggttaaaaacgtgaccacgagcattaataaacgccacgaaatgtggcgtttat-
ttattcaaaaagtatatctttcataa
aaagtgctaaggcagtagcagcaaaattgggataagtcccatggaatacggctgttttcgctgcaatttttaa-
ctttttcgtaaaaaaagatgtttctttgag
cgaacgatcaaaatatagcgttaaccggcaaaaaattattacattagaaaatagtttgtgtaatacttgtaac-
gctacatggagattaacttaatctagagg
gttttataccgtctctgaagctctcggtgaacattgttgcgaggcaggatgcgagaggttgtgttttgacatt-
accgataatgtgccgcgtgaacgcgtg
cgttatgcccgcccggaagcggcgttttcccgtccggggaatggcatggagctgcgccttatccagacgctga-
tcgcccatcatcgcggttctttagat
ctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggct-
taaaatgacccagttacctaccgcg
ggcccggttatccggcgctttgatatgtctgcccagtttacggcgctttatcgcatcagcgtggcgctgagtc-
aggaaagcaacaccgggcgcgcact
ggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtctgtttgataaagaa-
cgcaatgcactctttgtggaatccctgc
atggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttaccgcatgggggaaggcgtgatcggcgc-
ggtgatgagccagcgtcaggc
gctggtgttaccgcgcatttcagacgatcagcgttttctcgaccgcctgaatatttacgattacagcctgccg-
ttgattggcgtgccgatccccggtgcgg
ataatcagccatcgggcgtgctggtggcacagccgatggcgttgcacgaagaccggctgactgccagtacgcg-
ctttttagaaatggtcg 219
atgagcatcacggcgttatcagactcatttcctgagggaatatcgccagccgcttgtcgctgcaacatcc-
ttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgg-
gttttgcacttgagacacttttgggcg
agaaccaccgtctgctggtgaacatcactgatgcacaagctacctatgtcgaagaattaactaaaaaactgaa-
gatgcaggcattcgcgttaaagcc
gacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgttccttatatgttagttt-
gtggcgataaagaggtcgaagcaggca
aagttgctgttcgtacccgccgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgct-
ggcggaaatccgcagcagaagtctt
catcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaacaaag-
agattcgcgcgcaagaagttcgcc
tcacaggcgtcgatggcgagcagattggtattgtcagtctgaatgaagctcttgaaaaagctgaggaagcggg-
cgtcgatttagtagaaatcagtccg
aatgccgagccgccagtttgtcgaatcccgtctagaagctctcggtgaacattgttgcgaggcaggatgcgag-
ctggttgtgttttgacattaccgata
atggccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccggggaatggcatggagctg-
cgccttatccagacgctgatcgc
ccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggta-
cagcaggttatcaccggaggcttaaaa tga 220
tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttggtgcg-
aacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtg-
gctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaattgcct-
gcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgat-
gcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggtt-
ccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaaca-
tccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgcc-
agacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggtgaacatcactgatgcacaagctacctatgtcgaagaattaactaaaaaa-
ctgcaagatgcaggcattcgcgtta
aagccgacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgttccttatatgtt-
agtttgtggcgataaagaggtcgaagc
aggcaaagttgctgttcgtacccgccgcggcattagacttaggaagcatggatgttagcgaagtcgttgacaa-
actgctggcggaaatccgcagcaga
agtcttcatcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcatta-
acaaagagattcgcgcgcaagaagt
tcgcctcacaggcgtcgatggcgagcagattggtattgtcagtctgaatgaagctcttgaaaaagctgaggaa-
gcgggcgtcgatttagtagaaatca
gtccgaatgccgacccgccagtttgtcgaatcccgtctctgaagctctcggtgaacattgttgcgaggcagga-
tgcgagctggttgtgttttgacattac
cgataatgtgccgcgtgaacgggtgcgttatgcccgcccggagggcgttttcccgtccggggaatggcatgga-
gctgcgccttatccagacgctg
atcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgc-
cggtacagcaggttatcaccggaggct
taaaatgacccagttacctaccgcgggcccggttatccggcgctttgatatgtctgcccagtttacggcgctt-
tatcgcatcagcgtggcgctgagtcag
gaaagcaacaccgggcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggca-
tggtgtgtctgtttgataaagaacg
caatgcactctttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttac-
cgcatgggggaaggcgtgatcgg
cgcggtgatgagccagcgtcaggcgctggtgttaccgcgcatttcagacgatcagcgttttctcgaccgcctg-
aatatttacgattacagcctgccgttg
attggcgtgccgatccccggtgcggataatcagccatcgggcgtgctggtggcacagccgatggcgttgcacg-
aagaccggctgactgccagtacg cggtttttagaaatggtc 221
atgagcatcaccgcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatc-
cttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgg-
gttttgcacttgagacacttttgggcg
agaaccaccgtctgctggtgaacatcactgatgcacaagctacctatgtcgaagaattaactaaaaaactgca-
agatgcaggcattcgcgttaaagcc
gacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgttccttatatgttagtgg-
tggcgataaagaggtcgaagcaggca
aagttgctgttcgtacccgccgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgct-
ggcggaaatccgcagcagaagtctt
catcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaacaaag-
agattcgcgcgcaagaagttcgcc
tcacaggcgtcgatggcgagcagattggtattgtcagtctgaatgaagctcttgaaaaagctgaggaagcggg-
cgtcgatttagtagaaatcagtccg
aatgccgagccgccagtttgtcgaatcccgtctctgaagctctcggtgaacattgttgcgaggcaggatgcga-
gctggagtgttttgacattaccgata
atgtgccgcgtgaacgggtgcgttatgcccccccggaagcgccgttttcccgtccggggaatggcatggagct-
gcgccttatccagacgctgatcgc
ccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgcggtac-
agcaggttatcaccggacgcttaaaa tga 222
tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtg-
cgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtg-
gctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgccgttttgcacgaaaaatgcacataaattgcctg-
cgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgat-
gcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggtt-
ccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaaca-
tccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgcc-
agacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggtgaacatcactgatgcacaagctacctatgtcgaagaattaactaaaaaa-
ctgcaagatgcaggcattcgcgtta
aagccgacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgttccttatatgtt-
agtttgtggcgataaagaggtcgaagc
aggcaaagttgctgttcgtacccgccgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaa-
ctgctggcggaaatccgcagcaga
agtcttcatcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcatta-
acaaagagattcgcgcgcaagaagt
tcgcctcacaggcgtcgatggcgagcagattggtattgtcagtctgaatgaagctcttgaaaaagctgaggaa-
ggggcgtcgatttagtagaaatca
gtccgaatgccgagccgccagtttgtcgaatcccgtctctgaagctctcggtgaacattgttgcgaggcagga-
tgcgagctggttgtgttttgacattac
cgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagggcgttttcccgtccggggaatggcatgg-
agctgcgccttatccagacgctg
atcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgc-
cggtacagcaggttatcaccggaggct
taaaatgacccagttacctaccgcgggcccggttatccggcgctttgatatgtctgcccagtttacggcgctt-
tatcgcatcagcgtggcgctgagtcag
gaaagcaacaccgggcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggca-
tggtgtgtctgtttgataaagaacg
caatgcactctttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttac-
cgcatgggggaaggcgtgatcgg
cgcggtgatgagccaggtcaggcgctggtgttaccgcgcatttcagacgatcagcgttttctcgaccgcctga-
atatttacgattacagcctgccgttg
attggcgtgccgatccccggtgcggataatcagccatcgggcgtgctggtggcacagccgatggcgttgcacg-
aagaccggctgactgccagtacg cggtttttagaaatggtc 223
atggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacacaacttgcacgtcatc-
ctttattgctcgatgaactgctcgacccgc
gcacgctttaccagccgattgagccgggcgcttaccgcgacgaactccgtcagtatctgatgcgggtgccaac-
agaagacgaagaacagcagcttg
aagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccggt-
gatgaaagtcagtgaccatttaacc
taccttcccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggcagc-
ccgcgcatcttcagcaccgtgagg
ggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggatct-
ggtcttcctgctcgattgcgcgccgg
acgtgatgacggacggcgaacgcagcatcgacggacgtcagttttatcttcggctggcgcagcgcattatgca-
cttattcagcacccggacatcgca
ggcattctttacgaggttgatccgcgtctgcgaccttccggcgcatccggcatgctggtcagtaccattgaag-
cgtttgcagattatcaggccaatgaag
cctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatt-
taacgccacgcgtcgcgacattct
ttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatagg-
ggagtaaaaaagcccacgagtt
tgatctgaaagccgatccgggtggcatcacggatattgaattcattgcacaatacctggttctgcgtttcgcg-
catgatgagccgaagctgacgcgctg
gtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaagcgcgccatctg-
acgcaggcttatgtgacgctgcgcg
atgaaattcatcatctggcgttgcaggaacacagcgggaaagtggcgcggacagctttgctactgagcgcgcg-
cagatccgtgccagctgggcaa agtggctcggctga 224
cggtactggaacagaaatcggcggatgcgcaggaaatttgttatgacacggcctgtctgaagtgcaagtt-
agtgcttacttcctggctggcaacctcag
gctggacgccgtttattgatgataaatctgcgaagaaactggacgcttccttcaaacgttttgctgacatcat-
gctcggtcgtaccgcagggatctgaaa
gaagcctttgcgcagccactgacggaagaaggttatcgcgatcagctggcgcgcctgaaacgccagatcatta-
ccttccatttgcttgccggtgcttac
cctgaaaaagacgtcgatgcgtatattgccggctgggtggacctgcaacaggccatcgttcagcagcaacacg-
cctgggaggattcggcccgttctc
acgcggtgatgatggatgctttctggttaaacgggcaacctcgttaactgactgactagcctgggcaaactgc-
ccgggcttttttttgcaaggaatctgat
ttcatggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacacaacttgcacgtcatc-
ctttattgctcgatgaactgctcgaccc
gcgcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgcgggtgc-
caacagaagacgaagaacagcagct
tgaagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccg-
gtgatgaaagtcagtgaccatttaa
cctaccttgccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggca-
gcccgcgcatcttcagcaccgtga
ggggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggat-
ctggtcttcctgctcgattgcgcgcc
ggaggtgatgacggacggccaacgcagcatcgacggtcgtcagttttatcttcggctggcgcaggcattatgc-
acttattcagcacccggacatcgt
caggcattctttacgaggttgatccgcgtctccgaccttccggcgcatccggcatgctggtcagtaccattga-
aggtttgcagattatcaggccaatga
agcctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaa-
tttaacgccacgcgtcgcgacat
tctttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccat-
ctggggagtaaaaaagcccacga
gtttgatctgaaagccgatccgggtggcatcacggatattgaattcattgacaatacctggttctgcgtttcg-
cgcatgatgagccgaagctgacgcgc
tggtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaagagcgccatc-
tgacgcaggcttatgtgacgctgcg
cgatgaaattcatcatctggcgttgcaggaacacagcgggaaagtggccgcggacagctttgctactgagcgc-
gcgcagatccgtgccagctgggc
aaagtggctcggctgagggtttttattcggctaacaggcgcttgtgatattatccggcgcattgtatttaccc-
gatttgatttatctgttttggagtcttgggat
gaaagtgactttgcctgattttcaccgcgcaggtgtgctggttgtcggtgacgtaatgttagaccgttactgg-
tatggcccgaccaatcgtatttctccgga
agctccggtgccggtggtgaagctcagtaccattgaagagcggcctggcggtgcagctaacgtggcgatgaac-
atttcatctctgggcgcctcttcct
gtctgatcggcctgaccggcgtagacgacgctgcgcgtgccctcagtgagcgtctggcagaagtgattagtta-
actgcgatttcgtcgcactatccaca
catcctaccatcaccaaactgcgaattttgtcccgtaaccagcaactgatccgcctcgactttgaggaaggtt-
ttgaaggcgttgatctcgagccgatgct gaccaaaataga 225
atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgagcaacatcc-
ttcactgttttataccgtattgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgg-
gttttgcacttgagacacttttgggcg
agaaccaccgtctgctggtacagtaggcctctcaaaaatagataaacggctcatgtacgtgggccgtttattt-
tttctacccataatcgggaaccggtgt
tataatgccgcgccctcatattgtggggatttcttaatgacctatcctgggcctaaagagtagttgacattag-
cggagcactaacccgtctagaagctct
cggtgaacattgttgcgaggcaggatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacg-
ggtgcgttatgcccgcccggaagcg
gcgttttcccgtccggggaatggcatggagctgcgccttatccagacgctgatcgcccatcatcgcggttctt-
tagatctctcggtccgccctgatggcg
gcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggcttaaaatga 226
tgtttcgtctcgaggccgggcaactgagcggccccgttaaaccgacctgggctggcatctgttgttgtgc-
gaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacaggaatcattatcagcgccagt-
ggctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaattgcct-
gcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgat-
gataaaacaccccgtttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggtt-
caggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggcgaatatcgccagccgcttgtcgctgcaaca-
tccttcactgttttataccgtgcttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgcc-
agacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggtacagtagcgcctctcaaaaatagataaacggctcatgtacgtgggccgt-
ttattttttctacccataatcgggaac
cggtgttataatgccgcgccctcatattgtggggatttcttaatgacctatcctgggtcctaaagttgtagtt-
gacattagcggagcactaacccgtctctg
aagctctcggtgaacattgttgcgaggcaggatgcgagctggttgtgttttgacattaccgataatgtgccgc-
gtgaacgggtgcgttatgcccgcccg
gaagcggcgttttcccgtccggggaatggcatggagctgcgccttatccagacgctgatcgcccatcatcgcg-
gttctttagatctctcggtccgccct
gatggcggcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggcttaaaatgacccagtt-
acctaccgcgggcccggttatccg
gcgctttgatatgtctgcccagtttacggcgctttatcgcatcagcgtggcgctgagtcaggaaagcaacacc-
gggcgcgcactggcggcgatcctcg
aagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtctgtttgataaagaacgcaatgcactctt-
tgtggaatccctgcatggcatcgacggc
gaaaggaaaaaagagacccgccatgtccgttaccgcatgggggaaggcgtgatcggcgcggtgatgagccagc-
gtcaggcgctggtgttaccgc
gcattttagacgatcagcgttttctcgaccgcctgaatatttacgattacagcctgccgttgattggcgtgcc-
gatccccggtgcggataatcagccatcg
ggcgtgctggtggcacagccgatggcgttgcacgaagaccggctgactgccagtacgcggtttttagaaatgg-
tc 227
atggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtagcgacacaacttgcacgtcatc-
ctttattgctcgatgaactgctcgacccgc
gcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgggtgccaac-
agaagacgaagaacagcagcttg
aagccgtgcgccagttcaaacaggcccagcatttgcgtatagcagccggggatatttccggggcattgccggt-
gatgaaagtcagtgaccatttaacc
taccttgccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggcagc-
ccgcgcatcttcagcaccgtgagg
ggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggatct-
ggtcttcagctcgattgcgcgccgg
aggtgatgacggacggcgaacgcagcatcgacggacgtcagttttatcttcggctggcgcagcgcattatgca-
cttattcagcacccggacatcgtca
ggcattctttacgaggttgatccgcgtctgcgaccttccggcgcatccggcatgctggtcagtaccattgaag-
cgtttgcagattatcaggccaatgaag
cctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatt-
taacgccacgcgtcgcgacattct
ttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatctg-
gggagtaaaaaagcccacgagtt
tgatctgaaagccgatcccggtggcatcacggatattgaattcattgacaatacctggttctgcgtttcgcgc-
atcatgagccgaagctgacgcgctg
gtctgataacgtgcggatttttgtactgatggcacgatatgacatcatgcggaagaggaagcgcgccatctga-
cgcaggcttatgtgacgctgcgcg
atgaaattcatcatctggcgttgcacgaacacagcgggaaagtggccgcggacagctttgctactgagcgcgc-
gcagatccgtgccagctgggcaa agtggctcggctga 228
cggtactggaacagaaatcggcggatgcgcaggaaatttgttatgacacggcctgtctgaagtgcaagtt-
agtgcttacttcctggctggcaacctcag
gctggacgccgtttattgatgataaatctgcgaagaaactggacgcttccttcaaacgttttgctgacatcat-
gctcggtcgtaccgcagcggatctgaaa
gaagcctttgcgcagccactgacggaagaaggttatcgcgatcagctggcgcgcctgaaacgccagatcatta-
ccttccatttgcttgccggtgcttac
cctgaaaaagacgtcgatgcgtatattgccggctgggtggacctgcaacaggccatcgttcagcagcaacacg-
cctgggaggattcggcccgttctc
acgcggtgatgatggatgctttctggttacgggcaacctcgttaactgactgactagcctgggcaaactgccc-
gggcttttttttgcaaggaatctgat
ttcatggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacacaacttgcacgtcatc-
ctttattgctcgatgaactgctcgaccc
gcgcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgggtgcca-
acagaagacgaagaacagcagct
tgaagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccg-
gtgatgaaagtcagtgaccatttaa
cctaccttgccgaggccattctcgatgtcgtggtgcagaatgcgtgggaacaaatggtcgtaaaatacgggca-
gcccgcgcatcttcagcaccgtga
ggggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggat-
ctggtcttcctgctcgattgcgcgcc
ggaggtgatgacggacggcgaacgcagcatcgacggacgtcagttttcttcggaggcgcagcgcattatgcac-
ttattcagcacccggacatcgt
caggcattctttacgaggttgatccgcgtctgcgaccttccggcgcatccggcatgctggtcagtaccattga-
agcgtttgcagattatcaggccaatga
agcctggacgtgggagcatcaggcgctgtgtcgcgcgcgcgtggtttacggggatccgcaactgacacagcaa-
tttaacgccacgcgtagcgacat
tctttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccat-
ctggggagtaaaaaagcccacga
gtttgatctgaaagccgatccgggtggcatcacggatattgaattcattgcacaatacctggttctgcgtttc-
gcgcatgatgagccgaagctgacgcgc
tggtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaaggcgccatct-
gacgcaggcttatgtgacgctgcg
cgatgaaattcatcatctggcgttgcaggaacacagcgggaaagtggccgcggacagctttgctactgagcgc-
gcgcagatccgtgccagctgggc
aaagtggctcggctgagggtttttattcggctaacaggcgcttgtgatattatccggcgcattgtatttaccc-
gatttgatttatctgttttggagtcttgggat
gaaagtgactttgcctgattttcaccgcgcaggtgtgctggttgtcggtgacgtaatgttagaccgttactgg-
tatggcccgaccaatcgtatttctccgga
agctccggtgccggtggtgaaggtcagtaccattgaagagcggcctggcggtgcagctaacgtggcgatgaac-
attcatctctggacgcctcttcct
gtctgatcggcctgaccggcgtagacgacgctgcgcgtgccctcagtgagcgtctggcagaagtgaaagttaa-
ctgcgatttcgtcgcactatccaca
catcctaccatcaccaaactgcgaattttgtcccgtaaccagcaactgatccgcctcgactttgaggaaggtt-
ttgaaggcgttgatctcgagccgatgct gaccaaaataga 229
atgagcatcacggcgttatcagctgaatatcactgactcacaagctacctatgtcgaagaattaactaaa-
aaactgcaagatgcaggcattcgcgttaaa
gcgacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgtccttatatgttagtt-
tgtggcgataaagaggtcgaagcag
gcaaagttgctgttcgtactcgtcgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaact-
gctggcggaaatccgcagcagaagt
catcatcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaaca-
aagagattcgcgcgcaagaagttc
gcctcaccggcgtcgatggcgagcagattggtattgcagtctgaatgaagctcttgaaaaagctgaggaagcg-
ggcgtcgatttagtagaaatcagt
ccgaatgccgagccgccagtttgtcgaatctctttagatctctcggtccgccctgatggcggcaccttgctga-
cgttacgcctgccggtacagcaggtta tcaccggaggcttaaaatga 230
tgtttcgtcgaagccgggcaactgagcagccccgttgaaaccgaactgggctggcatctgttgttgtgcg-
aacaaattcgcctgcgcaacccttgc
cgaaagccgaggccttaacgcgggtgcgtcagcaactgattgcccggcaacagaatcattatcagcgccagtg-
gctgcaacaactgatcaacgcct
gagcctgttctccttcttgttggtgcagacgggttaatgcccgttttgcacgaaaaatgcacataaactgcct-
tcgctgccttataacagcgcatggaaatc
ctgcctcctgccttgtgccacgccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatg-
tttaaaacaccccgttcagatcaaccttt
gggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatatgtgatttgggttcc-
ggcattgcgcaataaaggggagaaa
gacatgagcatcacggcgttatcagctgaatatcactgactcacaagctacctatgtcgaagaattaactaaa-
aaactgcaggatgcaggcattcgcgtt
aaagccgacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgttccttatatgt-
tagtttgtggcgataaagaggtcgaag
caggcaaagtgctgttcgtactcgtcgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaa-
ctgctggcggagatccgcagcag
aagtcatcatcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcatt-
aacaaagagattcgcgcgcaagaa
gttcgcctcaccggcgtcgatggcgagcagattggtattgtcagtctgaatgaagacttgaaaaagctgagga-
agcgggcgtcgatttagtagaaatc
agtccgaatgccgagccgccagtttgtcgaatctctttagatctctcggtccgccctgatggcggcaccttgc-
tgacgttacgcctgccggtacagcag
gttatcaccggaggcttaaaatgacccagttacctaccgcgggcccggttatccggcgctttgatatgtctgc-
ccagtttacggcgctttatcgcatcagc
gtggcgctgagtcaggagagcaataccgcgcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcat-
ttatgcaatacggcatggtgtgtct
gttcgataaagaacgcaatgcactgtttgtgcaatccctgcatggcatcgacggcgaaaggaaaaaagaaacc-
cgccatgtccgttaccgcatgggg
gaaggcgtgatcggcgcggtgatgagccagcgtcaggcgtggtgttaccgcgcatttcagacgatcagcgttt-
tctcgaccgcctgaatatttacgat
tacagcctgccgctgattggtgtgccgatccccggtgcggataatcagcctgcgggtgtgctggtggcacagc-
cgatggcgttgcacgaagaccgg ctggctgccactacgcgctttttagaaatggtc 231
atgaccctgaatatgatgatggatgccggcggacatcatcgcgacaaacaatattaataccggcaaccac-
accggcaatttacgagactgcgcaggc
atcctttacccgtcaatttctgtcaaataaagtaaaagaggcagtctacttgaattacccccggctggttgag-
cgtttgttgaaaaaaagtaactgaaaa
tccgtagaatagcgccactctgatggttaattaacctattcaattaagaattatctggatgaatgtgccatta-
aatgcgcagcataatggtgcgttgtgcgg
gaaaactgcttttttttgaaagggttggtcagtagggaaacaactcacttcacaccccgaagggggaagttgc-
ctgaccctacgattcccgctatttcat tcactgaccggaggttcaaaatga 232
accggatacgagagaaaagtgtctacatttggttcggttgatattgaccggcgcatccgccagcccgccc-
agtttctggggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagat-
cccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccag-
tctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgg-
gttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgac-
acgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgcccgcggacatcatcgcgacaaacaatattaataccggcaaccac-
accggcaatttacgagactgcgca
ggcatcctttctcccgtcaatttctgtcaaataaagtaaaagaggcagtctacttgaattacccccggctggt-
tgagcgtttgttgaaaaaaagtaactgaa
aaatccgtagaatagcgccactctgatggttaattaacctattcaattaagaattatctggatgaatgtgcca-
ttaaatgcgcagattaatggtgcgttgtg
cgggaaaactgctttttttgaaagggttggtcagtagcggaaacaactcacttcacaccccgaagggggaagt-
tgcctgaccctacgattcccgctatt
tcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttgt-
cccagcagttcactgcgatgcagcgc
ataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgcacaatg-
acgcctttttgcagcacggcatgat
ctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatcccc-
ggcagctcgcaaatccgctatcgtcc
gggcgaagggctggtcgggacggtgctttcgcagggccaatcattagtgcttgcgcgcgttgctgacgatcag-
cgctttcttgaccggctcgggttgt
atgattacaacctgccgtttatcgccgtgccgctgatagggccagatgcgcagactttcggtggctgacggca-
caacccatggcgcgttacgaagag cgattacccgcctgcacccgctttctggaaacggtc 233
atgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaa-
aagcgcccttgtggcgctttttttatattccc
gcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatg-
gaagctcgctgttttaacacgcgttttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattct-
caacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggactgggcgcaactcac-
ttcacaccccgaagggggaagttgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga 234
accggatacgagagaaaagtgtctacatcggttcggttgatattgaccggcgcatccgccagcccgccca-
gtttctggtggatctgtttggcgattttgc
gggtcttgccggtgcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatc-
ccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccag-
tctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgg-
gttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgac-
acgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggccgtcctgtaataataaccgcacaattcggactgattaaaa-
aagcgcccttgtggcgctttttttatatt
cccgcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccg-
atggaagctcgctgttttaacacgcgttttt
taaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatat-
tctcaacctaaaaaagtttgtgtaatacttgt
aacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaac-
tcacttcacaccccgaagggggaagt
tgcctgaccctacgattcccgctatttcattcactgaccggaggttcataatgacccagcgaaccgagtcggg-
taataccgtctggcgcttcgatttgtc
ccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccag-
caagtgctgtgcgtattgcacaat
gacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgc-
aggaagccgatcagcagttaatcccc
ggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgtttcgcagggcaatcattagtg-
ctggcgcgcgttgctgacgatc
agcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatcgccgtgccgctgatagggccaga-
tgcgcagactttcggtgtgctgacggc
acaacccatggcgcgttacgaagagcgattacccgcctgcacccgctttctggaaacggtc 235
atgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaa-
aagcgcccttgtggcgcatttttttattccc
gcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatg-
gaagctcgctgttttaacacgcgtttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattct-
caacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactca-
cttcacaccccgaagggggaagttgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga 236
accggatacgagagaaaagtgtctacatcggttcggttgatattgaccggcgcatccgccagcccgccca-
gtttctggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagat-
cccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccag-
tctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgg-
gttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgac-
acgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaa-
aagcgcccttgtggcgctttttttatatt
cccgcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccg-
atggaagctcgctgttttaacacgcgttttt
taaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatat-
tctcaacctaaaaaagtttgtgtaatacttgt
aacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaac-
tcacttcacaccccgaagggggaagt
tgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcggg-
taataccgtctggcgatcgatttgtc
ccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccag-
caagtgctgtgcgtattgcacaat
gacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgc-
aggaagccgatcagcagttaatcccc
ggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaatcattag-
tgctggcgcgcgttgctgacgatc
agcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatcgccgtgccgctgatagggccaga-
tgcgcagactttcggtgtgctgacggc
acaacccatggcgcgttacgaagagcgattacccgcctgcacccgctttctggaaacggtc 237
atggcactgaaacacctcatttccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacc-
cgatcctgcttgatgaattgctcgacccg
aatacgctctatcaaccgacggcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccgg-
aagatgatgaagagcaacagcttg
aggcgctgcggcagtttaagcaggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagt-
aatgaaagtgagcgatcacttaac
ctggctggcggaagcgattattgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccag-
ccaacgcatctgcacgatcgcga
agggcgggttttgcggtggtcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatc-
tggtattcctgcacgactgcccga
tggatgtgatgaccgatggcgagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgat-
gcacctgtttagcacgcgcacgtcgt
ccggcatcctttatgaagttgatgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacgga-
atcgttcgccgattaccagcaaaacg
aagcctggacgtgggaacatcaggcgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccga-
atttgacgccattcgccgcgata
ttctgatgacgcctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgccca-
tcttggcaacaagcataaagacc
gcttcgatctgaaagccgatgaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctt-
tgcccatgacaagccgaaactgacgc
gctggtcggataatgtgcgcattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggc-
attgacgctggcgtacaccaca
ttgcgtgatgagctgcaccacctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccg-
agcgtgcgcttattaaaaccagctgg gacaagtggctggtggaa 238
gcgcaaagcgagtgctcacttacgtgatctgttgacacaatctgaagcgaccataacttctgccgtttca-
gcgaatacggcggtgtggagcgcacaatc
agccctggcgaagctggtgctcaccgagtggctagtgacgcagggctggcgaaccttccttgatgaaaaagcg-
caggccaaattcgccgactccttt
aaacgctttgctgacatccatctgtcacgcagcgccgccgagctgaaaaaaggcctttgcccaaccgctgggc-
gacagctatcgcgaccagttgccgc
gcctggcgcgtgatatcgactgcgcgttactgctggccgggcattacgatcgcgcgcgcgccgtggaatggct-
ggaaaactggcaggggcttcagc
acgccattgaaacgcgccagagagtcgaaatcgaacatttccgtaataccgcgattacccaggagccgttctg-
gttgcacagcggaaaacgttaacg
aaaggatatttcgcatggcactgaaacacctcatttccctgtgtgccgcgtcgccgatggttgccagtcagct-
ggcgcgctacccgatcctgcttgatga
attgctcgacccgaatacgctctatataccgacggcgatgaatgcctatcgcgatgagctgcgcatatacctg-
ctgcgcgtgccggaagatgatgaa
gagcaacagcttgaggcgctgcggcagtttaagcaggcgcagttgctgcgcgtggcggcggcggatattgccg-
gtacgttgccagtaatgaaagtg
agcgatcacttaacctggctggcggaagcgattattgatgcggtggtgcagcaagcctgggggcagatggtgg-
cgcgttatggccagccaacgcat
ctgcacgatcgcgaagggcgcggttttgcggtggtcggttatggcaagctgggcggctgggagctgggttaca-
gctccgatctggatctggtattcct
gcacgactgcccgatggatgtgatgaccgatggcgagcgtgaaatcgatggtcgccagttctatttgcgtctc-
gcgcagcgcgtgatgcacctgtttag
cacgcgcacgtcgtccggcatcctttatgaagttgatgcgcgtctgcgtccatctggcgctgcggggatgctg-
gtcactactacggaatcgttcgccga
ttaccagcaaaacgaagcctgcacgtgggaacatcaggcgctggcccgtgcgcgcgtggtgtacggcgatccg-
caactgaccgccgaatttgacg
ccattcgccgcgatattctgatgacgcctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcga-
gaaaatgcgtgcccatcttggca
acaagcataaagaccgcttcgatctgaaagccgatgaaggcggtatcaccgacatcgagtttatcgcccaata-
tctggtgctgcgctttgcccatgaca
aaccgaaactgacgcgctggtcggataatgtgcgcattctcgaagggctggcgcaaaacggcatcatggagga-
gcaggaagcgcaggcattgacg
ctggcgtacaccacattgcgtgatgagagcaccacctggcgctgcaagagttgccgggacatgtggcgctctc-
ctgttttgtcgccgagcgtgcgctt
attaaaaccagctgggacaagtggctggtggaaccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaa-
ttttgtatctctcaggagacaggaa
tgaaagtgacgagccagagtttaagcaagccggtgtaatggtggtgggtgatgtgatgctggatcgttactgg-
tatggcccaaccagccgtatctctc
cggaagcgccagtcccggttgttaaagtcgataccattgaagagcgtcctggcggcgcggcaaacgtggcgat-
gaatatcgcctcactgggcgcca
cggcgcgtctggttggcctgactggcattgacgatgcggcgcgcgcgctgagcaaagcgctggccgatgttaa-
cgttaaatgtgacttcgtttctgttc
cgacgcatcccaccatcactaagctgcgcgtgctgtcgcgtaaccagcagctgattcgcctggactttgaaga-
gggttttgaaggagtcgatccgcaa
ccgatgcatcgaacgcatcagccaggcgcttggtaatattggcgcgctggtgctgtcggatt 239
atgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcg-
aattgtggcaggatgcgttgcaggagga
ggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgatttt-
cgcaaagagttggataaacgcacc
attggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacg-
atgcgccagtaccgctgtcacgcc
tgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcact-
gaaacacctcatttccctgtgtgccgc
gtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctc-
tatcaaccgacggcgatgaatgccta
tcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcgg-
cagtttaagcaggcgcagttgct
gcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcg-
gaagcgattattgatgcggtggtg
cagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggtt-
ttgcggtggtcggttatggcaag
ctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactgcccgatggatgtga-
tgaccgatggcgagcgtgaaatcg
atggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcat-
cctttatgaagttgatacgcgtctgcgt
ccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctgga-
cgtgggaacatcaggcgctggcc
cgtgcacgcgtgggtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgac-
gcctcgcgacggcgcaacgctgc
aaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatct-
gaaagccgatgaaggcggtatca
ccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtc-
ggataatgtgcgcattctcgaagggc
tggcgcaaanggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatgag-
ctgcaccacctggcgctgcaa
gagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagt-
ggctggtggaaccgtgcgccccggc gtaa 240
atgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaa-
aagcgcccttgtggcgctttttttatattccc
gcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatg-
gaagctcgctgtttaacacgcgttttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattct-
caacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactca-
cttcacaccccgaagggggaagttgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga 241
accggatacgagagaaaagtgtctacatcgatcggttgatattgaccggcgcatccgccagcccgcccag-
tttctggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagat-
cccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccag-
tctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgg-
gttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccggccgacaaacggcctggtggcacaaattgtcagaactacgac-
acgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaa-
aagcgcccttgtggcgctttttttatatt
cccgcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccg-
atggaagctcgctgttttaacacgcgttttt
taaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatat-
tctcaacctaaaaaagtttgtgtaatacttgt
aacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaac-
tcacttcacaccccgaaggggcaagt
tgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcggg-
taataccgtctggcgcttcgatttgtc
ccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccag-
caagtgctgtgcgtattgcacaat
gacgcctttttgcagcacggcatgatctgtctgtacgacagccagcagccgattttgaatattgaagcgttgc-
aggaagccgatcagcagttaatcccc
ggcagctcgcaaatccgtatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaatcattagt-
gctggcgcgcgttgctgacgatc
agcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatcgccgtgccgctgatagggccaga-
tgcgcagactttcggtgtgctgacggc
acaacccatggcgcgttacgaagagcgattacccgcctgcacccgctttctggaaacggtc 242
gcgcaaagcgagtgctcacttacgtgatctgttgacacaatctgaagcgaccataacttctgccgtttca-
gcgaatacggcggtgtggagcgcacaatc
agccctggcgaagctggtgctcaccgagtggctagtgacgcagggctggcgaaccttccttgatgaaaaagcg-
caggccaaattcgccgactccttt
aaacgctttgctgacatccatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccaaccgctgggcg-
acagctatcgcgaccagttgccgc
gcctggcgcgtgatatcgactgcgcgttactgctggccgggcattacgatcgcgcgcgcgccgtggaatggct-
ggaaaactggcaggggcttcagc
acgccattgaaacgcgccagagagtcgaaatcgaacatttccgtaataccgcgattacccaggagccgttctg-
gttgcacagcggaaaacgttaacg
aaaggatatttcgcatgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctga-
gagctggcgcgaattgtggcaggatg
cgttgcaggaggaggattccacgcccgtgaggcgcatctctcagaggacgatcgccgccgcgtggtggcgctg-
attgccgattttcgcaaagagtt
ggataaacgcaccattggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgta-
tgacgcgcgacgatgcgccagta
ccgctgtcacgcctgacgccgctgacaccggaattattacccgcaccacttaccttgagctgctaagtgaatt-
tcccggcgcactgaaacacctcattt
ccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcga-
cccgaatacgctctatcaaccgacg
gcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagc-
ttgaggcgctgcggcagtttaag
caggcgcagttgctgcgcgtcgcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcact-
taacctggctggcggaagcgatta
ttgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcg-
cgaagggcgcggttttgcggtgg
tcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactg-
cccgatggatgtgatgaccgatggc
gagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgca-
cgtcgccggcatcctttatgaagttg
atgcgcgctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaa-
aacgaagcctggacgtgggaaca
tcaggcgctggcccgtgcgcgcgggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcg-
atattctgatgacgcctcgcgac
ggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaag-
accgcttcgatctgaaagccgat
gaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaac-
tgacgcgctggtcggataatgtgcg
cattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacacc-
acattgcgtgatgagctgcacc
acctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaac-
cagctgggacaagtggctggtggaa
ccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacaggaatgaaagtg-
acgctgccagagtttaagcaagccg
gtgtaatggtggtgggtgatgtgatgctggatcgttactggtatggcccaaccagccgtatctctccggaagc-
gccagtcccggttgttaaagtcgatac
cattgaagagcgtcctggcgccgcggcaaacgtggcgatgaatatcgcctcactgggcgccacggcgcgtctg-
gttggcctgactggcattgacga
tgcggcgcgcgcgctgagcaaagcgctggccgatgttaacgttaaatgtgacttcgtttctgttccgacgcat-
cccaccatcactaagctgcgcgtgct
gtcgcgtaaccagcagctgattcgcctggactttgaagagggttttgaaggagtcgatccgcaaccgatgcat-
gaacgcatcagccaggcgcttggta atattggcgcgctggtgctgtcggatt 243
atgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcg-
aattgtggcaggatgcgttgcaggagga
ggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgatttt-
cgcaaagagttggataaacgcacc
attggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacg-
atgcgccagtaccgctgcacgcc
tgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcact-
gattacacctcatttccctgtgtgccgc
gtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctc-
tatcaaccgacggcgatgaatgccta
tcggatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcggc-
agtttaagcaggcgcagttgct
gcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcg-
gaagcgattattgatgcggtggtg
cagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgacgatcgcgaagggcgcggttt-
gcggggtcggttatggcaag
ctgggcggctgggagctgggtacagctccgatctggatctggtattcctgcacgactgcccgatggatgtgat-
gaccgatggcgagcgtgaaatcg
atggtcgccagtctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgacgtcgtccggcatcc-
tttatgaagttgatgcgcgtctgcgt
ccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctgga-
cgtgggaacatcaggcgctggcc
cgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatga-
cgcctcgcgacggcgcaacgctgc
aaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatct-
gaaagccgatgaaggcggtatca
ccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtc-
ggataatgtgcgcattctcgaagggc
tggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatga-
gctgcaccacctgggctgcaa
gagttgccgggacatgtggcgctctcctgtttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtg-
gctggtggaaccgtgcgccccggc gtaa 244
gcgcaaagcgagtgctcacttacgtgatctgttgacacaatctgaagcgaccataacttctgccgtttca-
gcgaatacggcggtgtggagcgcacaatc
agccctggcgaagctggtgctcaccgagtggctagtgacgcagggctggcgaaccttccttgatgaaaaagcg-
caggccaaattcgccgactccttt
aaacgctttgctgacatccatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccaaccgctgggcg-
acagctatcgcgaccagttgccgc
gcctggcgcgtgatatcgactgcgcgttactgctggccgggcattacgatcgcgcgcgcgccgtggaatggct-
ggaaaactggcaggggcttcagc
acgccattgaaacgcgccagagagtcgaaatcgaacatttccgtaataccgcgattacccaggagccgttctg-
gttgcacagcggaaaacgttaacg
aaaggatatttcgcatgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctga-
gagctggcgcgaattgtggcaggatg
cgttgcaggaggaggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgct-
gattgccgattttcgcaaagagtt
ggataaacgcaccattggcccgcgagggcggaaggtactcgatcacttaatgccgcatctgctcagcgatgta-
tgctcgcgcgacgatgcgccagta
ccgctgtcacgcctgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaat-
ttcccggcgcactgaaacacctcattt
ccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcga-
cccgaatacgctctatcaaccgacg
gcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagc-
ttgaggcgctgcggcagtttaag
caggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcact-
taacctggctggcggaagcgatta
ttgatgcggtgggcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatagcacgatcgcg-
aagggcgcggttttgcggtgg
tcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcctgacgactgc-
ccgatggatgtgatgaccgatggc
gagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgca-
cgtcgtccggcatcctttatgaagttg
atgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagca-
aaacgaagcctggacgtgggaaca
tcagccgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgc-
gatattctgatgacgcctcgcgac
ggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagagaaaatgcgtgcccatcttggcaacaagcataa-
agaccgcttcgatctgaaagccgat
gaaggcggtatcaccgacatcgagtttatcgcccaatatctgggctgcgctttgcccatgacaagccgaaact-
gacgcgctggcggataatgtgcg
cattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacacc-
acattgcgtgatgagctgcacc
acctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaac-
cagctgggacaagtggctggtggaa
ccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacagcaatgaaagtg-
acgctgccagagtttaagcaagccg
gtgtaatggtggtgggtgatgtgatgctggatcgttactggtatggcccaaccagccgtatctctccggaagc-
gccagtcccggttgttaaagtcgatac
cattgaagagcgtcctggcggcgcggcaaacgtggcgatgaatatcgcctcactgggcgccacggcgcgtctg-
gttggcctgactggcattgacga
tgcggcgcgcgcgctgagcaaagcgctggccgatgttaacgtaaatgtgacttcgtttctgttccgacgcatc-
ccaccatcactaagctgcgcgtgct
gtcgcgtaaccagcagctgattcgcctggactttgaagagggttttgaaggagtcgatccgcaaccgatgcat-
gaacgcatcagccaggcgcttggta atattggcgcgctggtgctgcggatt 245
atgaccctgaatatgatgatggatgccggcggacatcatcgcgacaaacaatattaataccggcaaccac-
accggcaatttacgagactgcgcaggc
atcctttctcccgtcaatttctgtcaaataaagtaaaagaggcagtctacttgaattacccccggctggttca-
gcgtttgttgaaaaaaagtaactgaaaaa
tccgtagaatagcgccactctgatggtaattacctattcaattaagaattatctggatgaatgtgccattaaa-
tgcgcagcataatggtggttgtgcgg
gaaaactgcttttttttgaaagggttggtcagtagcggaaacaactcacttcacaccccgaagggggaagttg-
cctgaccctacgattcccgctatttcat tcactgaccggaggttcaaaatga 246
accggatacgagagaaaagtgtctacatcggttcggttgatattgaccggcgcatccgccagcccgccca-
gtttctggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaanataccaatatttgccataacacacgctcctgttgaaaaagagat-
cccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaacctgcaccagtttggttattaatgcaccagt-
ctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgg-
gttatcagccaaaggtgcactcttt
gcatggtatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgaca-
cgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggcggacatcatcgcgacaaacaatattaataccggcaaccac-
accggcaatttacgagactgcgca
ggcatcctttctcccgtcaatttctgcaaataaagtaaaagaggcagtctacttgaattacccccggctggtt-
gagcgtttgttgaaaaaaagtaactgaa
aaatccgtagaatagcgccactctgatggttaattaacctattcaattaagaattatctggatgaatgtgcca-
ttaaatgcgcagcataatggtgcgttgtg
cgggaaaactgcttttttttgaaagggttggtcagtagcggaaacaactcacttcacaccccgaagggggaag-
ttgcctgaccctacgattcccgctatt
tcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtaggcgcttcgatttgtc-
ccagcagttcactgcgatgcagcgc
ataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgacaatga-
cgcctttttgcagcacggcatgat
ctgctgtacgacagccaccaggcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatccccg-
gcagctcgcaaatccgctatcgtcc
gggcgaagggctggtcgggacggtgctttcgcagggccaatcattagtgctggcgcgcgttgctgacgatcag-
cgctttcttgaccggctcggttgt
atgattacaacctgccgtttatcgccgtgccgctgatagggccagatgcgcagactttcggtgtgctgacggc-
acaacccatggcgcgttacgaagag cgattacccgcctgcacccgctttctggaaacggtc 247
atgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaa-
aagcgcccttgtggcgctttttttatattccc
gcctccatttaaaataaaaaatccaatcggattcactatttaaactggccattatctaagatgaatccgatgg-
aagctcgctgttttaacacgcgttttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattct-
caacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactca-
cttcacaccccgaagggggaagttgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga 248
accggatacgagagaaaagtgctacatcggttcggttgatattgaccggcgcatccgccagcccgcccag-
tttctggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagat-
cccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccag-
tctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggtttgtcggg-
ttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgac-
acgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaa-
aagcgcccttgtggcgctttttttatatt
cccgcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccg-
atggaagctcgctgttttaacacgcttttt
taaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatat-
tctcaacctaaaaaagtttgtgtaatacttgt
aacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaac-
tcacttcacaccccgaagggggaagt
tgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcggg-
gtaataccgtctggcgcttcgatttgtc
ccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccag-
caagtgctgtgcgtattgcacaat
gacgccttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgca-
ggaagccgatcagcagttaatcccc
ggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaatcattag-
tgctggcgcgcgttgctgacgatc
agcgctttcttgaccggctcgggttgatgattacaacctgccgtttatcgccgtgccgctgatagggccagat-
gcgcagactttcggtgtgctgacggc
acaacccatggcgcgttacgaagagcgattcccgcctgcacccgctttctggaaacggtc 249
atggcactgaaacacctcattccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctaccc-
gatcctgcttgatgaattgctcgacccg
aatacgctctatcaaccgacggcgatgaatgcctatcgcgatgagagcgccaatacctgctgcgcgtgccgga-
agatgatgaagagcaacagcttg
aggcgctgcggcagtttaagcaggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagt-
aatgaaagtgagcgatcacttaac
ctggctggcggaggcgattattgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccag-
ccaacgcatctgcacgatcgcga
agggcgcggttttgcggtggtcggttatggcaagctgggcggctgggagctgggttacagctccgatctggat-
ctggtattcctgcacgactgcccga
tggatgtgatgaccgatggcgagcgtgaaatcgatggtcaccagttctatttgcgtctcgcgcagcgcgtgat-
gcacctgtttagcacgcgcacgtcgt
ccggcatcctttatgaagttgatgcgcgtctgtccatctggcgctgcggatgctggtcactactacggaatcg-
ttcgccgattaccagcaaaacg
aagcctggacgtgggaacatcaggcgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccga-
attgacgccattcgccgcgata
ttctgatgacgcctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgccca-
tcttggcaacaagcataaagacc
gcttcgatctgaaagccgatgaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctt-
tgcccatgacaagccgaaactgacgc
gctggtcggataatgtgcgcattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggc-
attgacgctggcgtacaccaca
ttgcgtgatgagctgcaccacctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccg-
agcgtgcgcttattaaaaccagctgg gacaagtggctggtggaa 250
gcgcaaagcgagtgctcacttacgtgatctgttgacacaatctgaagcgaccataacttctgccgtttca-
gcgaatacggcggtgtggagcgcacaatc
agccctggcgaagctggtgctcaccgagtggctagtgacgcagggctggcgaaccttccttgatgaaaaagcg-
caggccaaatcgccgactccttt
aaacgctttgctgacatccatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccaaccgctgggcg-
acagctatcgcgaccagttgccgc
gcctggcgcgtgatatcgactgcgcgttactgctggccgggcattacgatcgcgcgcgcgccgtggaatggct-
ggaaaactggcaggggcttcagc
acgccattgaaacgcgccagagagtcgaaatcgaacatttccgtaataccgcgattacccaggagccgttctg-
gttgcacagcggaaaacgtttacg
aaaggatatttcgcatggcactgaaacacctcatttccctgtgtgccgcgtcgccgatggttgccagtcagct-
ggcgcgctacccgatcctgcttgatga
attgctcgacccgaatacgctctatcaaccgacggcgatgaatgcctatcgcgatgagctgcgccaatacctg-
ctgcgcgtgccggaagatgatgaa
gagcaacagcttgaggcgctgcggcagtttaagcaggcgcagttgctgcgcgtggcggcgggatattgccggt-
acgttgccagtaatgaaagtg
agcgatcacttaacctggctggcggaagcgattattgatgcggtggtgcagcaagcctgggggcagatggtgg-
cgcgttatggccagccaacgcat
ctgcacgatcgcgaagggcgcggttttgcggtggtcggttatggcaagagggcggctgggagctgggttacag-
ctccgatctggatctggtattcct
gcacgactgcccgatggatgtgatgaccgatggcgagcgtgaaatcgatggtcgcagttctatttgcgtctcg-
cgcagcgcgtgatgcacctgttag
cacgcgcacgtcgtccggcatcctttatgaagttgatgcgcgtctgcgtccatctagcgctgcggggatgctg-
gtcactactacggaatcgttcgccga
ttaccagcaaaacgaagcctggacgtgggaacatcaggcgctggcccgtgcgcgtggtgtacggcgatccgca-
actgaccgccgaatttgacg
ccattcgccgcgatattctgatgacgcctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcga-
gaaaatgcgtgcccatcttggca
acaagcataaagaccgcttcgatctgaaagccgatgaaggcggtatcaccgacatcgagtttatcgcccaata-
tctggtgctgcgctttgcccatgaca
agccgaaactgacgcgctggtcggataatgtgcgcattctcgaacggctggcgcaaaacggcatcatggagga-
gcaggaagcgcaggcattcacg
ctggcgtacaccacattgcgtgatgagctgcaccacctggcgctgcaagagttgccgggacatgtggcgctct-
cctgttttgtcgccgagcgtgcgctt
attaaaaccagctgggacaagtggctggtggaaccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaa-
ttttgtatctctcaggagacaggaa
tgaaagtgacgctgccagagtttaagcaagccggtgtaatggtggtgggtgatgtgatgctggatcgttactg-
gtatggcccaaccagccgtatctctc
cggaaggccagtcccggttgttaaagtcgataccattgaagagcgtcctggcggcgcggcaaacgtggcgatg-
aatatcgcctcactgggcgcca
cggcgcgctggttggcctgactggcattgacgatgcggcgcgcgcgctgagcaaagcgctggccgatgttaac-
gttaaatgtgacttcgtttctgttc
cgacgcatcccaccatcactaagctgcgcgtgctgtcggtaaccagcagctgattcgcctggactttgaagag-
ggttttgaaggagtcgatccgcaa
ccgatgcatgaacgcatcagccaggcgcttggtaatattggcgcgctggtgctgtcggatt 251
tttcgctgaaggtgtgaccatgggccatcaggtgctggtgcagctggaaagtgttgccatcactatcgtg-
tggtctggcgtggtggcctttattggttaca
aactggcggacatgacggtaggcctgcgcgtaccggaagaacaagaacgtgaagggctggatgaaacagccac-
ggcgaaaacgcctataacgc ctga 252
tttcctttctgactctgcccgtccgggcgcactaacggcctgaaatactccctcttttcattcctggcac-
aacgattaaatgtagttgcgtgttagctgcg
gccattatcgaattcgactggagggggatctatgaagctggttaccgtggtgattaagccattcaaacttgaa-
gacgtgcgtgaagcgctttcttctattg
gtattcaagggttgaccgtaactgaagtgaaaggctttggccgtcagaagggtcacgctgagctgtaccgcgg-
tgcggaatatagcgttaatttcctgc
cgaaagtgaaaattgatgtggcgatcgctgacgatcaactcgatgaagtaatcgatgtgatcagcaaagcggc-
ctacaccggaaaaattggcgacgg
caaaattttcgttgctgagctgcaacgcgtcattcgtattcgtaccggcgaagccgacgaagcggcactgtaa-
tacaagacacacagtgatggggatc
ggtttcgctgaaggtgtgaccatgggccatcaggtgctggtgcagctggaaagtgttgccatcactatcgtgt-
ggtctggcgtggtggcctttattggtta
caaactggcggacatgacggtaggcctgcgcgtaccggaagaacaagaacgtgaagggctggatgtaaacagc-
cacggcgaaaacgcctataac
gcctgattgcgttgagttatctcctgagcataaaaaagcctccattcggaggcttttctttttttaagtttaa-
aggcggttagttgcgattgcgcatgacgcc
ttcctgcacgctggacgcgaccagcacaccctcttgcgtatagaactcgccgcgcacattaaccgcgagcgct-
ggaggctgacgtgctttccacactg
tagagcagccattcgttcatattaaacgggcgatggaaccacatggagtggtcaatggtggcaacctgcatac-
cgcgctcaaggaagcccacgccgt
gcggctgaagtgcaaccggcaggaagttaaagtctgaggcatatccaagcagatattgatgtacgcgaaaatc-
gtccacaccgtgccgtttgcgcg
gatccatacctggcgggtgggatcggcaacgtggcctttcagcgggttatgaaactcaaccgggcggatctcc-
agtggtttatcactaagaaacttctc tttggcctgcgg 253
atgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcg-
aattgtggcaggatgcgttgcaggagga
ggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgatttt-
cgcaaagagttggataaacgcacc
attggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacg-
atgcgccagtaccgctgtcacgcc
tgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcact-
gaaacacctcatttccctgtgtgccgc
gtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctc-
tatcaaccgacggcgatgaatgccta
tcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcgg-
cagtttaagcaggcgcagttgct
gcgcgggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcgg-
aagcgattattgatgcggtggtg
cagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggtt-
ttgcggtggtcggttatggcaag
ctgggcggctgggagctgggttacagatccgatctggatctggtattcctgcacgactgcccgatggatgtga-
tgaccgatggcgagcgtgaaatcg
atggtcgccagttctatttgcgtctcccgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcat-
cctttatgaagttgatgcgcgtctgcgt
ccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctgga-
cgtgggaacatcaggcgctggcc
cgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatga-
cgcctcgcgacggcgcaacgctgc
aaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatct-
gaaagccgatgaaggcggtatca
ccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtc-
ggataatgtgcgcattctcgaagggc
tggcgcaaaacggcatcatggacgagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatga-
gctgaccacctggcgctgcaa
gagttgccgggacatgggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtg-
gctggtggaaccgtgcgccccggc gtaa 254
gcgcaaagcgagtgctcacttacgtgatctgttgacacaatctgaaggaccataacttctgccgtttcag-
cgaatacggcggtgtggaggcacaatc
agccctggcgaagctggtgctcaccgagtggctagtgacgcagggctggcgaaccttccttgatgaaaaagcg-
caggccaaattcgccgactccttt
aaacgctttgctgacatccatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccaaccgctgggcg-
acagctatcgcgaccagttgccgc
gcctgccgcgtgatatcgactgcgcgttactgctggccgggcattacgatcgcgcgcgcgccgtggaatggct-
ggaaaactggcaggggcttcagc
acgccattgaaacgcgccagagagtcgaaatcgaacatttccgtaataccgcgattacccaggagccgttctg-
gttgcacagcggaaaacgttaacg
aaaggatatttcgcatgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctga-
gagctggcgcgaattgtggcaggatg
cgttgcaggaggaggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgct-
gattgccgattttcgcaaagagtt
ggataaacgcaccattcgcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgta-
tgctcgcgcgacgatgcgccagta
ccgctgtcacgcctgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaat-
ttcccggcgcactgaaacacctcattt
ccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcga-
cccgaatacgctctatcaaccgacg
gcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagc-
ttgaggcgtgggcagtttaag
caggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcact-
taacctggctggcggaagcgatta
ttgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcg-
cgaagggcgcggttttgcggtgg
tcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactg-
cccgatggatgtgatgaccgatggc
gagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgca-
cgtcgtccggcatcctttatgaagttg
atgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagca-
aaacgaagcctggacgtgggaaca
tcaggcgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgc-
gatattctgatgacgcctcgcgac
ggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaag-
accgcttcgatctgaaagccgat
gaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaac-
tgacgcgctggtcggataatgtgcg
cattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacacc-
acattgcgtgatgagctgcacc
acctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaac-
cagctgggacaagtggctggtggaa
ccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacaggaatgaaagtg-
acgctgccagagtttaagcaagccg
gtgtaatggtggtgggtgatgtgatgctggatcgttactggtatggcccaaccagccgtatctctccggaagc-
gccagtcccggttgttaaagtcgatac
cattgaagagcgtcctggcggcgcggcaaacgtggcgatgaatatcgcctcactgggcgccacggcgcgtctg-
gttggcctgactggcattgacga
tgcggcgcgcgcgctgagcaaagcgctggccgatgttaacgttaaatgtgacttcgtttctgttccgacgcat-
cccaccatcactaagctgcgcgtgct
gtcgcgtaaccagcagctgattcgcctggactttgaagagggttttgaaggagtcgatccgcaaccgatgcat-
gaacgcatcagccaggcgcttggta atattggcgcgctggtgctgtcggatt 255
atgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattggactgattaaaaa-
agcgcccttgtggcgattttttatattccc
gcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatg-
gaagctcgctgttttaacacgcgttttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattct-
caacctaaaaaagtttgtgtaatacttgtaac
gcttcatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactca-
cttcacaccccgaaggcgaagttgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga 256
accggatacgagagaaaagtgtctacatcggttcggttgatattgaccggcgcatccgccagcccgccca-
gtttctggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagat-
cccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccag-
tctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgg-
gttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgac-
acgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaa-
aagcgcccttgtggcgctttttttatatt
cccgcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccg-
atggaagctcgctgttttancacgcgttttt
taaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatat-
tcttcaacctaaaaaagtttgtgtaatacttgt
aacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaac-
tcacttcacaccccgaagggggaagt
tgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcggg-
taataccgtctggcgcttcgatttgtc
ccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccag-
caagtgctgtgcgtattgcacaat
gacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgc-
aggaagccgatcagcagttaatcccc
ggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaatcattag-
tgctggcgcgcgttgctgacgatc
agcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatcgccgtgccgctgatagggccaga-
tgcgcagactttcggtgtgctgacggc
acaacccatggcgcgttacgaagagcgattacccgcctgcacccgctttctggaaacggtc 257
tttcgctgaaggtgtgaccatgggccatcaggtgctggtgcagctggaaagtgttgccatcactatcgtg-
tggtctggcgtggtggcctttattggttaca
aactggcggacatgacggtaggcctgcgcgtaccggaagaacaagaacgtgaagggctggatgtaaacagcca-
cggcgaaaacgcctataacgc ctga 258
tttcctttctgactctgcccgtccgggcgcactaacggcctgaaatactccctcttttcattcctggcac-
aacgattgcaatgtctgttgcgtgttagctgcg
gccattatcgaattcgactggagggggatctatgaagctggttaccgtggtgattaagccattcaaacttgaa-
gacgtgcgtgaagcgctttcttctattg
gtattcaagggttgaccgtaactgaagtgaaaggctttggccgtcagaagggtcacgctgagctgtaccgcgg-
tgcggaatatagcgttaatttcctgc
cgaaagtgaaaattgatgtggcgatcgctgacgatcaactcgatgaagtaatcgatgtgatcagcaaagcggc-
ctacaccggaaaaattggcgacgg
caaaattttcgttgctgagctgcaacgcgtcattcgtattcgtaccggcgaagccgacgaagggcactgtaat-
acaagacacacagtgatggggatc
ggtttcgctgaaggtgtgaccatgggccatcaggtgctggtgcagctggaaagtgttgccatcactatcgtgt-
ggtctggcgtggtggcctttattggtta
caaactggcggacatgacggtaggcctgcgcgtaccggaagaacaagaacgtgaagggctggatgtaaacagc-
cacgacgaaacgcctataac
gcctgattgcgttgagttatctcctgagcataaaaaagcctccattcggaggcttttctttttttaagtttaa-
agcgcggttagttgcgattgcgcatgacgcc
ttcctgcacgctggacgcgaccagcacaccctcttgcgtatagaactcgccgcgcacaaaaccgcgagcgctg-
gaggctgacgtgctttccacactg
tagagcagccattcgttcatattaaacgggcgatggaaccacatggagtggtcaatggtggcaacctgcatac-
cgcgctcaaggaagcccacgccgt
gcggctgaagtgcaaccggcaggaagttaaagtctgaggcatatccaagcagatattgatgtacgcgaaaatc-
gtccggcaccgtgccgtttgcgcg
gatccatacctggcgggtggcatcggcaacgtggcctttcagcgggttatgaaactcaaccgggcggatctcc-
agtggtttatcactaagaaacttctc tttggcctgcgg 259
atgacctttaatatgatgcctggggtcactggagcgcttttatcggcatcctgaccgaagaatttgccgg-
tttcttcccgacctggctggcccctgttcagg
ttgtggtgatgaatatcactgattctcaagctgaatatgtcaacgaattgacccgtaaattgataaatgcggg-
cattcgtgtaaaagcggacttgagaaac
gagaagattggctttaaaatccgcgagcacactttacgtcgtgtcccttatatgttggtctgtggtgataaag-
aggtggaagcaggcaaagtggccgttc
gcacccgccgcggtaaagacctgggcagcctggacgtaaggaagtgattgagaagctgcaacaacagattcgc-
agccgcagtcttcaacaactgg
aggaataaggtattaaaggcggaatacgagttcaaacggcacgtccgaatcgtatcaatggcgagattcgcgc-
ccaggaagttcgcttaactggtctg
gaaggtgagcagctgggtattgcaatagaactaactacccgccctgaaggcggtacctgcctgaccctgcgat-
tcccgttatttcattcactgaccgga ggcccacgatga 260
ggtacgacaaaaacgtctccagcgacgtgcggttaatattgactggcgcatccgccacatcccccagttt-
ttgctggatcagtttggcgattttgcgggtt
tttcccgtgtcactgccaaaaaaaataccaatgttagccatgtcgcgctcctgttgagaaagaataaggccgc-
ctgcaaacggcggatatcccttctcct
gttgcgaaggctgtgccaggtttttttaaggccttctgtgcactgaaatgcgtgaaaaaatgactcttttttg-
tgcaggcaccgtcctctctccgctatccag
acctgctttgaaggcctctgagggccaaatcagggccaaaacacgaatcacgatcaatgtttcggcgcgttac-
ctgttcgaaaggtgcactctttgcat
ggttaatcacacccaatcaggcctgcggatgtcgggcgtttcacaacacaaaatgttgtaaatgcgacacagc-
cgggcctgaaaccaggagcgtgtg
atgacctttaatatgatgcctggggtcactggagcgctttatcggcatcctgaccgaagaatttgccggtttc-
ttcccgacctggctggcccctgttcagg
ttgtggtgatgaatatcactgattctcaagctgaatatgtcaacgaattgacccgtaaattgcaaaatgcggg-
cattcgtgtaaaagcggacttgagaaac
gagaagattggctttaaaatccgcgagcacactttacgtcgtgtcccttatatgttggtctgtggtgataaag-
aggtggaagcaggcaaagtggccgttc
gcacccgccgcggtaaagacctgggcagcctggacgtaagtgaagtgattgagaagctgcaacaagagattcg-
cagccgcagtcttcaacaactgg
aggaataaggtattaaaggcggaaaacgagttcaaacggcacgtccgaatcgtatcaatggcgagattcgcgc-
ccaggaagttcgcttaactggtctg
gaaggtgagcagctgggtattgcaatagaactaactacccgccctgaaggcggtacctgcctgaccctgcgat-
tcccgttatttcattcactgaccgga
ggcccacgatgacccaccgacccgagtcgggcaccaccgtctggcgttttgatctctcacagcaatttaccgc-
catgcagcgcatcagcgtggtgttg
agtcgcgcaaccgagataagccagacgctgcaggaggtgctgtgtgttctgcataatgacgcatttatgcaac-
acggcatgctgtgtctgtatgacaac
cagcaggaaattctgagtattgaagccttgcaggaggcagaccaacatctgatccccggcagctcgcaaattc-
gctatcgccctggcgaagggctgg
taggagccgtactgtcccagggacaatctcttgtgctgccgcgtgtcgccgacgatcaacgctttctcgacag-
gcttggcatctatgattacaacctgcc
gtttatcgccgtccccttaatggggccaggcgcgcagacgattggcgtgctcgccgcgcagccgatggcgcgt-
ctggaggagcggcttccttcctgt acgcgctttctggaaaccgtc 261
ttgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagc-
acagagagcttgctctcgggtgacgagt
ggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaatacc-
gcataacgtcgcaagaccaaaga
ggcggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctcacc-
taggcgacgatccctagctggtctg
agaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgc-
acaatgggcgcaagcctgatgc
agccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaagggagtaaggtta-
ataaccttattcattgacgttacccg
cagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattac-
tgggcgtaaaccgcacgcaggc
ggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcatccgaaactggcaggcttgagtct-
cgtagagggaggtagaattccagg
tgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactgacgc-
tcaggtgcgaaagcgtgggga
gcaaacaggattagataccctggtagtccacgccgtaaacgatgtctatttggaggttgtgcccttgaggcgt-
ggcttccggagctaacgcgttaaata
gaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagca-
tgtggtttaattcgatgcaacgc
gaagaaccttacctggtcttgacatccacagaactttccagagatggattggtgccttcgggaactgtgagac-
aggtgctgcatggctgtcgtcagctc
gtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggccggg-
aactcaaaggagactgccagtgataa
actggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggc-
gcatacaaagagaagcgacctcg
cgagagtaagcggacctcataaagtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcgga-
atcgctagtaatcgtggatcagaat
gccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagta-
ggtagcttaaccttcgggagggc
gcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatc-
acctcctt 262
ttgagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagca-
cagagagcttgctctcgggtgacgagt
ggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaatacc-
gcataacgtcgcaagaccaaaga
gggggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctcacc-
taggcgacgatccctagctggtctg
agaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattcc-
acaatgggcgcaagcctgatgc
agccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaaggnantanggtta-
ataacctgtgttnattgacgttacc
cgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaatt-
actgggcgtaaagcgcacgcag
gcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcatccgaaactggcaggcttgagt-
ctcgtagagggaggtagaattcca
ggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactgac-
gctcaggtgcgaaagcgtggg
gagcaaacaggattagataccctggtagtccacgccgtaaacgatgtctatttggaggttgtgcccttgaggc-
gtggcttccggagctaacgcgttaaa
tagaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggag-
catgtggtttaattcgatgcaac
gcgaagaaccttacctggtcttgacatccacagaacttagcagagatgctttggtgccttcgggaactgtgag-
acaggtgctgcatggctgtcgtcagc
tcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaaccatatcctttgttgccagcggttaggccgg-
gaactcaaaggagactgccagtgat
aaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatg-
gcgcatacaaagagaagcgacct
cgcgagagtaagcggacctcataaagtccgtcgtagtccggattggagtctgcaactcgactccatgaagtcg-
gaatcgctagtaatcgtggatcaga
atgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaag-
taggtagcttaaccttcgggaggg
cgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggat-
cacctcctt 263
atgaccatgcgtcaatgcgccatttacggcaaaggtgggatcggcaaatcgaccaccacacagaacctgg-
tcgccgcgctggcggagatgggtaaa
aaagtcatgattgtcggctgtgacccgaaagccgattccacgcgtttgatcctgcatgcgaaagcgcagaaca-
ccattatggagatggctgagaagt
cggctccgtggaagacctggagttagaagacgtgctgcaaatcggttacggcggcgtgcgctgcgcagagtcc-
ggcggcccggagccaggcgtg
ggctgtgccggtcgccgggtgatcaccgcgattaacttcctcgaagaagaaggcgcttacgtgccggatctcg-
attttgttttctacgacgtgctgggc
gacgtggtatgcggtggtttcgccatcccgattcgtgaaaacaaagcgcaggagatctacatcgtttgctctc-
gcgaaatgatggcgatgtacgccgc
caacaacatctccaaaggcatcgtgaaatacgccaaatccggtaaagtgcgcctcggcgggctgatttgtaac-
tcgcgccagaccgaccgtgaagat
gaactgatcattgcgctggcagaaaaactcggcacgcagatgatccactttgttccccgcgacaacattgtgc-
agcgtgcggaaatccgccgtatgac
ggttatcgaatatgacccgacctgcaatcaggcgaacgaatatcgcagccttgccagcaaaatcgtcaacaac-
accaaaatggtggtgcccaccccc
tgcaccatggatgaactggaagaactgctgatggagttcggcattatggatgtggaagacaccagcatcattg-
gtaaaaccgccgccgaagaaaacg ccgtctga 264
atgagcaatgcaacaggcgaacgcaacctggagataatcgagcaggtgctcgaggttttcccggagaaga-
cgcgcaaagaacgcagaaaacacat
gatggtgacggacccggagcaggaaagcgtcggtaagtgcatcatctctaaccgcaaatcgcagccaggcgtg-
atgaccgtgcgcggctgctcgta
tgccggttcgaaaggggtggtatttgggccaatcaaggatatggcgcatatctcgcatggcccaatcggctgc-
ggccaatactcccgcgccgggcgg
cggaactactacaccggcgtcagcggcgtggacagcttcggcacgctcaacttcacctccgattttcaggagc-
gcgacatcgtgtttggcggcgataa
aaagctcgccaaactgattgaagagctggaagagctgttcccgctgaccaaaggcatttcgattcagtcggaa-
tgcccggtcggcctgattggcgatg
acattgaggccgtcgcgaacgccagccgcaaagccatcaacaaaccggttattccggtgcgttgcgaaggctt-
tcgcggcgtgtcgcaatccctcgg
tcaccatattgccaacgatgtgatccgcgactgggtgctggataaccgcgaaggcaaaccgttcgaatccacc-
ccttacgatgtggcgatcatcggcg
attacaacatcggcggcgatgcctgggcttcgcgcattttgctcgaagagatgggcttgcgggggtggcacag-
tggtctggcgacggtacgctggt
ggagatggaaaacacgccgttcgtcaaactgaacctggtgcattgttaccgctcaatgaactacatctcgcgc-
catatggaggagaagcacggtattc
cgtggatggaatacaacttctttggtccgacgaaaatcgcggaatcgctgcgcaaaatcgccgaccagtttga-
cgacaccattcgcgccaacgccga
agcggtgatcgccagataccagccgcaaaacgacgccattatcgccaaatatcgcccgcgtctggaggggcgc-
aaagtgctgctttatatgggcgg
gctgcgtccgcgccatgtgattgccgcctatgaagacctgggaatggagatcatcgctgccgctatgagttcg-
gtcataacgatgattacgaccgca
ccttgccggatctgaaagagggcacgctgctgtttgagatgccagcagttatgagctggaggcgttcgtcaac-
gcgctgaaaccggatctcatcggtt
ccggcatcaaagagaagtacatctttcagaaaatgggcgtgccgtttcgccagatgcactcctgggattactc-
cggcccgtaccacggctatgacggc
ttcgccatcttcgcccgcgatatggatatgacgctcaacaaccccgcgtggggccagttgaccgcgccgtggc-
tgaaatccgcctga 265
atgagccagactgctgagaaaatacagaattgccatcccctgtttgaacaggatgcttaccagacgctgt-
ttgccggtaaacgggcactcgaagaggc
gcactcgccggagcgggtgcaggaagtgtttcaatggaccactaccccggaatatgaagcgctgaactttaaa-
cgcgaagcgctgactatcgaccc
ggcaaaagcctgccagccgctgggcgcggtgctctgttcgctggggtttgccaataccctaccgtatgtgcac-
ggttcacagggttgcgtggcctattt
ccgcacgtactttaaccgccactttaaagaaccggtggcctgcgtgtcggattcaatgacggaagacgcggcc-
gtgttcggcgggaataacaacctc
aacaccggcttacaaaacgccagcgcgctgtataaaccggagattatcgccgtctctaccacctgtatggcgg-
aagtgatcggtgatgatttgcaggc
ctttatcgccaacgccaaaaaagatggttttctcgatgccgccatccccgtgccctacgcgcacacccccagt-
tttatcggcagccatatcaccggctg
ggataacatgtttgaaggttttgcccggacctttacggcagaccatgaagctcagcccggcaaactttcacgc-
atcaacctggtgaccgggtttgaaac
ctatctcggcaatttccgcgtgctgaaacgcatgatggaacaaatggaggtgccggcgagtgtgctctccgat-
ccgtcggaagtgctggatactcccg
ccaacgggcattaccagatgtacgcgggcgggacgacgcagcaagagatgcgcgaggcgccggatgctatcga-
caccctgttgctgcagccctg
gcaactggtgaaaagcaaaaaagtggtgcaggagatgtggaatcagcccgccaccgaggtttctgttcccgtt-
gggctggcaggaacagacgaact
gttgatggcgattagccagttaaccggcaaggccattcccgattcactggcgctggagcgcgggcggctggtc-
gatatgatgctcgattcccacacct
ggttgcacggtaaaaaattcggcctgtttggcgatccggattttgtcatgggattgacccgtttcctgctgga-
gctgggctgcgaaccgaccgttatcctc
tgccacaacggtaacaaacgctggcagaaagcaatgaagaaaatgcttgacgcctcgccgtacggccaggaga-
gcgaagtgtttatcaactgcgatt
tgtggcatttccgctcgctgatgtttacccgccagccggattttatgattggcaactcgtacggcaagttcat-
tcagcgcgacaccttagccaaaggcga
gcagtttgaagttccgctgatccgcctcggttttcccctgttcgaccgccaccatctgcaccgccagaccacc-
tggggctacgagggcgccatgagca
ttctcactacccttgtgaatgcggtactggagagagtggacaaagagaccatcaagctcggcaaaaccgacta-
cagcttcgatatcttatccgttaa 266
atgaccctgaatatgatgatggatgccggcgcgcccgaggcaatcgccggtgcgctttcgcgacaccatc-
ctgggctgttttttaccatcgtgaagaa
gcgcccgtcgccatttcgctgactgatgccgacgcacgcattgtctatgccaacccggctttctgccgccaga-
ccggctatgaactagaagcgttgttg
cagcaaaatccccgcctgcttgcaagtcgccaaaccccacgggaaatctatcaggatatgtggcacaccttgt-
tacaacgccgaccgtggcgcgggc
aattgattaaccgccaccgcgacggcagcctgtatctggtcgagatcgatatcaccccggtgattaacccgtt-
tggcgaactggaacactacctggca
atgcagcgcgatatcagcgccagttatgcgctggagcagcggttgcgcaatcacatgacgctgaccgaagcgg-
tgctgaataacattccgccggcg
gtggttgtagtggatgaacgcgatcatgtggttatggataaccttgcctacaaaacgttctgtgccgactgcg-
gcggaaaagagctcctgagcgaactc
aatttttcagcccgaaaagcggagctggcaaacggccaggtcttaccggtggtgctgcgcggtgaggtgcgct-
ggttgtcggtgacctgctgggcgc
tgccgcgcgtcagcgaagaagccagtcgctactttattgataacaggctgacgcgcacgctggtggtgatcac-
cgacgacacccaacaacgccagc
agcaggaacagggccgacttgaccgccttaaacagcagatgaccaacggcaaactactggcagcgatccgcga-
aggcttgacgccgcgctgatc
cagcttaactgccccatcaatatgctggcggcggcgcgacgtttaaacggcagtgataacaacaatgtggcgc-
tcgacgccgcgtacgcgaaggt
gaagaggcgatggcgcggctgaaacgttgccgcccgtcgctggaactggaaagtgcggccgtctggccgctgc-
aacccttttttgacgatctgcgc
gcgctttatcacacccgctacgagcaggggaaaaatttgcaggtcacgctggattcccatcatctggtgggat-
ttggtcagcgtacgcaactgttagcct
gcctgagtctgtggctcgatcgcacgctggatattgccgccgggctgggtgatttcaccgcgcaaacgcagat-
ttacgcccgcgaagaagagggctg
gctctctttgtatatcactgacaatgtgccgctgatcccgctgcgccacacccactcgccggatgcgcttaac-
gctccgggaaaaggcatggagctgc
gcctgatccagacgctggtggcacaccaccacggcgcaatagaactcacttcacaccccgaagggggaagttg-
cctgaccctacgattcccgctatt tcattcactgaccggaggttcaaaatga 267
atgacccagcgaaccgagtcgggtaataccgtctggcgatcgatttggcccagcagttcactgcgatgca-
gcgcataagcgtggtactcagccggg
cgaccgaggtcgatcagacgctccagcaagtgctggcgtattgcacaatgacgcctttttgcagcacggcatg-
atctgtctgtacgacagccagcag
gcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatccccggcagctcgcaaatccgctatc-
gtccgggcgaagggctggtcggga
cggtgctttcgcagggccaatcattagtgctggcgcgcgttgctgacgatcagcgctttcttgaccggctcgg-
gttgtatgattacaacctgccgtttatc
gccgtgccgctgatagggccagatgcgcagactttcggtgtgctgacggcacaacccatggcgcgttacgaag-
agcgattacccgcctgcacccgc
tttctggaaacggtcgctaacctggtcgcgcaaaccgtgcgtttgatggcaccaccggcagtgcgcccttccc-
cgcgcgccgccataacacaggccg
ccagcccgaaatcctgcacggcctcacgcgcatttggttttgaaaatatggtcggtaacagtccggcgatgcg-
ccagaccatggagattatccgtcag
gtttcgcgctgggacaccaccgttctggtacgcggcgagagtggcaccggcaaggagctgattgccaacgcca-
tccaccaccattcgccgcgtgcc
ggtgcgccatttgtgaaattcaactgtgcggcgctgccggacacactgctggaaagcgaattgttcggtcacg-
agaaaggggcatttaccggcgcgg
tacgccagcgtaaaggccgttttgagctggccgatggcggcacgctgtttcttgacgagatcggcgagtgtag-
cgcctcgtttcaggctaagctgctg
cgcattttgcaggaaggcgaaatggaacgcgtcggcggcgacgagacattgcaagtgaatgtgcgcattattg-
ccgcgacgaaccgcaatcttgaa
gatgaagtccggctggggcactttcgcgaagatctctattatcgcctgaatgtgatgcccatcgccctgccgc-
cactacgcgaacgccaggaggacat
tgccgagctggcgcactttctggtgcgtaaaatcgcccataaccagagccgtacgctgcgcattagcgagggc-
gctatccgcctgctgatgagctaca
actggcccggtaatgtgcgcgaactggaaaactgccttgagcgctcagcggtgatgtcggagaacggtctgat-
cgatcgggatgtgattttgtttaatc
atcgcgaccagccagccaaaccgccagttatcagcgtctcgcatgatgataactggctcgataacaaccttga-
cgagcgccagcggctgattgcggc
gctggaaaaagcgggatgggtacaagccaaagccgcgcgcttgctggggatgacgccgcgccaggtcgcctat-
cgtattcagacgatggatataac cctgccaaggctataa 268
atgccgcaccacgcaggattgtcgcagcactggcaaacggtattttctcgtctgccggaatcgctcaccg-
cgcagccattgagcgcgcaggcgcagt
cagtgctcacttttagtgattttgttcaggacagcatcatcgcgcatcctgagtggctggcagagcttgaaag-
cgcgccgccgcctgcgaacgaatggc
aacactatgcgcaatggctgcaagcggcgctggatggcgtcaccgatgaagcctcgagatgcgcgcgctgcgg-
ctgtttcgccgtcgcatcatggt
gcgcatcgcctggagccaggcgttacagttggtggcggaagaagatatcctgcaacagcttagcgtgctggcg-
gaaaccctgatcgtcgccgcgcg
cgactggctttatgaggcctgctgccgtgagtggggaacgccgagcaatccacaaggcgtggcgcagccgatg-
ctggtactcggcatgggcaaact
gggtggcggcgaactcaatttctcatccgatatcgatttgattttcgcctgggacggaaaatggcgcaacgcg-
cggtggacgccgtgagctggataacg
cgcaatttttcactcgccttggtcaacggctgattaaagtcctcgaccagccaacgcaggatggctttgtcta-
ccgcgtcgatatgcgcttgcgcccgttt
ggcgacagcggcccgctggtgctgagctttgccgcgctggaagattactaccaggagcaggggcgcgattggg-
aacgctacgcgatggtgaaagc
gcgcattatgggcgataacgacggcgaccatgcgcgggagttgcgcgcaatgctgcgcccgtttgttttccgc-
cgttatatcgacttcagcgtgattca
gtccctgcgtaacatgaaaggcatgattgcccgcgaagtgcgtcgccgtggcctgaagacaacattaagctcg-
gcgcgggcgggatccgcgaaat
agaatttatcgtccaggttttccagctgattcgcggcggtcgcgagcctgcactgcaatcgcgttcactgttg-
ccgacgcttgctgccatagatcaactg
catctgctgccggatggcgacgcaacccggctgcgcgaggcgtatttgtggctgcgacggctggagaacctgc-
tgcaaagcatcaatgacgaacag
acacagacgctgccgggcgatgaactgaatcgcgcgcgcctcgcctggggaatgggcaaagatagctgggaag-
cgctctgcgaaacgctggaag
cgcatatgtcggcggtgcgtcagatatttaacgatctgattggcgatgatgaaacggattcgccggaagatgc-
gctttctgagagctggcgcgaattgt
ggcaggatgcgagcaggaggaggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtg-
gtggcgctgattgccgattttcg
caaagagttggataaacgcaccattggcccgcgagggoggcaggtactcgatcacttaatgccgcatctgctc-
agcgatgtatgctcgcgcgacgat
gcgccagtaccgctgtcacgcctgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgc-
taagtgaatttcccggcgcactgaaa
cacctcatttccctgtgtgccgcgtcgccgatggttgccagtcagaggcgcgctacccgatcctgcttgatga-
attgctcgacccgaatacgctctatc
aaccgacggcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaaga-
gcaacagcttgaggcgctgcgg
cagtttaagcaggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtga-
gcgatcacttaacctggctggcgg
aagcgattattgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatct-
gcacgatcgcgaagggcgcggtt
ttgcggtggtcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcct-
gcacgactgcccgatggatgtgatga
ccgatggcgagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttag-
cacgcgcacgtcgtccggcatccttt
atgaagttgatgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccga-
ttaccagcaaaacgaagcctggacg
tgggaacatcaggcgctggcccggcggcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccatt-
cgccgcgatattctgatgacgc
ctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaa-
gcataaagaccgcttcgatctga
aagccgatgaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaa-
gccgaaactgacgcgctggtcggat
aatgtgcgcattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctgg-
cgtacaccacattgcgtgatga
gctgcaccacctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgctt-
attaaaaccagctgggacaagtggc tggtggaaccgtgcgccccggcgtaa 269
attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtag-
cacagagagcttgactctcgggttgacga
gtggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaata-
ccgcataacgtcgcaagaccaaa
gagggggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctca-
cctaggcgacgatccctagctggt
ctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatat-
tgcacaatgggcgcaagcctga
tgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaagggagtaagg-
ttaataaccttgctcattgacgttat
ccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaat-
tactgggcgtaaagcgcacgca
ggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcatccgaaactggcaggcttgag-
tctcgtagagggaggtagaattcc
aggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactga-
cgctcaggtgcgaaagnnnnn
nnnnnnaacaggattagataccctggtagtccatgccgtaaacgatgtctactagccgttggggcctttgagg-
ctttagtggcgcagctaacgcgata
agtagaccgcctggggagtacggtcgcaagactaaanctcaaatgaattgacgggggcccgcacaagcggtgg-
agcatgtggtttaattcgatgca
acgcgaagaaccttacctggccttgacatagtaagaattttccagagatggattggtgccttcgggaacttac-
atacaggtgctgcatggctgtcgtcag
ctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcattagttgctacatttagttgg-
gcactctaatgagactgccggtgacaaa
ccggaggaaggtggggatgacgtcaagtcctcatggcccttataggtggggctacacacgtcatacaatggct-
ggtacaaagggttgccaacccgc
gagggggagctaatcccataaaaccagtcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaa-
tcgctagtaatcgtggatcagaat
gtcacggtgaatacgttcccgggtcttgtacacaccgcccgtcacaccatgggagcgggttctgccagaagta-
gttagcttaaccgcaaggagggcg
attaccacggcagggttcgtgactggggtgaagtcgtaacaaggtagccgtatcggaaggtgcggctgcatca-
cctccttt 270
atgaccatgcgtcaatgcgccatttacggcaaaggtgggatcggcaaatcgaccaccacacagaacctgg-
tcgccgcgctggcggagatgggtaaa
aaagtcatgattgtcggctgtgacccgaaagccgattccacgcgtttgatcctgcatgcgaaagcgcagaaca-
ccattatggagatggctgctgaagt
cggctccgtggaagacctggagttagaagacgtgctgcaaatcggttacggcggcgtgcgctgcgcagagtcc-
ggcggcccggagccaggcgtg
ggctgtgccggtcgcggggtgatcaccgcgattaacttcctcgaagaagaaggcgcttacgtgccggatctcg-
attttgttttctacgacgtgctgggc
gacgtggtatgcggtggtttcgccatgccgattcgtgaaaacaaagcgcaggagatctacatcgtttgctctg-
gcgaaatgatggcgatgtacgccgc
caacaacatctccaaaggcatcgtgaaatacgccaaatccggtaaagtgcgcctcggcgggctgatttgtaac-
tcgcgccagaccgaccgtgaagat
gaactgatcattgcgctggcagaaaaactcggcacgcagatgatccactttgttccccgcgacaacattgtgc-
agcgtgcggaaatccgccgtatgac
ggttatcgaatatgacccgacctgcaatcaggcgaacgaatatcgcagccttgccagcaaaatcgtcaacaac-
accatnatggtggtgcccaccccc
tgcaccatggatgaactggaagaactgctgatggagttcggcattatggatgtggaagacaccagcatcattg-
gtaaaaccgccgccgaagaaaacg ccgtctga 271
atggccgaaattctgcgcagtaaaaaaccgctggcggtcagcccgataaaaagggccagccgctgggggc-
gatcctcgcaagcctgggtgtcga
acagtgcataccgctggtacacggcgcacagggatgtagcgcgttcgcgaaggtgttctttattctacatttt-
cacgatccgatcccgctgcaatcgac
ggcgatggatccgacttccaccattatgggcgccgatgaaaacatttttaccgcgctcaatgtgctctgccag-
cgcaacgccgcgaaagccattgtgct
gctcagcaccgggctttcagaagcccagggcagcgacatttcgcgggtggtgcgccagtttcgtgatgatttt-
ccccggcataaaggcgttgcgctgc
tcaccgtcaacacacccgatttctacggctcgctggaaaacggctacagcgccgtgctggaaagcatgattga-
acagtgggtacccgcacagcccg
ccgccagcctgcgcaaccgccgtgtcaacctgctggtcagccatttactgacaccaggcgatatcgaactgtt-
gcgcagttatgttgaagccttcggcc
tgcaaccggtgattgtgccggatctgtcgctgtcgctggacgggcatctggcagacggtgatttttcgcctgt-
tacccaagggggaacatcgctgcgc
atgattgaacagatggggcaaaacctggccacctttgtgattggcgcctcgctgggccgtgcggcggcgttac-
tggcgcagcgcagccgtggcgag
gtgatcgccctgccgcatctgatgacgcttgcagcctgcgacacgtttattcatcgactgaaaaccctctccg-
ggcgcgatgtccccgcgtggattgag
cgccagcgcggccaagttcaggatgcgatgatcgattgccatatgtggctgcagggtgcggctatcgccatgg-
cagcagaaggcgatcacctggcg
gcatggtgcgatttcgcccgcagccagggcatgatccccggcccgattgtcgcaccggtcagccagccggggt-
tgcaaaatctgccggttgaaacc
gtggttatcggcgatctggaagatatgcaggatcggctttgcgcgacgcccgccgcgttactggtggccaatt-
ctcatgccgccgatctcgccacgca
gtttgatttgtcacttatccgcgccgggttcccggtgtatgaccggctgggggaatttcgtcgcctgcgccag-
gggtacagcggcattcgtgacacgct
gtttgagctgggaatgtgatgcgcgagcgccatcacocgcttgcaacctaccgctcgccgctgcgccagcacg-
ccgacgacaacgttacgcctgg agatctgtatgccgcatgttaa 272
atgaaaaacacaacattaaaaacagcgcttgcttcgctggcgttactgcctggcctggcgatggcggctc-
ccgctgtggcggataaagccgacaacg
gctttatgatgatttgcaccgcgctggtgctgtttatgaccattccgggcattgcgctgttctacggcggttt-
gatccgcggtaaaaacgtgctgtcgatgc
tgacgcaggttgccgtcaccttcgcactggtgtgcattctgtgggtggtgtatggctactcgctggcatttgg-
cgagggcaacagcttcttcgggagtttt
aactgggcgatgttgaaaaacatcgaactgaaagccgtgatgggcagcatttatcagtatatccacgtggcgt-
tccagggttccttcgcctgtatcaccg
ttggcctgattgtcggtgcactggctgagcgtattcgcttactgcggtgctgatttttgtggtggtatggctg-
acgctttcttacgtgccgattgcacacat
ggtggggggcggcggtctgctggcaacccacggtgcgctggatttcgcaggcggtacggttgttcacatcaac-
gctgcgattgcaggtctggtgggg
gcttacctgattggcaaacgcgtgggctttggcaaagaagcattcaaaccgcataacctgccgatggtcttca-
ctggcaccgctatcctgatgttggct
ggtttggtttcaacgccggctccgcaagctcggcgaacgaaattgctgcgctggccttcgtgaacactgtcgt-
tgccactgctgccgctattctggcgtg
ggtatttggcgaatgggcaatgcgcggcaagccgtctctgctcggtgcctgttctggtgccatcgcgggtctg-
gttggtatcacccccgcctgtggttat
gtgggtgtcggcggtgcgctgattgtgggtctgattgccggtctggctgggctgtggggcgttactgcgctga-
aacgtatgttgcgtgtcgatgacccg
tgtgacgtattcggtgtgcacggcgtgtgcggcatcgtgggctgtatcctgacgggtatcttcgcctctacgt-
cgctgggtggtgtcggtttcgctgaag
gtgtgaccatgggccatcaggtgctggtgcagctggaaagtgttgccatcactatcgtgtggtctggcgtggt-
ggcctttattggttacaaactggcgga
catgacggtaggcctgcgcgtaccggaagaacaagaacgtgaagggctggatgtaaacagccacggcgaaaac-
gcctataacgcctga 273
cgtcctgtaataataaccggacaattcggactgattaaaaaagcgcccttgtggcgctttttttatattc-
ccgcctccatttaaaataaaaaatccaatcgga
tttcactatttaaactggccattatctaagatgaatccgatggaagacgctgttttaacacgcgttttttaac-
cttttattgaaagtcggtgcttctttgagcga
acgatcaaatttaaatggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaacg-
ctacatggagattaactcaatctagagggt attaataatgaatcgtactaaactggtactgggcgc
274
ggacatcatcgcgacaaacaatattaataccggcaaccacaccggcaatttacgagactgcgcaggcatc-
ctttctcccgtcaatttctgtcaaataaag
taaaagaggcagtctacttgaattacccccggctggttgagcgtttgttgaaaaaaagtaactgaaaaatccg-
tagaatagcgccactctgatggttaatt
aacctattcaattaagaattatctggatgaatgtgccattaaatgcgcagcataatggtgcgttgtgcgggaa-
tactgcttttttttgaaagggttagtcagt agcggaaac 275
atgaccctgaatatgatgctcgataacgccgcgccggacgccatcgccggcgcgctgactcaacaacatc-
cggggagttttttaccatggtggaaca
ggcctcggtggccatctccctcaccgatgccagcgccaggatcatttacgccaacccggcgttttgccgccag-
accggctattcgctggcgcaattgtt
aaaccagaacccgcgcctgctggccagcagccagacgccgcgcgagatctatcaggagatgtggcataccctg-
accagcgtcagccctggcgcg
gtcagctgattaatcagcgtcgggacggcggcctgtacctggtggagattgacatcaccccggtgcttagccc-
gcaaggggaactggagcattatct
ggcgatgcagcgggatatcagcgtcagctacaccctcgaacagcggctgcgcaaccatatgaccctgatggag-
gcggtgctgaataatatccccgc
cgccgtggtagtggtggacgagcaggatcgggtggtgatggacaacctcgcctacaaaaccttctgcgctgac-
tgcggcggccgggagctgctcac
cgagctgcaggtctcccctggccggatgacgcccggcgtggaggcgatcctgccggtggcgctgcgcggggcc-
gcgcgctggctgtcggtaacc
tgctggccgttgcccggcgtcagtgaagaggccagccgctactttatcgacagcgcgctggcgcggaccctgg-
tggtgatcgccgactgtacccag
cagcgtcagcagcaggagcaagggcgccttgaccggctgaagcagcaaatgaccgccggcaagctgctggcgg-
cgatccgcgagtcgctggac
gccgcgctgatccagctgaactgcccgattaatatgctggcggcagcccgtcggctgaacggcgagggaagcg-
ggaatgtggcgctggaggccg
cctggcgtgaaggggaagaggcgatggcgcggctccagcgctgtcgcccatcgctggaactcgaaaaccccgc-
cgtctggccgctgcagccctttt
tcgacgatctgtgcgccctctaccgtacacgcttcgatcccgacgggctgcaggtcgacatggcctcaccgca-
tctgatcggctttggccagcgcacc
ccactgctggcgtgcttaagcctgtggctcgatcgcaccctggccctcgccgccgaactcccctccgtgccgc-
tggcgatgcagctctacgccgagg
agaacgacggctggctgtcgctgtatctgactgacaacgtaccgctgctgcagggcgctacgctcactccccc-
gacgcgctgaactcgccgggca
aaggcatggagctgaggctgatccagaccctggtggcgcaccatcgcggggccattgagctggcttcccgacc-
gcagggcggcacctgcctgacc
ctgcgtttcccgctgtttaacaccctgaccggaggtgaagcatga 276
atgatccctgaatccgacccggacaccaccgtcagacgcttcgacctctctcagcagttcaccgccatgc-
agcggataagcgtggtgctgagccggg
ccaccgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgatgcctttatgcagcacgggat-
gatctgcctgtacgacagcgagc
aggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgcagatccgcta-
tcgccccggcgagcgactggt
ggggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgttttctcgaccgc-
ctgagcctctacgattacgatctg
ccgtttatcgccgaccgttgatgcggcccaacgcccggccaataggggtgctggcggcccagccgatggcgcg-
ccaggaagagcggctgccgg
cctgcacccgttttctcgaaaccgtcgccaacctcgtcgcccagaccatccggctgatgatccttccggcctc-
acccgccctgtcgagccgccagccg
ccgaaggtggaacggccgccggcctgctcgtcgtcgcgcggcgtgggccttgacaatatggtcggcaagagcc-
cggcgatgcgccagatcgtgg
aggtgatccgtcaggtttcgcgctgggacaccaccgtgctggtacgcggcgaaagcggcaccgggaaagagct-
gatcgccaacgccatccatcac
cattcgccacgggctggcgccgccttcgtcaaatttaactgcgcggcgctgccggacaccctgctggaaagcg-
aactgttcggccatgagaaaggc
gcctttaccggggcggtgcgtcagcgtaaaggacgttttgagctggcggatggcggcaccctgttcctcgatg-
agattggtgaaagcagcgcctcgtt
ccaggccaagagctgcgtatcctccaggagggggagatggagcgggtcggcggcgatgagaccctgcgggtga-
atgtccgcatcatcgccgcc
accaaccgtcacctggaggaggaggtccggctgggccatttccgcgaggatctctactatcgtctgaacgtga-
tgcccatcgccctgcccccgctgc
gcgagcgtcaggaggacatcgccgagctggcgcacttcctggtgcgcaaaatcggccagcatcaggggcgcac-
gctgcggatcagcgagggcg
cgatccgcctgctgatggagtacagctggccgggtaacgttcgcgaactggagaactgcctcgaacgatcggc-
ggtgatgtcggagagtggcctga
tcgatcgcgacgtgatcctcttcactcaccaggatcgtcccgcaaagccctgcctgccagcgggccagcggaa-
gacagctggctggacaacagcc
tggacgaacgtcagcgactgatcgccgcgctggaaaaagccggctgggtgcaggccaaggcggcacggctgct-
ggggatgacgccgcgccagg
tcgcttatcggatccagatcatggatatcaccctgccgcgtctgtag 277
attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggcag-
cgggaagtagcttgctactttgccggc
gagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaa-
taccgcatgacctcgaaagagc
aaagtgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggtaatggc-
tcacctaggcgacgatccctagct
ggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaa-
tattgcacaatgggcgcaagc
ctgatgcagccatgccgcggtgtgaagaaggccttagggtttgtaaagcactttcagcgaggaggaaggcatc-
atacttaatacgtgtggtgattgtcg
ttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcg-
gaattactgggcgtaaagcgcac
gcaggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagcta-
gagtcttgtagaggggggtagaattc
caggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactg-
acgctcaggtgcgaaagcgtg
gggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgag-
gcgtggcttcggagctaacgcgtt
aagtcgaccgcctggggagtacggccgcaagtttaaaactcaaatgaattgacgggggcccgcacaaagcggt-
ggagcatgtggtttaattcgatgc
aacgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaactct-
gagacaggtgctgcatggctgtcgt
cagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtaa-
tggtgggaactcaaaggagactgccg
gtgataaaccggaggaaggtggggatgacgtcaagtcatcattgcccttacgagtagggctacacacgtgcta-
caatggcatatacaaagagaagc
gaactcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatga-
agtcggaatcgctagtaatcgtaga
tcagaatgctacggtgaatacgttccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaa-
gaagtaggtagcttaaccttcggg
agggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggtt-
ggatcacctcctt 278
attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggcat-
cgggaagtagcttgctactttgccggcg
agcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaat-
accgcatgacctcgaaagagca
aagtgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggtaatggct-
nacctaggcgacgatccctagctg
gtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaat-
attgcacaatcggcgcaagcct
gatgcagccatgccgcgtgtgtgaagaaggccttaggggttaaagcactttcagcgaggaggaaggcatcata-
cttaatacgtgtggtgattgacgtt
actcgcagaagaagcaccggctaactccgtgccagcagacgggtaatacggagggtgcaagcgttaatcggaa-
ttactgggcgtaaagcgcacg
caggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagctag-
agtcttgtagaggggggtagaattcca
ggtgtagggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagtctgacg-
ctcaggtgcgaaagcgtggg
gagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgaggc-
gtggcttccggagctaacgcgttaa
gtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtgga-
gcatgtggtttaattcgatgcaa
cgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaactctga-
gacaggtgctgcatggctgtcgtca
gctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtgatg-
gtgggaactcaaaggagactgccggt
gataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctaca-
atggcatatacaaagagaagcga
actcgcgagagcaagggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagt-
cggaatcgctagtaatcgtagatca
gaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacacattgggagtgggttgcaaaaga-
agtaggtagcttaaccttcgggag
ggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttgg-
atcacctcctt 279
attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggcag-
gggaagtagcttgctactttgccggc
gagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaa-
taccgcatgacctcgaaagagc
aaagttgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggttaatg-
gctcacctaggcgacgatccctagct
ggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaa-
tattgcacaatgggcgcaagc
ctgatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaaggcanc-
atacttaatacgtgtggtgattgac
gttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatc-
ggaattactgggcgtaaagcgca
cgcaggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagct-
agagtcttgtagaggggggtagaatt
ccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagact-
gacgctcaggtgcgaaaggcgt
ggggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttga-
ggcgtggcttccggagctaacgcgt
taagtcgaccgcctggggagtacggccgcaaggttaaactcaaatgaattgacgggggcccgcacaagcggtg-
gagcatgtggtttaattcgatgc
aacgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaactct-
gagacaggtgctgcatggagtcgt
cagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtga-
tggtgggaactcaaaggagactgccg
gtgataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgcta-
caatggcatatacaaagagaagc
gaactcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtagcaactcgactccatgaa-
gtcggaatcgctagtaatcgtaga
tcagaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaa-
agaagtaggtagcttaaccttcggg
agggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggtt-
ggatcacctcctt 280
attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcgacan-
cgggaagtagcttgctactttgccggc
gagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaa-
taccgcatgacctcgaaagagc
aaagtgggggatcttcggacctcacgccatcggatgtgcctagatgggattagctagtaggtgaggtaatggc-
ttacctaggcgacgatccctagctg
gctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagggggaatat-
tgcacaatgggcgcaagcct
gatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaaggcatcac-
acttaatacgtgggtgattgacgtt
actcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcgga-
attactgggcgtaaagcgcacg
caggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagctag-
agtcttgtagagggggtgagaattcca
ggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgac-
gctcaggtgcgaaagcgtggg
gagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgaggc-
gtggcttccggagctaacgcgttaa
gtcgaccgactggggagtacggccgaaaggtttaaaactcaaatgaattgacgggggcccgcacaagcggtgg-
agcatgtggtttaattcgatgcaa
cgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaaggccttcgggaactctgag-
acaggtgctgcatggctgtcgtca
gctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtgatg-
gtgggaactcaaaggagactgccggt
gataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacggctacaa-
tggcatatacaaagagaagcga
actcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaag-
tcggaatcgctagtaatcgtagatca
gaatgctactgtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaaga-
agtaggtagcttaaccttcgggag
ggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttgg-
atcacctcctt 281
attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggcag-
cgggaagaagcttgctactttgccggc
gagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaa-
taccgcatgacctcgaaagagc
aaagtgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagaggtgaggtaatggct-
cacctaggcgacgatccctagct
ggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaa-
tattgcacaatgggcgcaagc
ctgatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaaggcatc-
acacttaatacgtgtgttgattgacg
ttactcgcagaagaagcaccggctaactccgtgccagcagccgcgattaatacggagggtgcaagcgttaatc-
ggaattactgggcgtaaagcgcac
gcaggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagcta-
gagtcttgtagaggggggtagaattc
caggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactg-
acgctcaggtgctgaaagcgtg
gggagcaaacaggattagataccaggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgagg-
cgtggcttccggagctaacgcgtt
aagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtg-
gagcatgtggtttaattcgatgc
aacgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaactct-
gagacaggtgctgcatggctgtcgt
cagctcgtgttgtgaaatgttgggttaagtacgcaacgagcgcaacccttatcctttgttgccagcacgtgat-
ggtgggaactcaaaggagactgccg
gtgataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgcta-
caatggcatatacaaagagaagc
gaactcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatga-
agtcggaatcgctagtaatcgtaga
tcagaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaa-
agaagtaggtagcttaaccttcggg
agggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggtt-
ggatcacctcctt 282
gtagctaataccgcatgacctcgaaagagcaaagtgggggatcttcggacctcacgccatcggatgtgcc-
cagatgggattagctagtaggtgaggt
aatggctcacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaactgagacacggtc-
cagactcctacgggaggcagca
gtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtgtgaagaaggccttagggttgt-
aaagcactttcagcgaggaggaa
ggcatcanacttaatacgtgtgntgattgacgttactcgcagaagaagcaccggctaactccgtgccagcagc-
cgcggtaatacggagggtgcaagc
gttaatcggaattactgcgcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgagcttaa-
cttgggaactgcatttgaaactggca
agctagagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccg-
gtggcgaaggcggccccctggac
aaagactgacgctcaggtgcgaaagcgtgggagcaaacaggattagataccctggtagtccacgctaaacgat-
gtcgacttggaggttgtgccc
ttgaggcgtggcttccggagctaacgcgttaagtcgaccgcctggggagtacggccgcaaggttaaaactcaa-
atgaattgacgggggcccgcaca
agcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctactcttgacatccagagaatttgc-
cagagatggcgaagtgccttcgggaa
ctctgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgc-
aacccttatcctttgttgccagcacgt natggtgggaactcaaaggagactgccggtgataaac
283
attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgaagtcgagcggcagc-
gggaagtagcttgctactttgccggc
gagcggcggacgggtgagtaatgtcctgatggaggggataactactggaacggtagctaataccgcacctcga-
aagagcaaagtgggggatcttcg
gacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggtaatggctcacctaggcgacgatc-
cctagctggtctgagaggatgacc
agccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgc-
aagcctgatgcagccatgccgc
gtgtgtgaagaaggccttagggttctaaagcactttcagcgagcaggaaggcatcatacttaatacgtgtggt-
gattgacgttactcgcagaagaagca
ccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcttsatcggaattactgggcgtaaagc-
gcacgcaggcggttgttaagtca
gatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagctagagtcttgtttgaggggggt-
agaattccaggtgtagcggtgaaatgc
gtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgg-
gggagcaaacaggattaata
ccctggtagtccacgctgtaacgatgtcgacttggaggttgtgccctgaggcgtggcttccggagctaacgcg-
ttaagtcgaccgcctggggagtacg
gccgcaaggtaaaactaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaa-
cgcgaagaaccttacctactctt
gacatccagagaatttgccagagatggcgaagtgccttcgggaactctgagacaggtgctgcatggctgtcgt-
cagctcgtgttgtgaaatgttgggtt
aagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtaatggtgggaactcaaaggagactgccg-
gtgataaaccggaggaaggtggg
gatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatatacaaagagaagc-
gaactcgcgagagcaagcggacc
tcataaagtatgtcgtagtccggattggagtctgcgactcgactccatgaagtcggaatcgctagaatcgtag-
atcagaatgctacggtgaatacgttcc
cgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaacatcgggag-
ggcgcttaccactttgtgattcatg
actggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt 284
atgaccatgcgtcaatgcgctatctacggtaaaggcggtatcggtaaatccaccaccacccagaatctcg-
tcgcggccctcgccgagatgggtaaga
aagtgatgatcgtcggctgcgatccgaaagcggattccacccgtctgatcctccacgctaaagcccagaacac-
catcatggagatggcggcggaagt
gggctcggtcgaggatctggagctcgaagacgttctgcaaatcggctatggcgatgtccgttgcgccgaatcc-
ggcggcccggagccaggcgtcg
gctgcgccggacgcggggtgatcaccgccatcaacttcctcgaggaaggcgcctatgaagaagatttggattt-
cgtcttctatgacgtcctcggc
gacgtggtctgcggcggcttcgctatgccgatccgcgaaaacaaagcccaggagatctacatcgtctgctccg-
gcgagatgatggcgatgtatgccg
ccaacaatatctccaaagggatcgtgaagtacgccaaatccggcaaggtgcgcctcggcggcctgatctgtaa-
ctcgcgcaaaaccgaccgggaag
acgaactgatcatcgccctggcggagaagcttggcacgcagatgatccacttcgttccccgcgacaacattgt-
gcagcgcgcggagatccgccgga
tgacggtgatcgagtacgacccgacctgtcagcaggcgaggaatatcgtcaactggcgcagaagatcgtcaat-
aacaccaaaaaagtggtgccga
cgccgtgcaccatggacgagctggaatcgctgctgatggagttcggcatcatggaagaagaagacaccagcat-
cattggtaaaaccgccgctgaag aaaacgcggcctga 285
atggttaggaaaagtagaagtaaaaatacaaatatagaactaactgaacatgaccatttattaataagtc-
aaataaaaaagcttaaaacacaaaccacttg
cttttttaataataaaggaggggttgggaagactacattagtagcaaatttaggagcagagctatcaataaac-
tttagtgcaaaagttcttattgtggatgcc
gaccctcaatgtaatctcacgcagtatgtattaagtgatgaagaaactcaggacttatatgggcaagaaaatc-
cagatagtatttatacagtaataagacc
actatcctttggtaaaggatatgaaagtgacctccctataaggcatgtagagaatttcggttttgacataatt-
gtcggtgaccctagacttgctttacaggaa
gaccttttagctggagactggcgagatgccaaaggcggtgggatgcgaggaattaggacaacttttgtatttg-
cagagttaattaagaaagctcgtgag
ctaaattatgattttgttttctttgacatgggaccatcattaggcgcaatcaacagggcagtattactggcaa-
tggaattctttgtcgtcccaatttcaatcga
tgtattttcactatgggctattaaaaatattggctccacggtttcaatatggaaaaaagaattagacacaggg-
attcggctctcagaggaacctagcgaatt
atcacaattatcttcctcaaggaaaactaaagtttctcggttacgtcacccaacaacataaagaacgctctgg-
atacgatacaattcagcttgagaatactg
aggaagaaataaaatcgaaacgtcgggtaaaggcgtatgaagacattggagaggtgtttccttctaaaattac-
tgagcatctttctaaactttatgatca
aaagatatgaacccacaccttggagatatacgtcatttaggtagtttagctccgaaatcacaatcacaacacg-
ttccgatgatatcagtgtctggtacagg
aaattacaccagacttagaaaaagcgcgcgtgaactttatcgagatattgcaagaagatacttagagaacatt-
cagactgctaatggcgagaaatag 286
atgaagggaaaggaaattctggcgctgctggacgaacccgcctgcgagcacaaccagaagcaaaaatccg-
gctgcagcgcccctaagcccggcg
ctaccgccggcggttgcgccttcgacggcgcgcagataacgctcctgcccatcgccgacgtcgcgcacctggt-
gcacggccccatcggctgcgcg
ggcagctcgtgggataaccgcggcagcgtcagcgccggcccggccctcaaccggctcggctttaccaccgatc-
ttaacgaacaggatgtgattatg
ggccgcggcgaacgccgcctgttccacgccgtgcgtcacatcgtcgaccgctatcatccggcggcggtcttta-
tctacaacacctgcgtaccggcga
tggagggcgatgacatcgaggcggtctgccaggccgcacagaccgccaccggcgtcccggtcatcgctattga-
cgccgccggtttctacggcagta
aaaatcttggcaaccgaatggcgggcgacgtgatgctcaggcaggtgattggccagcgcgaaccggccccgtg-
gccagacaacacgccctttgcc
ccggcccagcgccacgatatcggcctgattggcgaattcaatatcgccggcgagttctggcaggtccagccgc-
tgacgacgagctggagatccgc
gtcctcggcaccctctccggcgacggccgctttgccgagatccagaccctgcaccgggcgcaggccaatatgc-
tggtgtgctcgcgcgcgctgatc
aacgtcgcccgggggctggagctgcgctacggcacgccgtggtttgaaggcagcttctacgggatccgcgcca-
cctccgacgccttgcgccagct
ggcgacgctgctgggggatgacgacctgcgccgccgcaccgaggcgctgatcgcccgcgaagagcaggcggcg-
gagcaggctcttgcgccgt
ggcgtgagcagctccgcgggcgcaaatgctgctctataccggcggcgtgaaatcctggtcggtggtatcggcc-
ctgcaggatctcggcatgaccg
tggtggccaccggcacgcgcaaatccaccgaggaggacaaacagcggatccgtgagctgatgggcgacgaggc-
ggtgatgcttgaggagggca
atgcccgcaccctgctcgacgtggtgtaccgctatcaggccgacctgatgatcgccggcggacgcaatatgta-
caccgcctggaaagcccggctgc
cgtttctcgatatcaatcaggagcgcgagcacgcctacgccggctatcagggcatcatcaccctcgcccgcca-
gctctgtctgaccctcgccagcccc gtctggccgcaaacgcatacccgcgccccgtggcgctag
287
atgaccaacgcaacaggcgaacgtaaccttgcgctcatccaggaagtcctggaggtgtttcccgaaaccg-
cgcgcaaagagcgcagaaagcacat
gatgatcagcgatccgagatggagagcgtcggcaagtgcattatctcgaaccgtaaatcgcagcccggggtga-
tgaccgtgcgcggctgcgccta
tgcgggctcgaaaggggtggtgtttgggccaatcaaagacatggcccatatctcgcacggccccatcggctgc-
ggccagtattcccgcgccggacg
gcgcaactactataccggcgtcagcggtgtcgacagcttcggcaccctgaacttcacctctgattttcaggag-
cgcgatattgttttcggcggcgataaa
aagctgaccaaactgatcgaagagatggagagctgttcccgctgaccaaagggatcaccatccagtcggagtg-
cccggtgggcctgatcggcgat
gacatcagcgccgtagccaacgccagcagcaaggcgctggataaaccggtgatcccggtgcgctgcgaaggct-
ttcgcggcgtatcgcaatcgct
gggccaccatatcgccaacgacgtggtgcgcgactgggtgctgaacaatcgcgaagggcagccgtttgccagc-
accccgtacgatgttgccatcatt
ggcgattacaacatcggcggcgacgcctgggcctcgcgcattctgctggaagagatggggctgcgcgtagtgg-
cgcagtggtccggcgacggca
ccctggtggagatggagagcaccccattcgttaagcttaacctcgtccactgctaccgttcgatgaactatat-
cgcccgccatatggaggagaaacatc
agatcccatggatggaatataacttcttcggcccgaccaaaatcgccgaatcgctgcgcaagatcgccgatca-
atttgatgacaccattcgcgccaatg
cggaagcggtgatcgccaaatatgaggggcagatggcggccatcatcgccaaatatcgcccgcggctggaggg-
gcgcaaagtgctgctgtacatg
ggggggctgaggccgcgccacgtcatcggcgcctatgaggatctcgggatggagatcatcgccgccggctacg-
agtttgcccataacgatgattac
gaccgcaccctgacggacctgaaagagggcaccctgctgtttgacgatgccagcagatatgagctggaggcct-
tcgtcaaagcgctgaaacctgac
ctcatcggctccgggatcaaagagaaatatatcttccagaaaatgggggtgcgttccgccagatgcactcctg-
ggactattccggcccctatcacgg
ctatgacggcttcgccatctttgcccgcgatatggatatgaccctgaacaatccggcgtggaacgaactgact-
gccccgtggctgaagtctgcgtga
288
atggcagatattatccgcagtgaaaaaccgctggcggtgagcccgattaaaaccgggcaaccgctcgggg-
cgatcctcgccagcctcgggctggc
ccaggccatcccgctggccacggcgcccagggctgcagcgccttcgccaaagttttctttattcagcatttcc-
atgacccggtgccgctgcagtcgac
ggccatggatccgaccgccacgatcatgggggccgacggcaatatcttcaccgcgctcgacaccctctgccag-
cgccacagcccgcaggccatcg
tgctgctcagcaccggtctggcggaagcgcagggcagcgatatcgcccgggtggtgcgccagtttcgcgaggc-
gcatccgcgccataacggcgtg
gcgatcctcaccgtcaataccccggatttttttggctctatggaaaacggctacagcgcggtgatcgagagcg-
tgatcgagaagtgggtcgcgccgac
gccgcgtccgggcagcggccccggcgggtcaacctgctggtcagccacctctgttcgccaggggatatcgaat-
ggctgggccgctgcgtggag
gcctttggcctgcagccggtgatcctgccggacctctcgcagtcaatggatggccacctcggtgaaggggatt-
ttacgcccctgacccagggcggcg
cctcgctgcgccagattgcccagatgggccagagtagggcagcttcgccattggcgtgtcgctccagagggcg-
gcatcgatcctgacccaacgca
gccgcggcgacgtgatcgccctgccgatctgatgaccctcgaccattgcgatacctttatccatcagctggcg-
aagatgtccggacgccgcgtacc
ggcctggattgagcgccagcgtggccagctgcaggatgcgatgatcgactgccatatgtggcttcagggccag-
cgcatggcgatggcggcggagg
gcgacctgctggcggcgtggtgtgatttcgcccgcagccaggggatgcagcccggcccgctggtcgcccccac-
cagccaccccagcctgcgcca
gctgccggcgagcaagtcgtgccgggggatcttgaggatctgcagcagctgctgagccaccaacccgccgatc-
tgaggtggctaactctcacgc
ccgcgatctggcggagcagtttgccctgccgctgatccgcgtcggttttcccctcttcgaccggctcggtgag-
tttcgtcgagtccgccaggggtacgc
cggtatgcgagatacgctgtttgaactggccaatctgctgcgcgaccgccatcaccacaccgccctctaccgc-
tcgccgcttcgccagggcgccgac ccccagccggcttcaggagacgcttatgccgcccattaa
289
atgagccaaacgatcgataaaattcacagctgttatccgatatttgaacaggatgaataccagaccctgt-
tccagaataaaaagacccttgaagaggcg
cacgacgcgcagcgtgtgcaggaggtttttgcctggaccaccaccgccgagtatgaagcgctgaacttccagc-
gcgaggcgctgaccgtcgaccc
ggccaaagcctgccagccgctcggcgccgtactctgcgcgctggggttcgccggcaccctgccctacgtgcac-
ggctcccagggctgcgtcgcct
atttcgcacctactttaaccgccattttaaagagccggtcgcctgcgtctccgactccatgaccgaggacgcg-
gcggtgttcggcggcaacaacaac
atgaatctgggcctgcacaatgccagcgcgctgtataaacccgagattatcgccgtctccaccacctgtatgg-
ccgaggtgatcggcgacgatctgca
ggcgtttatcgccaacgccaaaaaagagggatttgttgacgaccgcatcgccattccttacgcccataccccc-
agctttatcggcagccatgtcaccgg
ctgggacaatatgttcgaagggttcgcgaagacctttaccgctgactacgccgggaagccgggcaaacagaaa-
aagctcaatctggtgaccggattt
gagacctatctcggcaacttccgcgtgctgaagcggatgatggcgcagatggatgtcccgtgcagcctgctct-
ccgacccatcagaggtgctcgaca
cccccgccgacggccattaccggatgtacgccggcggcaccagccagcaggagatcaaaaccgcgccggacgc-
cattgacaccctgctgctgca
gccgtggcagatggtgaaaagcaaaaaggtggttcaggagatgtggaaccagcccgccaccgaggtggccgtt-
ccgctgggcctggacgccacc
gacgcgctgctgatgaccgtcagtcagctgaccggcaaaccgatcgccgacgctctgaccctggagagaggcc-
ggctggtcgacatgatgctggat
tcccacacctggctgcatggcaaaaaattcggcctctacggcgatccggatttcgtgatggggctgacgcgct-
tcctgctggagctgggctgcgagcc
gacggtgatcctcagtcataacgccaataaacgctggcaaaaagcgatgaagaaaatgctcgatgcctcgccg-
tacggtcaggaaagcgaagtgttc
atcaactgcgacctgtggcacttccggtcgctgatgttcacccgtcagccggactttatgatcggtaactcct-
acggcaagtttatccagcgcgataccc
tggcaaagggcaaagccttcgaagtgccgatgatccgtctgggctttccgctgttcgaccgccatcatctgca-
ccgccagaccacctggggctatgaa
ggcgcaatgaacatcgtcacgacgctggtgaacgccgtgctggaaaaactggaccacgacaccagccagttgg-
gcaaaaccgattacagcttcgac ctcgttcgttaa 290
atgatgccgctttctccgcaattacagcagcactggcagacggtcgctgaccgtctgccagaggattttc-
ccattgccgaactgagcccacaggccag
gtcggtcatggcgttcagcgattttgtcgaacagagtgtgatcgcccagccgggctggctgaatgagcttgag-
gactcctcgccggaggcggaagag
tggcggcattacgaggcctggctgcaggatcgcctgcaggccgtcactgacgaagggggttgatgcgagagct-
gcgtctcttccgccgccagatg
atggtccgcatcgcctgggcgcaggcgctgtcgctggtgagcgaagaagagactctgcagcagctgagcgtcc-
tggcggagaccctgattgtcgcc
gcccgcgactggctgtacgccgcctgctgtaaggagtggggaacgccatgcaatgccgagggccagccgcagc-
cgctgctgatcctcgggatgg
gaaagctgggcggcggcgagctgaacttctcttccgatatcgatctgatctttgcctggcctgagcatggcgc-
cacccgcggcggccgccgcgagct
ggataacgcccagttctttacccgtctggggcagcggctgatcaaggcccttgaccagccgacgcaggacggc-
tttgtctatcgggttgacatgcgcc
tgcggccgtttggcgacagtgggccgctggtactcagttttgcggcgctggaagattattaccaggagaaggg-
tcgggactgggaacgctatgcgat
ggtgaaagcgcggatcatggccgataacgacggcgtgtacgccagcgacttgcgcgcgatgctccgtcctttc-
gtcttccgccgttatatcgacttca
gcgtgatccagtcgctgcgtaacatgaaaggcatgatcgcccgcgaagtgcggcgtcgcgggctgaaagacaa-
catcaagctcggcgccggcgg
gatccgtgaaattgagtttatcgttcaggctttcaactgatccgcggtggtcgcgaacctgcactgcagcagc-
gcgccctgctgccgacgctggcgg
cgattgatgagctacatctgctgccggaaggcgacgcggcgctgctgcgcgaggcctatctgttcctgcgccg-
gctggaaaacctgctgcaaagcat
caacgatgagcagacccagaccctgccgcaggatgaacttaaccgcgccaggctggcgtgggggatgcatacc-
gaagactgggagacgctgagc
gcgcagctggcgagccagatggccaacgtgcggcgagtgtttaatgaactgatcggcgatgatgaggatcagt-
ccccggatgagcaactggccga
gtactggcgcgagctgtggcagcatgcgctggaagaagatgacgccagcccggcgctggcgcatttaaacgat-
accgaccgccgtagcgtgctgg
cgctgattgccgattttcgtaaagagctggatcggcgcaccatcggcccgcgcggccgccaggtgctggatca-
gcttgatgccgcatctgctgagcga
aatctgctcgcgcgccgatgcgccgctgcctctggcgcggatcacgccgctgttgaccgggatcgtcacccgt-
accacctatcttgagctgctgagc
gaattccccggcgcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgc-
gccacccgctgctgctggatga
gctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctg-
ctgcgcgtgccggaagaggatga
agagcagcagatggaggcgttgcgccagtttaagcaggcgcagcagctgcatatcgcggcggcggatatcgct-
ggtaccctgccggtgatgaagg
tcagcgatcacttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggt-
cgctcgctacggccagccgaccc
acctgcacgatcgccagggtcgcggcttcgccgtcgtcggctacgctaagcttggcggctgggagctgggcta-
cagctccgatctcgatctggtgttc
ctccatgactgcccggcggaggtgatgaccgacggcgagcgggagattgacggccgtcagttctacctgcggc-
tggcccagcggatcatgcacct
gttcagcacccgcacctcgtccggtattctctacgaagttgacgcccggagcgccttctggcgcggcggggat-
gaggtcaccaccgccgacgc
gtttgctgactatcagcagaacgaagcaggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatg-
gcgacccggcgctgcaggcgc
gctttgacgccattcgtcgcgatatcctgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcga-
gatgcgcgagaagatgcgcgcc
caccttggcaacaaacatcccgatcgttttgatatcaaagccgatgccggcgggatcaccgatattgaattta-
ttactcagtatctggtcctacgctatgcc
agtgacaagccgaagctgacccgctggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatca-
tggaggaggaggaggcgcgcgc
cttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggccctgcaggagcagccgggacacgtg-
gcgccagaggccttcagccggga
gcgtcagcaggtcagcgccagctggcagaagtggctgatggcttaa 291
agtctgaactcatcctgcggcagtcggtgagacgtatttttgaccaaagagtgatctacatcacggaatt-
ttgtggttgttgctgcttaaaagggcaaatct
acccttagaatcaactgttatatcagggggattcagagagatattaggaatttgcacaagcgcacaatttaac-
cacatcatgataacgccatgtaaaaca
aagataaaaaaacaaaatgcagtgacttacatcgcaagcaaggcattttcttatccaattgctcaaagtttgg-
cctttcatatcgcaacgaaaatgcgtaat
atacgcgcccttgcggacatcagtatggtcattcctagttcatgcgcatcggacaccaccagcttacaaattg-
cctgattgcggccccgatggccggtat
cactgaccgaccatttcgtgccttatgtcatgcgatgggggctgg 292
tgaacatcactgatgcacaagctacctatgacgaagaattaactaaaaaactgcaagatgaggcattcgc-
gttaaagccgacttgagaaatgagaag
attggctttaaaattcgcgaacacacgctacgccgtgttcatatatgttagtttgtggcgataaagaggtcga-
agcaggcaaagttgctgttcgtacccg
ccgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgctggcggaaatccgcagcaga-
agtcttcatcaactggaggaataa
agtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaacaaagagattcgcgcgcaagaag-
ttcgcctcacaggcgtcgatggcg
agcagattggtattgtcagtagaatgaagctcttgaaaaagctgaggaaggggcgtcgatttagtagaaatca-
gtccgaatgccgagccgccagttt gtcgaatc 293
tacagtagcgcctctcaaaaatagataaacggctcatgtacgtgggccgtttattttttctacctataat-
cgggaaccggtgttataatgccgcgccctcat
attgtggggattcttaatgacctatcctgggtcctaaagttgtagttgacattagcggagcactaac
294
aattttttttcacaaagcgtagcgttattgaatcgcacattttaaactgttggccgctgtggaagcgaat-
attcgtgaaagtgcggttttaaggcctttttctt
tgactctctgtcgttacaaagttaatatgcgcgccct 295
ttaaaaacgtgaccacgagcattaataaacgccacgaaatgtggcgtttatttattcaaaaagtatctct-
ttcataaaaagtgctaaatgcagtagcagca
aaattgggataagtcccatggaatacggctgttttcgctgcaatttttaactttttcgtaaaaaaagatgttt-
ctttgagcgaacgatcaaaatatagcgttaa
ccggcaaaaaattattctcattagaaaatagtttgtgtaatacttgtaacgctacatggagattaacttaatc-
tagagggttttata 296
atggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacacaacttgcacgtcatc-
ctttattgctcgatgaactgctcgacccgc
gcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgggtgccaac-
agnagacgaagaacagcagcttg
aagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccggt-
gatgaaagtcagtgaccatttaacc
taccttgccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggcagc-
ccgcgcatcttcagcaccgtgagg
ggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctggttatagctcagatctggatctg-
gtcttcctgctcgattgcgcgccgg
aggtgatgacggacggcgaacgcagcatcgacggacgtcagttttatcttcggctggcgcagcgcattatgca-
cttattcagcacccggacatcgtca
ggcattctttacgaggttgatccgcgtctgcgaccttccggcgcatccggcatgctggtcagtaccattgaag-
cgtttgcagattatcaggccaatgaag
cctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatt-
taacgccacgcgtcgcgacattct
ttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatctg-
gggagtaaaaaagcccacgagtt
tgactgaaagccgatccgggtggcatcacggatattgaattcattgcacaatacctggttctgcgtttcgcgc-
atgatgagccgagctgacgcgctg
gtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaagcgcgccatctg-
acgcaggcttatgtgacgctgcgog
atgaaattcatcatctggcgttgcaggaacacagcgggaaagtggccgcggacagctttgctactgagcgcgc-
gcagatccgtgccagctgggcga agtggctcggctga 297
cggtactggaacagaaatcggcggatgcgcaggaaatttgttatgacacggcctgtctgaagtgcaagtt-
agtgcttacttcctggctggcaacctcag
gctggacgccgtttattgatgataaatctgcgaagaaactggacgcttccttcaaacgttttgctgacatcat-
gctcggtcgtaccgcagcggatctgaaa
gaggcctttgcgcagccactgacggaagaaggttatcgcgatcagctggcgcgcctgaaacgccagatcatta-
ccttccatttgatgccggtgcttac
cctgaaaaagacgtcgatgcgtatattgccggctgggtggacctgcaacaggccatcgttcagcagcaacacg-
cctgggaggattcggcccgtctc
acgcggtgatgatggatgctttctggttaaacgggcaacctcgttaactgactgactagcctgggcaaactgc-
ccgggcttttttttgcaaggaatctgat
ttcatggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacacaacttgcacgtcatc-
ctttattgctcgatgaactgctcgaccc
gcgcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgggtgcca-
acagaagacgaagaacagcagct
tgaagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccg-
gtgatgaangtcagtgaccatttaa
cctaccttgccgaggccattctcgatgtcgggtgcagcatgctgtgggaacaaatggtcgtaaaatacgggca-
gcccgcgcatcttcagcaccgtga
ggggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggat-
ctggtcttcctgctcgattgcgcgcc
ggaggtgatgacggacggcgaacgcagcatcgacggacgtcagttttatcttcggctggcgcagcgcattatg-
cacttattcagcacccggacatcgt
caggcattctttacgaggttgatccgcgtctgcgaccttccggcgcatccggcatgctggtcagtaccattga-
agcgtttgcagattatcaggccaatga
agcctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaa-
tttaacgccacgcgtcgcgacat
tctttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccat-
ctggggagtaaaaaagcccacga
gtttgatagaaagccgatccgggtggcatcacggatattgaattcattgcacaatacctggttctgcgtttcg-
cgcatgatgagccgaagctgacgcgc
tggtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaagcgcgccatc-
tgacgcaggcttatgtgacgctgcg
cgatgaaattcatcatctggcgttgcaggaacacagcgggaaagtggccgcggacagctttgctactgagcgc-
gcgcagatccgtgccagctgggc
aaagtggctcggctgagggtttttattcggctaacaggcgcttgtgatattatccggcgcattgtatttaccc-
gatttgatttatctgttttggagtcttgggat
gaaagtgactttgcagattttcaccgcgcaggtgtgctggttgtcggtgacgtaatgttagaccgttactggt-
atggcccgaccaatcgtatttctccgga
agctccggtgccggtggtgaaggtcagtaccattgaagagcggcctggcggtgcagctaacgtggcgatgaac-
atttcatctctgggcgcctcttcct
gtctgatcggcctgaccggcgtagacgacgctgcgcgtgccctcagtgagcgtctggcagaagtgaaagttaa-
ctgcgttttcgtcgcactatccaca
catcctaccatcaccaaactgcgaattttgtcccgtaaccagcaactgatccgcctcgactttgaggaaggtt-
ttgaaggcgttgatctcgagccgatgct gaccaaaataga 298
atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatc-
cttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgg-
gttttgcacttgagacacttttgggcg
agaaccaccgtctgctggagtctgaactcatcctgcgatgggggctgggccgtctctgaagctctcggtgaac-
attgttgcgaggcaggatgcgagct
ggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagggcgttttcc-
cgtccggggaatggcatggagctgc
gccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcacctt-
gctgacgttacgcctgccggtacagca ggttatcaccggaggcttaaaatga 299
tgtttcgtctcgaggccgggcaactgagggccccgttgaaaccgacctgggctggcatctgttgttgtgc-
gaacaaattcgcctgccgcaaccttgc
cgaaagcccaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtg-
gctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaattgcct-
gcgttgccttataacaggcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgat-
gcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaacgcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggtt-
ccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgacgggaatatcgccagccgcttgtcgctgcaaca-
tccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgcc-
agacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggagtctgaactcatcctgcgatgggggctgggccgtctctgaagctctcgg-
tgaacattgttgcgaggcaggatg
cgagctggttgtgttttcacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggc-
gttttcccgtccggggaatggcatcg
agctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcgg-
caccttgctgacgttacgcctgccggt
acagcaggttatcaccggaggcttaaaatgacccagttacctaccgcgcgcccggttatccggcgctttgata-
tgtctgcccagtttacggcgctttatc
gcatcagcgtggcgctgagtcaggaaagcaacaccgggcgcgcactggcggcgatcctcgaagtgcttcacga-
tcatgcatttatgcaatacggcat
ggtgtgtctgtttgataaagaacgcaatgcactctttgtggaatccctgcatggcatcgacggcgaaaggaaa-
aaagagacccgccatgtccgttacc
gcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggcgctggtgttaccgcgcatttcagacga-
tcagcgttttctcgaccccctgaa
tatttacgattacagcctgccgttgattggcgtgccgatccccggtgcggataatcagccatcgggcgtgctg-
gtggcacagccgatggcgttgcacg aagaccggctgactgccagtacgcggtttttagaaatggtc
300
atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatc-
cttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgg-
gttttgcacttgagacacttttgggcg
agaaccaccgtctgctggttaaagcctgccggtacagcaggttatcaccggaggcttaaatga 301
tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtg-
cgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtg-
gagcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaatttgcc-
tgcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgat-
gcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatctgtgtgatttgggtt-
ccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaaca-
tccttcactgttttataccgtggttgaa
caattcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgc-
cagacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggttaaagcctgccggtacagcaggttatcaccggaggcttaaaatgaccca-
gttacctaccgcgggcccggttat
ccggcgctttgatatgtctgcccagtttacggcgctttatcgcatcagcgtggcgctgagtcaggaaagcaac-
accgggcgcgcactggcggcgatc
ctcgaagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtctgtttgataaagaacgcaatgcac-
tctttgtggaatccctgcatggcatcgac
ggcgaaaggaaaaaagagacccgccatgtccgttaccgcatgggggaaggcgtgatcggcgcggtgatgagcc-
agcgtcaggcgctggtgttac
cgcgcatttcagacgatcagcgttttctcgaccgcctgaatatttacgattacagcctgccgttgattggcgt-
gccgatccccggtgcggataatcagcc
atcgggcgtgctggtggcacagccgatggcgttgcacgaagaccggctgactgccagtacgcggtttttagaa-
atggtc 302
atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatc-
cttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgg-
gttttgcacttgagacacttttgggcg
agaaccaccgtctgctggagtctgaactcatcctgcggcagtcggtgagacgtatttttgaccaaagagtgat-
ctacatcacggaattttgtggttgttgc
tgcttaaaagggcaaatctacccttagaatcaactgttatatcagggggattcagagagatattaggaatttg-
cacaagcgcacaatttaaccacatcatg
ataacgccatgtaaaacaaagataaaaaaacaaaatgcagtgacttacatcgcaagcaaggcattttcttatc-
caattgctcaaagtttggcccttcatatc
gcaacgaaaatgcgtaatatacgcgcccttgcggacatcagtatggtcattcctagttcatgcgcatcggaca-
ccaccagcttacaaattgcctgattgc
ggccccgatggccggtatcactgaccgaccatttcgtgccttatgtcatgcgatgggggctggcccgtctctg-
aagctctcggtgaacattgttgcgag
gcaggatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccg-
gaagcggcgttttcccgtccgggga
atggcatggagagcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccct-
gatggcggcaccttgctgacgttacg cctgccggtacagcaggttatcaccggaggcttaaaatga
303
tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtg-
cgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtg-
gctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgagacgggttaatgcccgttttgcacggaaaatgcacataaattgcctg-
cgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgat-
gcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtcatttgggtt-
ccgccattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaaca-
tccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccccc-
agacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggagtctgaactcatcctgcggcagtcggtgagacgtatttttgaccaaaga-
gtgatctacatcacggaattttgtggt
tgttgctgcttaaaagggcaaatctacccttagaatcaactgttatatcagggggattcagagagatattagg-
aatttgcacaagcgcacaatttaaccac
atcatgataacgccatgtaaaacaaagataaaaaaacaaaatgcagtgacttacatcgcaagcaaggcatttt-
cttatccaattgctcaaagtttggccttt
catatcgcaacgaaaatgcgtaatatacgcgcccttgcggacatcagtatggtcattcctagttcatgcgcat-
cggacaccaccagcttacaaattgcct
gattgcggccccgatggccggtatcactgaccgaccatttcgtgccttatgtcatgcgatgggggctgggccg-
tctctgaagctctcggtgaacattgtt
gcgaggcaggatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgccc-
gcccggaagcggcgttttcccgtcc
ggggaatggcatggagctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtc-
cgccctgatggcggcaccttgctgac
gttacgcctgccggtacagcaggttatcaccggaggcttaaaatgacccagttacctaccgcgggcccggtta-
tccggcgctttgatatgtctgcccag
tttacggcgctttatcgcatcagcgtggcgctgagtcacgaaagcaacaccgggcgcgcactggcgccgatcc-
tcgaagtgcttcacgatcatgcatt
tatgcaatacggcatggtgtgctgtttgataaagaacgcaatgcactctttgtggaatccctgcatggcatcg-
acggcgaaaggaaaaaagagaccc
gccatgtccgttaccgcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggcgctggtgttacc-
gcgcatttcagacgatcagcgttt
tctcgaccgcctgaatatatttacgattacagcctgccgttgattggcgtgccgatccccggtgcggataatc-
agccatcgggcgtgctggtggcacagcc
gatggcgttgcacgaagaccggctgactgccagtacgcggtttttagaaatggtc
[0433] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. For example, if the range 10-15 is disclosed, then
11, 12, 13, and 14 are also disclosed. All methods described herein
can be performed in any suitable order unless otherwise indicated
herein or otherwise clearly contradicted by context. The use of any
and all examples, or exemplary language (e.g., "such as") provided
herein, is intended merely to better illuminate the invention and
does not pose a limitation on the scope of the invention unless
otherwise claimed. No language in the specification should be
construed as indicating any non-claimed element as essential to the
practice of the invention.
CLAUSES
[0434] 1. A method of increasing nitrogen fixation in a
non-leguminous plant, comprising: [0435] a. applying to the plant a
plurality of non-intergeneric bacteria, said plurality comprising
non-intergeneric bacteria that: [0436] i. have an average
colonization ability per unit of plant root tissue of at least
about 1.0.times.10.sup.4 bacterial cells per gram of fresh weight
of plant root tissue; and/or [0437] ii. produce fixed N of at least
about 1.times.10.sup.4' mmol N per bacterial cell per hour, and
[0438] wherein the plurality of non-intergeneric bacteria, in
planta, produce 1% or more of the fixed nitrogen in the plant, and
[0439] wherein each member of the plurality of non-intergeneric
bacteria comprises at least one genetic variation introduced into
at least one gene, or non-coding polynucleotide, of the nitrogen
fixation or assimilation genetic regulatory network, such that the
non-intergeneric bacteria are capable of fixing atmospheric
nitrogen in the presence of exogenous nitrogen. [0440] 2. The
method according to clause 1, wherein the plurality of
non-intergeneric bacteria comprise bacteria that: have an average
colonization ability per unit of plant root tissue of at least
about 1.0.times.10.sup.4 bacterial cells per gram of fresh weight
of plant root tissue. [0441] 3. The method according to clause 1,
wherein the plurality of non-intergeneric bacteria comprise
bacteria that: produce fixed N of at least about 1.times.10.sup.-17
mmol N per bacterial cell per hour. [0442] 4. The method according
to clause 1, wherein the plurality of non-intergeneric bacteria
comprise bacteria that: have an average colonization ability per
unit of plant root tissue of at least about 1.0.times.10.sup.4
bacterial cells per gram of fresh weight of plant root tissue and
produce fixed N of at least about 1.times.10.sup.-17 mmol N per
bacterial cell per hour. [0443] 5. The method according to clause
1, wherein the at least one genetic variation comprises an
introduced control sequence operably linked to the at least one
gene of the nitrogen fixation or assimilation genetic regulatory
network. [0444] 6. The method according to clause 1, wherein the at
least one genetic variation comprises a promoter operably linked to
the at least one gene of the nitrogen fixation or assimilation
genetic regulatory network. [0445] 7. The method according to
clause 1, wherein the at least one genetic variation comprises an
inducible promoter operably linked to the at least one gene of the
nitrogen fixation or assimilation genetic regulatory network.
[0446] 8. The method according to clause 1, wherein the plurality
of non-intergeneric bacteria do not comprise a constitutive
promoter operably linked to a gene of the nitrogen fixation or
assimilation genetic regulatory network. [0447] 9. The method
according to clause 1, wherein the plurality of non-intergeneric
bacteria do not comprise a constitutive promoter operably linked to
a gene in the nif gene cluster. [0448] 10. The method according to
clause 1, wherein the plurality of non-intergeneric bacteria, in
planta, excrete nitrogen-containing products of nitrogen fixation.
[0449] 11. The method according to clause 1, wherein the plurality
of non-intergeneric bacteria applied to the plant do not stimulate
an increase in the uptake of exogenous non-atmospheric nitrogen.
[0450] 12. The method according to clause 1, wherein the plant is
grown in soil from a field which has been administered at least
about 50 lbs of nitrogen-containing fertilizer per acre, and
wherein the nitrogen-containing fertilizer comprises at least about
5% nitrogen by weight. [0451] 13. The method according to clause 1,
wherein the plant is grown in soil from a field which has been
administered at least about 50 lbs of nitrogen-containing
fertilizer per acre, and wherein the nitrogen-containing fertilizer
comprises at least about 5% nitrogen by weight, and wherein the
nitrogen-containing fertilizer comprises ammonium or an ammonium
containing molecule. [0452] 14. The method according to clause 1,
wherein the exogenous nitrogen is selected from fertilizer
comprising one or more of: glutamine, ammonia, ammonium, urea,
nitrate, nitrite, ammonium-containing molecules, nitrate-containing
molecules, and nitrite-containing molecules. [0453] 15. The method
according to clause 1, wherein the plurality of non-intergeneric
bacteria, in plan/a, produce 5% or more of the fixed nitrogen in
the plant. [0454] 16. The method according to clause 1, wherein the
plurality of non-intergeneric bacteria, in planta, produce 10% or
more of the fixed nitrogen in the plant. [0455] 17. The method
according to clause 1, wherein the plurality of non-intergeneric
bacteria, in planta, fix atmospheric nitrogen in
non-nitrogen-limiting conditions. [0456] 18. The method according
to clause 1, wherein the plurality of non-intergeneric bacteria, in
planta, excrete nitrogen-containing products of nitrogen fixation.
[0457] 19. The method according to clause 1, wherein the fixed
nitrogen produced by the plurality of non-intergeneric bacteria is
measured through dilution of enriched fertilizer by atmospheric N2
gas in plant tissue. [0458] 20. The method according to clause 1,
wherein the at least one gene, or non-coding polynucleotide, of the
nitrogen fixation or assimilation genetic regulatory network are
selected from the group consisting of: nifA, nifL, ntrB, ntrC,
polynucleotide encoding glutamine synthetase, glnA, glnB, glnK,
drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ,
nifD, nifK, nifY, nifE, nifL, nifV, nifW, nifZ, nifM, nifF, nifB,
nifQ, and a gene associated with biosynthesis of a nitrogenase
enzyme. [0459] 21. The method according to clause 1, wherein the at
least one genetic variation is a mutation that results in one or
more of: increased expression or activity of NifA or glutaminase;
decreased expression or activity of NifL, NtrB, glutamine
synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing
activity of GlnE; or decreased uridylyl-removing activity of GlnD.
[0460] 22. The method according to clause 1, wherein the at least
one genetic variation is selected from: (A) a knock-out mutation;
(B) alters or abolishes a regulatory sequence of a target gene; (C)
comprises the insertion of a heterologous regulatory sequence; or
(D) a domain deletion. [0461] 23. The method according to clause 1,
wherein the at least one genetic variation is a mutated nifL gene
that has been altered to comprise a heterologous promoter inserted
into said nifL gene. [0462] 24. The method according to clause 1,
wherein the at least one genetic variation is a mutated glnE gene
that results in a truncated GlnE protein lacking an
adenylyl-removing (AR) domain. [0463] 25. The method according to
clause 1, wherein the at least one genetic variation is a mutated
amtB gene that results in the lack of expression of said amtB gene.
[0464] 26. The method according to clause 1, wherein the at least
one genetic variation is selected from: a mutated nifL gene that
has been altered to comprise a heterologous promoter inserted into
said nifL gene; a mutated glnE gene that results in a truncated
GlnE protein lacking an adenylyl-removing (AR) domain; a mutated
amtB gene that results in the lack of expression of said amtB gene;
and combinations thereof. [0465] 27. The method according to clause
1, wherein the at least one genetic variation is a mutated nifL
gene that has been altered to comprise a heterologous promoter
inserted into said nifL gene and a mutated glnE gene that results
in a truncated GlnE protein lacking an adenylyl-removing (AR)
domain. [0466] 28. The method according to clause 1, wherein the at
least one genetic variation is a mutated nifL gene that has been
altered to comprise a heterologous promoter inserted into said nifL
gene and a mutated glnE gene that results in a truncated GlnE
protein lacking an adenylyl-removing (AR) domain and a mutated amtB
gene that results in the lack of expression of said amtB gene.
[0467] 29. The method according to clause 1, wherein the plant
comprises the seed, stalk, flower, fruit, leaves, or rhizome.
[0468] 30. The method according to clause 1, wherein the plurality
of non-intergeneric bacteria are formulated into a composition.
[0469] 31. The method according to clause 1, wherein the plurality
of non-intergeneric bacteria are formulated into a composition
comprising an agriculturally acceptable carrier. [0470] 32. The
method according to clause 1, wherein the plurality of
non-intergeneric bacteria are applied into furrows in which seeds
of said plant are planted. [0471] 33. The method according to
clause 1, wherein the plurality of non-intergeneric bacteria are
formulated into a liquid in-furrow composition comprising bacteria
at a concentration of about 1.times.10.sup.7 to about
1.times.10.sup.10 du per milliliter. [0472] 34. The method
according to clause 1, wherein the plurality of non-intergeneric
bacteria are applied onto a seed of said plant. [0473] 35. The
method according to clause 1, wherein the plurality of
non-intergeneric bacteria are formulated into a seed coating and
are applied onto a seed of said plant. [0474] 36. The method
according to clause 1, wherein the plurality of non-intergeneric
bacteria are formulated into a seed coating and are applied onto a
seed of said plant, at a concentration of about 1.times.10.sup.5 to
about 1.times.10.sup.7 cfu per seed. [0475] 37. The method
according to clause 1, wherein the plant is a cereal crop. [0476]
38. The method according to clause 1, wherein the plant is selected
from the group consisting of: corn, rice, wheat, barley, sorghum,
millet, oat, rye, and triticale. [0477] 39. The method according to
clause 1, wherein the plant is corn. [0478] 40. The method
according to clause 1, wherein the plant is an agricultural crop
plant. [0479] 41. The method according to clause 1, wherein the
plant is a genetically modified organism. [0480] 42. The method
according to clause 1, wherein the plant is not a genetically
modified organism. [0481] 43. The method according to clause 1,
wherein the plant has been genetically engineered or bred for
efficient nitrogen use. [0482] 44. The method according to clause
1, wherein the plurality of non-intergeneric bacteria comprise at
least two different species of bacteria. [0483] 45. The method
according to clause 1, wherein the plurality of non-intergeneric
bacteria comprise at least two different strains of the same
species of bacteria. [0484] 46. The method according to clause 1,
wherein the plurality of non-intergeneric bacteria comprise
bacteria selected from: Rahnella aquatilis, Klebsiella variicola,
Achromobacter spiritinus, Achromobacter marplatensis,
Microbacterium murale, Kluyvera intermedia, Kosakonia
pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia
sacchari, and combinations thereof. [0485] 47. The method according
to clause 1, wherein the plurality of non-intergeneric bacteria are
endophytic, epiphytic, or rhizospheric. [0486] 48. The method
according to clause 1, wherein the plurality of non-intergeneric
bacteria comprise bacteria selected from: a bacteria deposited as
PTA-122293, a bacteria deposited as PTA-122294, a bacteria
deposited as NCMA 201701002, a bacteria deposited as NCMA
201708004, a bacteria deposited as NCMA 201708003, a bacteria
deposited as NCMA 201708002, a bacteria deposited as NCMA
201712001, a bacteria deposited as NCMA 201712002, and combinations
thereof. [0487] 49. A bacterial composition, comprising: [0488] at
least one genetically engineered bacterial strain that fixes
atmospheric nitrogen in an agricultural system that has been
fertilized with more than 20 lbs of Nitrogen per acre. [0489] 50. A
bacterial composition, comprising: [0490] at least one bacterial
strain that has been bred to fix atmospheric nitrogen in an
agricultural system that has been fertilized with more than 20 lbs
of Nitrogen per acre. [0491] 51. The bacterial composition of
clause 49 or clause 50, wherein said fertilizer is a chemical
fertilizer selected from the group consisting of anhydrous ammonia,
ammonia sulfate, urea, diammonium phosphate, urea-form, UAN (urea
ammonium nitrate) monoammonium phosphate, ammonium nitrate,
nitrogen solutions, calcium nitrate, potassium nitrate, and sodium
nitrate. [0492] 52. A bacterial composition, comprising: [0493] at
least one genetically engineered bacterial strain that fixes
atmospheric nitrogen, the at least one genetically engineered
bacterial strain comprising exogenously added DNA wherein said
exogenously added DNA shares at least 80% identity to a
corresponding native bacterial strain. [0494] 53. The bacterial
composition of clause 52, wherein said exogenously added DNA shares
at least 85% identity to a corresponding native bacterial strain.
[0495] 54. The bacterial composition of clause 52, wherein said
exogenously added DNA shares at least 90% identity to a
corresponding native bacterial strain. [0496] 55. The bacterial
composition of clause 52, wherein said exogenously added DNA shares
at least 95% identity to a corresponding native bacterial strain.
[0497] 56. The bacterial composition of clause 52, wherein said
exogenously added DNA shares at least 99% identity to a
corresponding native bacterial strain. [0498] 57. The bacterial
composition of clause 52, wherein said exogenously added DNA is
derived from a same bacterial strain as said corresponding native
bacterial strain. [0499] 58. The bacterial composition of any of
the preceding clauses, wherein said bacterial composition is a
fertilizing composition. [0500] 59. The bacterial composition of
any of the preceding clauses, wherein said at least one genetically
engineered bacterial strain comprises at least one variation in a
gene or intergenic region within 10,000 bp of a gene selected from
the group consisting of: nifA, nifL, ntrB, ntrC, glnA, glnB, glnK,
draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN,
nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ. [0501]
60. The bacterial composition of any of the preceding clauses,
further comprising at least one additional component selected from
the group consisting of a tackifier, a microbial stabilizer, a
fungicide, an antibacterial agent, a preservative, a stabilizer, a
surfactant, an anti-complex agent, an herbicide, a nematicide, an
insecticide, a plant growth regulator, a fertilizer, a rodenticide,
a dessicant, a bactericide, and a nutrient. [0502] 61. The
bacterial composition of any of the preceding clauses, further
comprising at least one additional component selected from the
group consisting of a colorant, primer, pellet, disinfectant, plant
growth regulator, safener, and a nematicide. [0503] 62. The
bacterial composition of any of the preceding clauses, wherein said
bacterial composition is formulated to be applied to a field.
[0504] 63. The bacterial composition of clause 62, wherein said
bacterial composition is formulated to be applied in-furrow.
[0505] 64. The bacterial composition of clause 62, wherein said
bacterial composition is formulated to be applied as a seed
coating, seed dressing, or seed treatment. [0506] 65. The bacterial
composition of clause 61 or clause 62, wherein said bacterial
composition is formulated to be applied at, prior to, or post
planting of the seed. [0507] 66. A seed composition comprising a
seed of a plant that is inoculated with a bacterial composition of
any of the preceding clauses. [0508] 67. A method of growing a crop
using a plurality of seeds having a seed composition of clause 66.
[0509] 68. The method of clause 67, further comprising harvesting
said crop. [0510] 69. A method of applying a bacterial composition
of any of the preceding clauses to a field. [0511] 70. The method
of clause 69, wherein said bacterial composition is applied to said
field in a form selected from the group consisting of a liquid
form, a dry form, a granule, a powder, and a pellet. [0512] 71. The
method of clause 69, wherein said bacterial composition is applied
to said field as a seed coating, seed dressing, or seed treatment.
[0513] 72. The method of clause 69, wherein said bacterial
composition is applied to said field as an in-furrow treatment.
[0514] 73. The method of clause 69, wherein said bacterial
composition is applied to said field at, prior to, or post planting
of the seed. [0515] 74. A fertilizer composition comprising a
bacterial composition of any of the preceding clauses. [0516] 75.
The fertilizer composition of clause 74, wherein said fertilizer
composition is a seed coating, seed dressing, or seed treatment
composition. [0517] 76. The fertilizer composition of clause 74,
wherein said fertilizer composition is an in-furrow composition.
[0518] 77. The fertilizer composition of clause 74, wherein said
fertilizer composition is provided to a crop at, prior to, or post
planting. [0519] 78. The fertilizer composition of clause 74,
further comprising a porous carrier. [0520] 79. The fertilizer
composition of clause 74, further comprising an additional
synergistic component that, when combined with said bacterial
composition, increases a fertilizing benefit of said fertilizer
composition to a crop that is beyond a cumulative benefit of its
individual components. [0521] 80. A method of maintaining soil
nitrogen levels, comprising: [0522] planting, in soil of a field, a
crop inoculated by a genetically engineered bacterium that fixes
atmospheric nitrogen; and [0523] harvesting said crop, wherein no
more than 90% of a nitrogen dose required for producing said crop
is administered to said soil of said field between planting and
harvesting. [0524] 81. The method of clause 80, wherein no more
than 80% of a nitrogen dose required for producing said crop is
administered to said soil of said field between planting and
harvesting. [0525] 82. The method of clause 80, wherein no more
than 70% of a nitrogen dose required for producing said crop is
administered to said soil of said field between planting and
harvesting. [0526] 83. The method of clause 80, wherein no more
than 60% of a nitrogen dose required for producing said crop is
administered to said soil of said field between planting and
harvesting. [0527] 84. The method of clause 80, wherein no more
than 50% of a nitrogen dose required for producing said crop is
administered to said soil of said field between planting and
harvesting. [0528] 85. The method of clause 80, wherein no more
than 40% of a nitrogen dose required for producing said crop is
administered to said soil of said field between planting and
harvesting. [0529] 86. The method of clause 80, wherein said
genetically engineered bacterium comprises a bacterial composition
of any of the preceding clauses. [0530] 87. The method of clause
80, wherein said genetically engineered bacterium consists of a
bacterial composition of any of the preceding clauses. [0531] 88. A
method of delivering a probiotic supplement to a crop plant,
comprising: [0532] coating a crop seed with a seed coating, seed
treatment, or seed dressing, wherein said seed coating, seed
dressing, or seed treatment comprises living representatives of
said probiotic; and [0533] applying, in soil of a field, said crop
seeds. [0534] 89. The method of clause 88, wherein said seed
coating, seed dressing, or seed treatment is applied in a single
layer to said crop seed. [0535] 90. The method of clause 88,
wherein said seed coating is applied in multiple layers to said
crop seed. [0536] 91. The method of clause 88, wherein said seed
coating is applied in a blend to said crop seed. [0537] 92. The
method of clause 88, wherein said crop seed is non-nodulating.
[0538] 93. The method of clause 88, wherein said seed coating
comprises a bacterial composition of any of the preceding clauses.
[0539] 94. The method of any of the proceeding clauses, wherein the
genetically engineered bacterial strain is a genetically engineered
Gram-positive bacterial strain. [0540] 95. The method of clause 94,
wherein the genetically engineered Gram-positive bacterial strain
has an altered expression level of a regulator of a Nif cluster.
[0541] 96. The method of clause 94, wherein the genetically
engineered Gram-positive bacterial strain expresses a decreased
amount of a negative regulator of a Nif cluster. [0542] 97. The
method of clause 94, wherein the genetically engineered bacterial
strain expresses a decreased amount of GlnR. [0543] 98. The method
of any one of the proceeding clauses, wherein the genome of the
genetically engineered bacterial strain encodes a polypeptide with
at least 75% identity to a sequence from the Zehr lab NifH
database. [0544] 99. The method of any one of the proceeding
clauses, wherein the genome of the genetically engineered bacterial
strain encodes a polypeptide with at least 85% identity to a
sequence from the Zehr lab NifH database. [0545] 100. The method of
any one of the proceeding clauses, wherein the genome of the
genetically engineered bacterial strain encodes a polypeptide with
at least 75% identity to a sequence from the Buckley lab NifH
database. [0546] 101. The method of any one of the proceeding
clauses, wherein the genome of the genetically engineered bacterial
strain encodes a polypeptide with at least 85% identity to a
sequence from the Buckley lab NifH database. [0547] 102. A method
of breeding microbial strains to improve specific traits of
agronomic relevance, comprising: [0548] providing a plurality of
microbial strains that have the ability to colonize a desired crop;
[0549] improving regulatory networks influencing the trait through
intragenomic rearrangement; [0550] assessing microbial strains
within the plurality of microbial strains to determine a measure of
the trait; and [0551] selecting one or more microbial strains of
the plurality of microbial strains that generate an improvement in
the trait in the presence of the desired crop. [0552] 103. The
method of clause 102, wherein the specific trait which is improved
is the ability of the microbial strain to fix nitrogen. [0553] 104.
The method of clause 103, wherein the specific trait which is
improved is the ability of the microbial strain to fix atmospheric
nitrogen in the presence of N-fertilized growing conditions. [0554]
105. A method of breeding microbial strains to improve specific
traits of agronomic relevance, comprising: [0555] providing a
plurality of microbial strains that have the ability to colonize a
desired crop; [0556] introducing genetic diversity into the
plurality of microbial strains; [0557] assessing microbial strains
within the plurality of microbial strains to determine a measure of
the trait; and [0558] selecting one or more microbial strains of
the plurality of microbial strains that generate an improvement in
the trait in the presence of the desired crop. [0559] 106. The
method of clause 105, wherein the specific trait which is improved
is the ability of the microbial strain to fix nitrogen. [0560] 107.
The method of clause 106, wherein the specific trait which is
improved is the ability of the microbial strain to fix atmospheric
nitrogen in the presence of N-fertilized growing conditions. [0561]
108. A method of increasing the amount of atmospheric derived
nitrogen in a non-leguminous plant, comprising: [0562] exposing
said non-leguminous plant to engineered non-intergeneric microbes,
said engineered non-intergeneric microbes comprising at least one
genetic variation introduced into a nitrogen fixation genetic
regulatory network or at least one genetic variation introduced
into a nitrogen assimilation genetic regulatory network. [0563]
109. The method of clause 108, wherein said engineered
non-intergeneric microbes comprise at least one genetic variation
introduced into said nitrogen fixation genetic regulatory network.
[0564] 110. The method of clause 108, wherein said engineered
non-intergeneric microbes comprise at least one genetic variation
introduced into said nitrogen assimilation genetic regulatory
network. [0565] 111. The method of clause 108, wherein said
engineered non-intergeneric microbes comprise at least one genetic
variation introduced into said nitrogen fixation genetic regulatory
network and at least one genetic variation introduced into said
nitrogen assimilation genetic regulatory network. [0566] 112. The
method of clause 108, wherein said engineered non-intergeneric
microbes are applied into furrows in which seeds of said
non-leguminous plant are planted. [0567] 113. The method of clause
108, wherein said engineered non-intergeneric microbes are coated
onto a seed of said non-leguminous plant. [0568] 114. The method of
clause 108, wherein said non-leguminous plant is a non-leguminous
agricultural crop plant selected from the group consisting of
sorghum, canola, tomato, strawberry, barley, rice, corn, wheat,
potato, millet, cereals, grains, and maize. [0569] 115. The method
of clause 108, wherein said engineered non-intergeneric microbes
colonize at least a root of said non-leguminous plant such that
said engineered non-intergeneric microbes are present in said
non-leguminous plant in an amount of at least 10.sup.5 colony
forming units per gram fresh weight of tissue. [0570] 116. The
method of clause 108, wherein said engineered non-intergeneric
microbes are capable of fixing atmospheric nitrogen in
non-nitrogen-limiting conditions. [0571] 117. The method of clause
108, wherein said engineered non-intergeneric microbes, in planta,
excrete nitrogen-containing products of nitrogen fixation. [0572]
118. The method of clause 108, wherein said at least one genetic
variation is introduced into a gene selected from the group
consisting of nifA, nut, ntrB, ntrC, polynucleotide encoding
glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide
encoding glutaminase, glnD, glnE, nifJ, nifH, nit)), nifK, nifY,
nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ,
and a gene associated with biosynthesis of a nitrogenase enzyme.
[0573] 119. The method of clause 108, wherein said engineered
non-intergeneric microbes, in planta, produce at least 1% of fixed
nitrogen in said non-leguminous plant. [0574] 120. The method of
clause 119, wherein said fixed nitrogen in said non-leguminous
plant produced by said engineered non-intergeneric microbes is
measured by dilution of 15N in crops grown in fields treated with
fertilizer containing 1.2% 15N. [0575] 121. The method of clause
119, wherein said engineered non-intergeneric microbes, in planta,
produce 5% or more of the fixed nitrogen in said non-leguminous
plant. [0576] 122. The method of clause 108, wherein said
non-intergeneric microbes are engineered using at least one type of
engineering selected from the group consisting of directed
mutagenesis, random mutagenesis, and directed evolution. [0577]
123. A method of increasing an amount of atmospheric derived
nitrogen in a corn plant, comprising [0578] exposing said corn
plant to engineered non-intergeneric microbes comprising engineered
genetic variations within at least two genes selected from the
group consisting of nifL, glnB, glnE, and amtB. [0579] 124. The
method of clause 123, wherein said engineered non-intergeneric
microbes, in planta, excrete nitrogen-containing products of
nitrogen fixation. [0580] 125. The method of clause 123, wherein
said engineered non-intergeneric microbes are applied into furrows
in which seeds of said corn plant are planted. [0581] 126. The
method of clause 123, wherein said engineered non-intergeneric
microbes are coated onto a seed of said corn plant. [0582] 127. A
method of increasing an amount of atmospheric derived nitrogen in a
corn plant, comprising: [0583] exposing said corn plant to
engineered non-intergeneric microbes comprising at least one
genetic variation introduced into a nitrogen fixation genetic
regulatory network and at least one genetic variation introduced
into a nitrogen assimilation genetic regulatory network, wherein
said engineered non-intergeneric microbes, in planta, produce at
least 5% of fixed nitrogen in said corn plant as measured by
dilution of 15N in crops grown in fields treated with fertilizer
containing 1.2% 15N. [0584] 128. A method of increasing nitrogen
fixation in a non-leguminous plant, comprising: [0585] a. applying
to the plant a plurality of non-intergeneric bacteria, said
plurality comprising non-intergeneric bacteria that: [0586] i. have
an average colonization ability per unit of plant root tissue of at
least about 1.0.times.10.sup.4 bacterial cells per gram of fresh
weight of plant root tissue; and [0587] ii. produce fixed N of at
least about 1.times.10.sup.-17 mmol N per bacterial cell per hour,
and [0588] wherein the product of (i) the average colonization
ability per unit of plant root tissue and (ii) produced fixed N per
bacterial cell per hour, is at least about 2.5.times.10.sup.4 mmol
N per gram of fresh weight of plant root tissue per hour, and
[0589] wherein the plurality of non-intergeneric bacteria, in
planta, produce 1% or more of the fixed nitrogen in the plant, and
wherein each member of the plurality of non-intergeneric bacteria
comprises at least one genetic variation introduced into at least
one gene, or non-coding polynucleotide, of the nitrogen fixation or
assimilation genetic regulatory network, such that the
non-intergeneric bacteria are capable of fixing atmospheric
nitrogen in the presence of exogenous nitrogen. [0590] 129. The
method according to clause 128, wherein the at least one genetic
variation comprises an introduced control sequence operably linked
to the at least one gene of the nitrogen fixation or assimilation
genetic regulatory network. [0591] 130. The method according to
clause 128, wherein the at least one genetic variation comprises a
promoter operably linked to the at least one gene of the nitrogen
fixation or assimilation genetic regulatory network. [0592] 131.
The method according to clause 128, wherein the at least one
genetic variation comprises an inducible promoter operably linked
to the at least one gene of the nitrogen fixation or assimilation
genetic regulatory network.
[0593] 132. The method according to clause 128, wherein the
plurality of non-intergeneric bacteria do not comprise a
constitutive promoter operably linked to a gene of the nitrogen
fixation or assimilation genetic regulatory network. [0594] 133.
The method according to clause 128, wherein the plurality of
non-intergeneric bacteria do not comprise a constitutive promoter
operably linked to a gene in the nif gene cluster. [0595] 134. The
method according to clause 128, wherein the plurality of
non-intergeneric bacteria, in planta, excrete the
nitrogen-containing products of nitrogen fixation. [0596] 135. The
method according to clause 128, wherein the plurality of
non-intergeneric bacteria applied to the plant do not stimulate an
increase in the uptake of exogenous non-atmospheric nitrogen.
[0597] 136. The method according to clause 128, wherein the plant
is grown in soil from a field which has been administered at least
about 50 lbs of nitrogen-containing fertilizer per acre, and
wherein the nitrogen-containing fertilizer comprises at least about
5% nitrogen by weight. [0598] 137. The method according to clause
128, wherein the plant is grown in soil from a field which has been
administered at least about 50 lbs of nitrogen-containing
fertilizer per acre, and wherein the nitrogen-containing fertilizer
comprises at least about 5% nitrogen by weight, and wherein the
nitrogen-containing fertilizer comprises ammonium or an ammonium
containing molecule. [0599] 138. The method according to clause
128, wherein the exogenous nitrogen is selected from fertilizer
comprising one or more of: glutamine, ammonia, ammonium, urea,
nitrate, nitrite, ammonium-containing molecules, nitrate-containing
molecules, and nitrite-containing molecules. [0600] 139. The method
according to clause 128, wherein the plurality of non-intergeneric
bacteria, in planta, produce 5% or more of the fixed nitrogen in
the plant. [0601] 140. The method according to clause 128, wherein
the plurality of non-intergeneric bacteria, in planta, produce 10%
or more of the fixed nitrogen in the plant. [0602] 141. The method
according to clause 128, wherein the plurality of non-intergeneric
bacteria, in planta, fix atmospheric nitrogen in
non-nitrogen-limiting conditions. [0603] 142. The method according
to clause 128, wherein the plurality of non-intergeneric bacteria,
in planta, excrete nitrogen-containing products of nitrogen
fixation. [0604] 143. The method according to clause 128, wherein
the fixed nitrogen produced by the plurality of non-intergeneric
bacteria is measured through dilution of enriched fertilizer by
atmospheric N2 gas in plant tissue. [0605] 144. The method
according to clause 128, wherein the at least one gene, or
non-coding polynucleotide, of the nitrogen fixation or assimilation
genetic regulatory network are selected from the group consisting
of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine
synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding
glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN,
nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifQ, nifA, and a gene
associated with biosynthesis of a nitrogenase enzyme. [0606] 145.
The method according to clause 128, wherein the at least one
genetic variation is a mutation that results in one or more of:
increased expression or activity of NifA or glutaminase; decreased
expression or activity of NifL, NtrB, glutamine synthetase, GlnB,
GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or
decreased uridylyl-removing activity of GlnD. [0607] 146. The
method according to clause 128, wherein the at least one genetic
variation is selected from: (A) a knock-out mutation; (B) alters or
abolishes a regulatory sequence of a target gene; (C) comprises the
insertion of a heterologous regulatory sequence; or (D) a domain
deletion. [0608] 147. The method according to clause 128, wherein
the at least one genetic variation is a mutated nifL gene that has
been altered to comprise a heterologous promoter inserted into said
nifL gene. [0609] 148. The method according to clause 128, wherein
the at least one genetic variation is a mutated glnE gene that
results in a truncated GlnE protein lacking an adenylyl-removing
(AR) domain. [0610] 149. The method according to clause 128,
wherein the at least one genetic variation is a mutated amtB gene
that results in the lack of expression of said amtB gene. [0611]
150. The method according to clause 128, wherein the at least one
genetic variation is selected from: a mutated nifL gene that has
been altered to comprise a heterologous promoter inserted into said
n/IL gene; a mutated glnE gene that results in a truncated GlnE
protein lacking an adenylyl-removing (AR) domain; a mutated amtB
gene that results in the lack of expression of said amtB gene; and
combinations thereof. [0612] 151. The method according to clause
128, wherein the at least one genetic variation is a mutated nifL
gene that has been altered to comprise a heterologous promoter
inserted into said nifL gene and a mutated glnE gene that results
in a truncated GlnE protein lacking an adenylyl-removing (AR)
domain. [0613] 152. The method according to clause 128, wherein the
at least one genetic variation is a mutated nifL gene that has been
altered to comprise a heterologous promoter inserted into said nifL
gene and a mutated glnE gene that results in a truncated GlnE
protein lacking an adenylyl-removing (AR) domain and a mutated amtB
gene that results in the lack of expression of said amtB gene.
[0614] 153. The method according to clause 128, wherein the plant
comprises the seed, stalk, flower, fruit, leaves, or rhizome.
[0615] 154. The method according to clause 128, wherein the
plurality of non-intergeneric bacteria are formulated into a
composition. [0616] 155. The method according to clause 128,
wherein the plurality of non-intergeneric bacteria are formulated
into a composition comprising an agriculturally acceptable carrier.
[0617] 156. The method according to clause 128, wherein the
plurality of non-intergeneric bacteria are applied into furrows in
which seeds of said plant are planted. [0618] 157. The method
according to clause 128, wherein the plurality of non-intergeneric
bacteria are formulated into a liquid in-furrow composition
comprising bacteria at a concentration of about 1.times.10.sup.7 to
about 1.times.10.sup.10 cfu per milliliter. [0619] 158. The method
according to clause 128, wherein the plurality of non-intergeneric
bacteria are applied onto a seed of said plant. [0620] 159. The
method according to clause 128, wherein the plurality of
non-intergeneric bacteria are formulated into a seed coating and
are applied onto a seed of said plant. [0621] 160. The method
according to clause 128, wherein the plurality of non-intergeneric
bacteria are formulated into a seed coating and are applied onto a
seed of said plant, at a concentration of about 1.times.10.sup.5 to
about 1.times.10.sup.7 cfu per seed. [0622] 161. The method
according to clause 128, wherein the plant is a cereal crop. [0623]
162. The method according to clause 128, wherein the plant is
selected from the group consisting of: corn, rice, wheat, barley,
sorghum, millet, oat, rye, and triticale. [0624] 163. The method
according to clause 128, wherein the plant is corn. [0625] 164. The
method according to clause 128, wherein the plant is an
agricultural crop plant. [0626] 165. The method according to clause
128, wherein the plant is a genetically modified organism. [0627]
166. The method according to clause 128, wherein the plant is not a
genetically modified organism. [0628] 167. The method according to
clause 128, wherein the plant has been genetically engineered or
bred for efficient nitrogen use. [0629] 168. The method according
to clause 128, wherein the plurality of non-intergeneric bacteria
comprise at least two different species of bacteria. [0630] 169.
The method according to clause 128, wherein the plurality of
non-intergeneric bacteria comprise at least two different strains
of the same species of bacteria. [0631] 170. The method according
to clause 128, wherein the plurality of non-intergeneric bacteria
comprise bacteria selected from: Rahnella aquatilis, Klebsiella
variicola, Achromobacter spiritinus, Achromobacter marplatensis,
Microbacterium murale, Kluyvera intermedia, Kosakonia
pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia
sacchari, and combinations thereof. [0632] 171. The method
according to clause 128, wherein the plurality of non-intergeneric
bacteria are endophytic, epiphytic, or rhizospheric. [0633] 172.
The method according to clause 128, wherein the plurality of
non-intergeneric bacteria comprise bacteria selected from: a
bacteria deposited as PTA-122293, a bacteria deposited as
PTA-122294, a bacteria deposited as NCMA 201701002, a bacteria
deposited as NCMA 201708004, a bacteria deposited as NCMA
201708003, a bacteria deposited as NCMA 201708002, a bacteria
deposited as NCMA 201712001, a bacteria deposited as NCMA
201712002, and combinations thereof. [0634] 173. A method of
increasing nitrogen fixation in a non-leguminous plant, comprising:
[0635] a. applying to the plant a plurality of bacteria, said
plurality comprising bacteria that: [0636] i. have an average
colonization ability per unit of plant root tissue of at least
about 1.0.times.10.sup.4 bacterial cells per gram of fresh weight
of plant root tissue; and/or [0637] ii. produce fixed N of at least
about 1.times.10.sup.-17 mmol N per bacterial cell per hour, and
[0638] wherein the plurality of bacteria, in planta, produce 1% or
more of the fixed nitrogen in the plant. [0639] 174. The method
according to clause 173, wherein the plurality of bacteria comprise
bacteria that: have an average colonization ability per unit of
plant root tissue of at least about 1.0.times.10.sup.4 bacterial
cells per gram of fresh weight of plant root tissue. [0640] 175.
The method according to clause 173, wherein the plurality of
bacteria comprise bacteria that: produce fixed N of at least about
173.times.10.sup.-17 mmol N per bacterial cell per hour. [0641]
176. The method according to clause 173, wherein the plurality of
bacteria comprise bacteria that: have an average colonization
ability per unit of plant root tissue of at least about
1.0.times.10.sup.4 bacterial cells per gram of fresh weight of
plant root tissue and produce fixed N of at least about
1.times.10.sup.-17 mmol N per bacterial cell per hour. [0642] 177.
The method according to clause 173, wherein the plurality of
bacteria comprise bacteria that: have an average colonization
ability per unit of plant root tissue of at least about
1.0.times.10.sup.4 bacterial cells per gram of fresh weight of
plant root tissue and produce fixed N of at least about
1.times.10.sup.-17 mmol N per bacterial cell per hour, and wherein
the product of (i) the average colonization ability per unit of
plant root tissue and (ii) produced fixed N per bacterial cell per
hour, is at least about 2.5.times.10.sup.-8 mmol N per gram of
fresh weight of plant root tissue per hour. [0643] 178. The method
according to clause 173, wherein the plurality of bacteria, in
planta, excrete the nitrogen-containing products of nitrogen
fixation. [0644] 179. The method according to clause 173, wherein
the plurality of bacteria applied to the plant do not stimulate an
increase in the uptake of exogenous non-atmospheric nitrogen.
[0645] 180. The method according to clause 173, wherein the plant
comprises the seed, stalk, flower, fruit, leaves, or rhizome.
[0646] 181. The method according to clause 173, wherein the
plurality of bacteria are formulated into a composition. [0647]
182. The method according to clause 173, wherein the plurality of
bacteria are formulated into a composition comprising an
agriculturally acceptable carrier. [0648] 183. The method according
to clause 173, wherein the plurality of bacteria are applied into
furrows in which seeds of said plant are planted. [0649] 184. The
method according to clause 173, wherein the plurality of bacteria
are formulated into a liquid in-furrow composition comprising
bacteria at a concentration of about 1.times.10.sup.7 to about
1.times.10.sup.10 cfu per milliliter. [0650] 185. The method
according to clause 173, wherein the plurality of bacteria are
applied onto a seed of said plant. [0651] 186. The method according
to clause 173, wherein the plurality of bacteria are formulated
into a seed coating and are applied onto a seed of said plant.
[0652] 187. The method according to clause 173, wherein the
plurality of bacteria are formulated into a seed coating and are
applied onto a seed of said plant, at a concentration of about
1.times.10.sup.5 to about 1.times.10.sup.7 cfu per seed. [0653]
188. The method according to clause 173, wherein the plant is a
cereal crop. [0654] 189. The method according to clause 173,
wherein the plant is selected from the group consisting of: corn,
rice, wheat, barley, sorghum, millet, oat, rye, and triticale.
[0655] 190. The method according to clause 173, wherein the plant
is corn. [0656] 191. The method according to clause 173, wherein
the plant is an agricultural crop plant. [0657] 192. The method
according to clause 173, wherein the plant is a genetically
modified organism. [0658] 193. The method according to clause 173,
wherein the plant is not a genetically modified organism. [0659]
194. The method according to clause 173, wherein the plant has been
genetically engineered or bred for efficient nitrogen use. [0660]
195. The method according to clause 173, wherein the plurality of
bacteria comprise at least two different species of bacteria.
[0661] 196. The method according to clause 173, wherein the
plurality of bacteria comprise at least two different strains of
the same species of bacteria. [0662] 197. The method according to
clause 173, wherein the plurality of bacteria comprise bacteria
selected from: Rahnella aquatilis, Klebsiella variicola,
Achromobacter spiritinus, Achromobacter marplatensis,
Microbacterium murale, Kluyvera intermedia, Kosakonia
pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia
sacchari, and combinations thereof. [0663] 198. The method
according to clause 173, wherein the plurality of bacteria are
endophytic, epiphytic, or rhizospheric. [0664] 199. The method
according to clause 173, wherein the plurality of bacteria are
selected from: a bacteria deposited as NCMA 201701003, a bacteria
deposited as NCMA 201701001, and a bacteria deposited as NCMA
201708001. [0665] 200. A non-intergeneric bacterial population
capable of increasing nitrogen fixation in a non-leguminous plant,
comprising: [0666] a. a plurality of non-intergeneric bacteria,
that: [0667] i. have an average colonization ability per unit of
plant root tissue of at least about 1.0.times.10.sup.4 bacterial
cells per gram of fresh weight of plant root tissue; and/or [0668]
ii. produce fixed N of at least about 1
.times.10.sup.-17 mmol N per bacterial cell per hour, and [0669]
wherein the plurality of non-intergeneric bacteria, in planta,
produce 1% or more of the fixed nitrogen in a plant grown in the
presence of the plurality of non-intergeneric bacteria, and [0670]
wherein each member of the plurality of non-intergeneric bacteria
comprises at least one genetic variation introduced into at least
one gene, or non-coding polynucleotide, of the nitrogen fixation or
assimilation genetic regulatory network, such that the
non-intergeneric bacteria are capable of fixing atmospheric
nitrogen in the presence of exogenous nitrogen. [0671] 201. The
non-intergeneric bacterial population according to clause 200,
wherein the plurality of non-intergeneric bacteria comprise
bacteria that: have an average colonization ability per unit of
plant root tissue of at least about 1.0.times.10.sup.4 bacterial
cells per gram of fresh weight of plant root tissue. [0672] 202.
The non-intergeneric bacterial population according to clause 200,
wherein the plurality of non-intergeneric bacteria comprise
bacteria that: produce fixed N of at least about 1.times.10.sup.-17
mmol N per bacterial cell per hour. [0673] 203. The
non-intergeneric bacterial population according to clause 200,
wherein the plurality of non-intergeneric bacteria comprise
bacteria that: have an average colonization ability per unit of
plant root tissue of at least about 1.0.times.10.sup.4 bacterial
cells per gam of fresh weight of plant root tissue and produce
fixed N of at least about 1.times.10.sup.-17 mmol N per bacterial
cell per hour. [0674] 204. The non-intergeneric bacterial
population according to clause 200, wherein the at least one
genetic variation comprises an introduced control sequence operably
linked to the at least one gene of the nitrogen fixation or
assimilation genetic regulatory network. [0675] 205. The
non-intergeneric bacterial population according to clause 200,
wherein the at least one genetic variation comprises a promoter
operably linked to the at least one gene of the nitrogen fixation
or assimilation genetic regulatory network. [0676] 206. The
non-intergeneric bacterial population according to clause 200,
wherein the at least one genetic variation comprises an inducible
promoter operably linked to the at least one gene of the nitrogen
fixation or assimilation genetic regulatory network. [0677] 207.
The non-intergeneric bacterial population according to clause 200,
wherein the plurality of non-intergeneric bacteria do not comprise
a constitutive promoter operably linked to a gene of the nitrogen
fixation or assimilation genetic regulatory network. [0678] 208.
The non-intergeneric bacterial population according to clause 200,
wherein the plurality of non-intergeneric bacteria do not comprise
a constitutive promoter operably linked to a gene in the nif gene
cluster. [0679] 209. The non-intergeneric bacterial population
according to clause 200, wherein the plurality of non-intergeneric
bacteria, in planta, excrete nitrogen-containing products of
nitrogen fixation. [0680] 210. The non-intergeneric bacterial
population according to clause 200, wherein the plurality of
non-intergeneric bacteria applied to the plant do not stimulate an
increase in the uptake of exogenous non-atmospheric nitrogen.
[0681] 211. The non-intergeneric bacterial population according to
clause 200, wherein the plant is grown in soil from a field which
has been administered at least about 50 lbs of nitrogen-containing
fertilizer per acre, and wherein the nitrogen-containing fertilizer
comprises at least about 5% nitrogen by weight. [0682] 212. The
non-intergeneric bacterial population according to clause 200,
wherein the plant is grown in soil from a field which has been
administered at least about 50 lbs of nitrogen-containing
fertilizer per acre, and wherein the nitrogen-containing fertilizer
comprises at least about 5% nitrogen by weight, and wherein the
nitrogen-containing fertilizer comprises ammonium or an ammonium
containing molecule. [0683] 213. The non-intergeneric bacterial
population according to clause 200, wherein the exogenous nitrogen
is selected from fertilizer comprising one or more of: glutamine,
ammonia, ammonium, urea, nitrate, nitrite, ammonium-containing
molecules, nitrate-containing molecules, and nitrite-containing
molecules. [0684] 214. The non-intergeneric bacterial population
according to clause 200, wherein the plurality of non-intergeneric
bacteria, in planta, produce 5% or more of the fixed nitrogen in
the plant. [0685] 215. The non-intergeneric bacterial population
according to clause 200, wherein the plurality of non-intergeneric
bacteria, in planta, produce 10% or more of the fixed nitrogen in
the plant. [0686] 216. The non-intergeneric bacterial population
according to clause 200, wherein the plurality of non-intergeneric
bacteria, in planta, fix atmospheric nitrogen in
non-nitrogen-limiting conditions. [0687] 217. The non-intergeneric
bacterial population according to clause 200, wherein the plurality
of non-intergeneric bacteria, in planta, excrete
nitrogen-containing products of nitrogen fixation. [0688] 218. The
non-intergeneric bacterial population according to clause 200,
wherein the fixed nitrogen produced by the plurality of
non-intergeneric bacteria is measured through dilution of enriched
fertilizer by atmospheric N.sub.2 gas in plant tissue. [0689] 219.
The non-intergeneric bacterial population according to clause 200,
wherein the at least one gene, or non-coding polynucleotide, of the
nitrogen fixation or assimilation genetic regulatory network are
selected from the group consisting of: nifA, nifL, ntrB, ntrC,
polynucleotide encoding glutamine synthetase, glnA, glnB, glnK,
drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ,
nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ,
nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of
a nitrogenase enzyme. [0690] 220. The non-intergeneric bacterial
population according to clause 200, wherein the at least one
genetic variation is a mutation that results in one or more of:
increased expression or activity of NifA or glutaminase; decreased
expression or activity of NifL, NtrB, glutamine synthetase, GlnB,
GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or
decreased uridylyl-removing activity of GlnD. [0691] 221. The
non-intergeneric bacterial population according to clause 200,
wherein the at least one genetic variation is selected from: (A) a
knock-out mutation; (B) alters or abolishes a regulatory sequence
of a target gene; (C) comprises the insertion of a heterologous
regulatory sequence; or (D) a domain deletion. [0692] 222. The
non-intergeneric bacterial population according to clause 200,
wherein the at least one genetic variation is a mutated/tiff, gene
that has been altered to comprise a heterologous promoter inserted
into said nifL gene. [0693] 223. The non-intergeneric bacterial
population according to clause 200, wherein the at least one
genetic variation is a mutated glnE gene that results in a
truncated GlnE protein lacking an adenylyl-removing (AR) domain.
[0694] 224. The non-intergeneric bacterial population according to
clause 200, wherein the at least one genetic variation is a mutated
amtB gene that results in the lack of expression of said amtB gene.
[0695] 225. The non-intergeneric bacterial population according to
clause 200, wherein the at least one genetic variation is selected
from: a mutated nifL gene that has been altered to comprise a
heterologous promoter inserted into said nifL gene; a mutated glnE
gene that results in a truncated GlnE protein lacking an
adenylyl-removing (AR) domain; a mutated amtB gene that results in
the lack of expression of said amtB gene; and combinations thereof.
[0696] 226. The non-intergeneric bacterial population according to
clause 200, wherein the at least one genetic variation is a mutated
nifL gene that has been altered to comprise a heterologous promoter
inserted into said nifL gene and a mutated glnE gene that results
in a truncated GlnE protein lacking an adenylyl-removing (AR)
domain. [0697] 227. The non-intergeneric bacterial population
according to clause 200, wherein the at least one genetic variation
is a mutated nifL gene that has been altered to comprise a
heterologous promoter inserted into said nifL gene and a mutated
glnE gene that results in a truncated GlnE protein lacking an
adenylyl-removing (AR) domain and a mutated amtB gene that results
in the lack of expression of said amtB gene. [0698] 228. The
non-intergeneric bacterial population according to clause 200,
wherein the plant comprises the seed, stalk, flower, fruit, leaves,
or rhizome. [0699] 229. The non-intergeneric bacterial population
according to clause 200, wherein the plurality of non-intergeneric
bacteria are formulated into a composition. [0700] 230. The
non-intergeneric bacterial population according to clause 200,
wherein the plurality of non-intergeneric bacteria are formulated
into a composition comprising an agriculturally acceptable carrier.
[0701] 231. The non-intergeneric bacterial population according to
clause 200, wherein the plurality of non-intergeneric bacteria are
formulated into a liquid in-furrow composition comprising bacteria
at a concentration of about 1.times.10.sup.7 to about
1.times.10.sup.10 cfu per milliliter. [0702] 232. The
non-intergeneric bacterial population according to clause 200,
wherein the plurality of non-intergeneric bacteria are formulated
into a seed coating. [0703] 233. The non-intergeneric bacterial
population according to clause 200, wherein the plant is a cereal
crop. [0704] 234. The non-intergeneric bacterial population
according to clause 200, wherein the plant is selected from the
group consisting of: corn, rice, wheat, barley, sorghum, millet,
oat, rye, and triticale. [0705] 235. The non-intergeneric bacterial
population according to clause 200, wherein the plant is corn.
[0706] 236. The non-intergeneric bacterial population according to
clause 200, wherein the plant is an agricultural crop plant. [0707]
237. The non-intergeneric bacterial population according to clause
200, wherein the plant is a genetically modified organism. [0708]
238. The non-intergeneric bacterial population according to clause
200, wherein the plant is not a genetically modified organism.
[0709] 239. The non-intergeneric bacterial population according to
clause 200, wherein the plant has been genetically engineered or
bred for efficient nitrogen use. [0710] 240. The non-intergeneric
bacterial population according to clause 200, wherein the plurality
of non-intergeneric bacteria comprise at least two different
species of bacteria. [0711] 241. The non-intergeneric bacterial
population according to clause 200, wherein the plurality of
non-intergeneric bacteria comprise at least two different strains
of the same species of bacteria. [0712] 242. The non-intergeneric
bacterial population according to clause 200, wherein the plurality
of non-intergeneric bacteria comprise bacteria selected from:
Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus,
Achromobacter marplatensis, Microbacterium morale, Kluyvera
intermedia, Kosakonia pseudosacchari, Enterobacter sp.,
Azospirillum lipoferum, Kosakonia sacchari, and combinations
thereof. [0713] 243. The non-intergeneric bacterial population
according to clause 200, wherein the plurality of non-intergeneric
bacteria are endophytic, epiphytic, or rhizospheric. [0714] 244.
The non-intergeneric bacterial population according to clause 200,
wherein the plurality of non-intergeneric bacteria comprise
bacteria selected from: a bacteria deposited as PTA-122293, a
bacteria deposited as PTA-122294, a bacteria deposited as NCMA
201701002, a bacteria deposited as NCMA 201708004, a bacteria
deposited as NCMA 201708003, a bacteria deposited as NCMA
201708002, a bacteria deposited as NCMA 201712001, a bacteria
deposited as NCMA 201712002, and combinations thereof [0715] 245. A
bacterial population capable of increasing nitrogen fixation in a
non-leguminous plant, comprising: [0716] a. a plurality of
bacteria, that: [0717] i. have an average colonization ability per
unit of plant root tissue of at least about 1.0.times.10.sup.4
bacterial cells per gram of fresh weight of plant root tissue;
and/or [0718] ii. produce fixed N of at least about
1.times.10.sup.-17 mmol N per bacterial cell per hour, and [0719]
wherein the plurality of bacteria, in planta, produce 1% or more of
the fixed nitrogen in die plant. [0720] 246. The bacterial
population according to clause 245, wherein the plurality of
bacteria comprise bacteria that: have an average colonization
ability per unit of plant root tissue of at least about
1.0.times.10.sup.4 bacterial cells per gram of fresh weight of
plant root tissue. [0721] 247. The bacterial population according
to clause 245, wherein the plurality of bacteria comprise bacteria
that: produce fixed N of at least about 245.times.10.sup.-17 mmol N
per bacterial cell per hour. [0722] 248. The bacterial population
according to clause 245, wherein the plurality of bacteria comprise
bacteria that: have an average colonization ability per unit of
plant root tissue of at least about 1.0.times.10.sup.4 bacterial
cells per gram of fresh weight of plant root tissue and produce
fixed N of at least about 1.times.10.sup.-17 mmol N per bacterial
cell per hour. [0723] 249. The bacterial population according to
clause 245, wherein the plurality of bacteria comprise bacteria
that: have an average colonization ability per unit of plant root
tissue of at least about 1.0.times.10.sup.4 bacterial cells per
gram of fresh weight of plant root tissue and produce fixed N of at
least about 1.times.10.sup.-17 mmol N per bacterial cell per hour,
and wherein the product of (i) the average colonization ability per
unit of plant root tissue and (ii) produced fixed N per bacterial
cell per hour, is at least about 2.5.times.mmol N per gram of fresh
weight of plant root tissue per hour. [0724] 250. The bacterial
population according to clause 245, wherein the plurality of
bacteria, in planta, excrete the nitrogen-containing products of
nitrogen fixation. [0725] 251. The bacterial population according
to clause 245, wherein the plurality of bacteria applied to the
plant do not stimulate an increase in the uptake of exogenous
non-atmospheric nitrogen. [0726] 252. The bacterial population
according to clause 245, wherein the plant comprises the seed,
stalk, flower, fruit, leaves, or rhizome. [0727] 253. The bacterial
population according to clause 245, wherein the plurality of
bacteria are formulated into a composition. [0728] 254. The
bacterial population according to clause 245, wherein the plurality
of bacteria are formulated into a composition comprising an
agriculturally acceptable carrier. [0729] 255. The bacterial
population according to clause 245, wherein the plurality of
bacteria are applied into furrows in which seeds of said plant are
planted.
[0730] 256. The bacterial population according to clause 245,
wherein the plurality of bacteria are formulated into a liquid
in-furrow composition comprising bacteria at a concentration of
about 1.times.10.sup.7 to about 1.times.10.sup.10 cfu per
milliliter. [0731] 257. The bacterial population according to
clause 245, wherein the plurality of bacteria are applied onto a
seed of said plant. [0732] 258. The bacterial population according
to clause 245, wherein the plurality of bacteria are formulated
into a seed coating and are applied onto a seed of said plant.
[0733] 259. The bacterial population according to clause 245,
wherein the plurality of bacteria are formulated into a seed
coating and are applied onto a seed of said plant, at a
concentration of about 1.times.10.sup.5 to about 1.times.10.sup.7
cfu per seed. [0734] 260. The bacterial population according to
clause 245, wherein the plant is a cereal crop. [0735] 261. The
bacterial population according to clause 245, wherein the plant is
selected from the group consisting of: corn, rice, wheat, barley,
sorghum, millet, oat, rye, and triticale. [0736] 262. The bacterial
population according to clause 245, wherein the plant is corn.
[0737] 263. The bacterial population according to clause 245,
wherein the plant is an agricultural crop plant. [0738] 264. The
bacterial population according to clause 245, wherein the plant is
a genetically modified organism. [0739] 265. The bacterial
population according to clause 245, wherein the plant is not a
genetically modified organism. [0740] 266. The bacterial population
according to clause 245, wherein the plant has been genetically
engineered or bred for efficient nitrogen use. [0741] 267. The
bacterial population according to clause 245, wherein the plurality
of bacteria comprise at least two different species of bacteria.
[0742] 268. The bacterial population according to clause 245,
wherein the plurality of bacteria comprise at least two different
strains of the same species of bacteria. [0743] 269. The bacterial
population according to clause 245, wherein the plurality of
bacteria comprise bacteria selected from: Rahnella aquatilis,
Klebsiella variicola, Achromobacter spiritinus, Achromobacter
marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia
pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia
sacchari, and combinations thereof. [0744] 270. The bacterial
population according to clause 245, wherein the plurality of
bacteria are endophytic, epiphytic, or rhizospheric. [0745] 271.
The bacterial population according to clause 245, wherein the
plurality of bacteria are selected from: a bacteria deposited as
NCMA 201701003, a bacteria deposited as NCMA 201701001, and a
bacteria deposited as NCMA 201708001. [0746] 272. A bacterium that:
[0747] i. has an average colonization ability per unit of plant
root tissue of at least about 1.0.times.10.sup.4 bacterial cells
per gram of fresh weight of plant root tissue; and/or [0748] ii.
produces fixed N of at least about 1.times.10.sup.-17 mmol N per
bacterial cell per hour. [0749] 273. A non-intergeneric bacterium,
comprising: at least one genetic variation introduced into at least
one gene, or non-coding polynucleotide, of the nitrogen fixation or
assimilation genetic regulatory network, such that the
non-intergeneric bacterium is capable of fixing atmospheric
nitrogen in the presence of exogenous nitrogen, and wherein said
bacterium: [0750] i. has an average colonization ability per unit
of plant root tissue of at least about 1.0.times.10.sup.4 bacterial
cells per gram of fresh weight of plant root tissue; and/or [0751]
ii. produces fixed N of at least about 1.times.10.sup.-17 mmol N
per bacterial cell per hour. [0752] 274. The non-intergeneric
bacterium according to clause 273, wherein the bacterium has an
average colonization ability per unit of plant root tissue of at
least about 1.0.times.10.sup.4 bacterial cells per gram of fresh
weight of plant root tissue. [0753] 275. The non-intergeneric
bacterium according to clause 273, wherein the bacterium produces
fixed N of at least about 1.times.10.sup.-17 mmol N per bacterial
cell per hour. [0754] 276. The non-intergeneric bacterium according
to clause 273, wherein the bacterium has an average colonization
ability per unit of plant root tissue of at least about
1.0.times.10.sup.4 bacterial cells per gram of fresh weight of
plant root tissue and produces fixed N of at least about
1.times.10.sup.-17 mmol N per bacterial cell per hour. [0755] 277.
The non-intergeneric bacterium according to clause 273, wherein the
bacterium has an average colonization ability per unit of plant
root tissue of at least about 1.0.times.10.sup.4 bacterial cells
per grain of fresh weight of plant root tissue and produces fixed N
of at least about 1.times.10.sup.-17 mmol N per bacterial cell per
hour, and wherein the product of (i) the average colonization
ability per unit of plant root tissue and (ii) produced fixed N per
bacterial cell per hour, is at least about 2.5.times.10.sup.-8 mmol
N per gram of fresh weight of plant root tissue per hour. [0756]
278. The non-intergeneric bacterium according to clause 273,
wherein the at least one genetic variation comprises an introduced
control sequence operably linked to the at least one gene of the
nitrogen fixation or assimilation genetic regulatory network.
[0757] 279. The non-intergeneric bacterium according to clause 273,
wherein the at least one genetic variation comprises a promoter
operably linked to the at least one gene of the nitrogen fixation
or assimilation genetic regulatory network. [0758] 280. The
non-intergeneric bacterium according to clause 273, wherein the at
least one genetic variation comprises an inducible promoter
operably linked to the at least one gene of the nitrogen fixation
or assimilation genetic regulatory network. [0759] 281. The
non-intergeneric bacterium according to clause 273, wherein the
bacterium does not comprise a constitutive promoter operably linked
to a gene of the nitrogen fixation or assimilation genetic
regulatory network. [0760] 282. The non-intergeneric bacterium
according to clause 273, wherein the bacterium does not comprise a
constitutive promoter operably linked to a gene in the nif gene
cluster. [0761] 283. The non-intergeneric bacterium according to
clause 273, wherein the bacterium, in planta, excretes the
nitrogen-containing products of nitrogen fixation. [0762] 284. The
non-intergeneric bacterium according to clause 273, wherein the at
least one gene, or non-coding polynucleotide, of the nitrogen
fixation or assimilation genetic regulatory network are selected
from the group consisting of: nifA, nifL, ntrB, ntrC,
polynucleotide encoding glutamine synthetase, glnA, glnB, glnK,
drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ,
nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ,
nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of
a nitrogenase enzyme. [0763] 285. The non-intergeneric bacterium
according to clause 273, wherein the at least one genetic variation
is a mutation that results in one or more of: increased expression
or activity of NifA or glutaminase; decreased expression or
activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT,
AmtB; decreased adenylyl-removing activity of GlnE; or decreased
uridylyl-removing activity of GlnD. [0764] 286. The
non-intergeneric bacterium according to clause 273, wherein the at
least one genetic variation is selected from: (A) a knock-out
mutation; (B) alters or abolishes a regulatory sequence of a target
gene; (C) comprises the insertion of a heterologous regulatory
sequence; or (D) a domain deletion. [0765] 287. The
non-intergeneric bacterium according to clause 273, wherein the at
least one genetic variation is a mutated MIL gene that has been
altered to comprise a heterologous promoter inserted into said nifL
gene. [0766] 288. The non-intergeneric bacterium according to
clause 273, wherein the at least one genetic variation is a mutated
glnE gene that results in a truncated GlnE protein lacking an
adenylyl-removing (AR) domain. [0767] 289. The non-intergeneric
bacterium according to clause 273, wherein the at least one genetic
variation is a mutated amtB gene that results in the lack of
expression of said amtB gene. [0768] 290. The non-intergeneric
bacterium according to clause 273, wherein the at least one genetic
variation is selected from: a mutated nifL gene that has been
altered to comprise a heterologous promoter inserted into said nifL
gene; a mutated glnE gene that results in a truncated GlnE protein
lacking an adenylyl-removing (AR) domain; a mutated amtB gene that
results in the lack of expression of said amtB gene; and
combinations thereof. [0769] 291. The non-intergeneric bacterium
according to clause 273, wherein the at least one genetic variation
is a mutated nifL gene that has been altered to comprise a
heterologous promoter inserted into said nifL gene and a mutated
glnE gene that results in a truncated GlnE protein lacking an
adenylyl-removing (AR) domain. [0770] 292. The non-intergeneric
bacterium according to clause 273, wherein the at least one genetic
variation is a mutated nifL gene that has been altered to comprise
a heterologous promoter inserted into said tuft gene and a mutated
glnE gene that results in a truncated GlnE protein lacking an
adenylyl-removing (AR) domain and a mutated amtB gene that results
in the lack of expression of said amtB gene. [0771] 293. The
non-intergeneric bacterium according to clause 273, formulated into
a composition. [0772] 294. The non-intergeneric bacterium according
to clause 273, formulated into a composition comprising an
agriculturally acceptable carrier. [0773] 295. The non-intergeneric
bacterium according to clause 273, formulated into a liquid
in-furrow composition. [0774] 296. The non-intergeneric bacterium
according to clause 273, formulated into a seed coating. [0775]
297. The non-intergeneric bacterium according to clause 273,
wherein said bacterium is selected from: Rahnella aquatilis,
Klebsiella variicola, Achromobacter spiritinus, Achromobacter
marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia
pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia
sacchari, and combinations thereof. [0776] 298. The
non-intergeneric bacterium according to clause 273, wherein said
bacterium is endophytic, epiphytic, or rhizospheric. [0777] 299.
The non-intergeneric bacterium according to clause 273, wherein
said bacterium is selected from: a bacteria deposited as
PTA-122293, a bacteria deposited as PTA-122294, a bacteria
deposited as NCMA 201701002, a bacteria deposited as NCMA
201708004, a bacteria deposited as NCMA 201708003, a bacteria
deposited as NCMA 201708002, a bacteria deposited as NCMA
201712001, a bacteria deposited as NCMA 201712002, and combinations
thereof. [0778] 300. A method of increasing nitrogen fixation in a
plant, comprising administering to the plant an effective amount of
a composition comprising: [0779] i. a purified population of
bacteria that comprises bacteria with a 16S nucleic acid sequence
that is at least about 97% identical to a nucleic acid sequence
selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136,
149, 157, 167, 261, 262, 269, 277-283; [0780] ii. a purified
population of bacteria that comprises bacteria with a nucleic acid
sequence that is at least about 90% identical to a nucleic acid
sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135,
137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276,
284-295; and/or [0781] iii. a purified population of bacteria that
comprises bacteria with a nucleic acid sequence that is at least
about 90% identical to a nucleic acid sequence selected from SEQ ID
NOs: 177-260, 296-303; and [0782] wherein the plant administered
the effective amount of the composition exhibits an increase in
nitrogen fixation, as compared to a plant not having been
administered the composition. [0783] 301. The method of clause 300,
wherein the composition comprises: a purified population of
bacteria that comprises bacteria with a 16S nucleic acid sequence
that is at least about 97% identical to a nucleic acid sequence
selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136,
149, 157, 167, 261, 262, 269, and 277-283. [0784] 302. The method
of clause 300, wherein the composition comprises: a purified
population of bacteria that comprises bacteria with a 16S nucleic
acid sequence that is at least about 99% identical to a nucleic
acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123,
124, 136, 149, 157, 167, 261, 262, 269, and 277-283. [0785] 303.
The method of clause 300, wherein the composition comprises: a
purified population of bacteria that comprises bacteria with a 165
nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121,
122, 123, 124, 136, 149, 157, 167, 261, 262, 269, and 277-283.
[0786] 304. The method of clause 300, wherein the composition
comprises: a purified population of bacteria that comprises
bacteria with a nucleic acid sequence that is at least about 90%
identical to a nucleic acid sequence selected from SEQ ID NOs:
86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166,
168-176, 263-268, 270-274, 275, 276, and 284-295. [0787] 305. The
method of clause 300, wherein the composition comprises: a purified
population of bacteria that comprises bacteria with a nucleic acid
sequence that is at least about 95% identical to a nucleic acid
sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135,
137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and
284-295. [0788] 306. The method of clause 300, wherein the
composition comprises: a purified population of bacteria that
comprises bacteria with a nucleic acid sequence that is at least
about 99% identical to a nucleic acid sequence selected from SEQ ID
NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166,
168-176, 263-268, 270-274, 275, 276, and 284-295. [0789] 307. The
method of clause 300, wherein the composition comprises: a purified
population of bacteria that comprises bacteria with a nucleic acid
sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135,
137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and
284-295. [0790] 308. The method of clause 300, wherein the
composition comprises: a purified population of bacteria that
comprises bacteria with a nucleic acid sequence that is at least
about 90% identical to a nucleic acid sequence selected from SEQ ID
NOs: 177-260 and 296-303. [0791] 309. The method of clause 300,
wherein the composition comprises: a purified population of
bacteria that comprises bacteria with a nucleic acid sequence that
is at least about 95% identical to a nucleic acid sequence selected
from SEQ ID NOs: 177-260 and 296-303. [0792] 310. The method of
clause 300, wherein the composition comprises: a purified
population of bacteria that comprises bacteria with a nucleic acid
sequence that is at least about 99% identical to a nucleic acid
sequence selected from SEQ ID NOs: 177-260 and 296-303.
[0793] 311. The method of clause 300, wherein the composition
comprises: a purified population of bacteria that comprises
bacteria with a nucleic acid sequence selected from SEQ ID NOs:
177-260 and 296-303. [0794] 312. The method of clause 300, wherein
the composition comprises: a purified population of bacteria that
comprise bacteria with at least one genetic variation introduced
into a gene selected from the group consisting of nifA, nifL, ntrB,
ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB,
glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE,
nifJ, nifH, nifH, nifD, nifY, nifE, nifN, nifU, nifS, nifV, nifW,
nifZ, nifM, nifB, nifQ, and a gene associated with biosynthesis of
a nitrogenase enzyme. [0795] 313. The method of clause 300, wherein
the composition comprises: a purified population of bacteria that
comprise bacteria with at least one genetic variation introduced
into a gene selected from the group consisting of nifA, nifL, ntrB,
ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB,
glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE,
nifJ, nifH, nifD, nifK, nifE, nifN, nifU, nifS, nifV, nifW, nifZ,
nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of
a nitrogenase enzyme, wherein the genetic variation (A) is a
knock-out mutation; (B) alters or abolishes a regulatory sequence
of a target gene; (C) comprises the insertion of a heterologous
regulatory sequence; or (D) a domain deletion. [0796] 314. The
method of clause 300, wherein the composition comprises: a purified
population of bacteria that comprise bacteria with at least one
mutation that results in one or more of: increased expression or
activity of NifA or glutaminase; decreased expression or activity
of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, or AmtB;
decreased adenylyl-removing activity of GlnE; or decreased
uridylyl-removing activity of GlnD. [0797] 315. The method of
clause 300, wherein the composition comprises: a purified
population of bacteria that comprise bacteria with a mutated MIL
gene that has been altered to comprise a heterologous promoter
inserted into said nifL gene. [0798] 316. The method of clause 300,
wherein the composition comprises: a purified population of
bacteria that comprise bacteria with a mutated glnE gene that
results in a truncated GlnE protein lacking an adenylyl-removing
(AR) domain. [0799] 317. The method of clause 300, wherein the
composition comprises: a purified population of bacteria that
comprise bacteria with a mutated amtB gene that results in the lack
of expression of said amtB gene. [0800] 318. The method of clause
300, wherein the composition comprises: a purified population of
bacteria that comprise bacteria with at least one of the following
genetic alterations: a mutated nifL gene that has been altered to
comprise a heterologous promoter inserted into said nifL gene; a
mutated glnE gene that results in a truncated GlnE protein lacking
an adenylyl-removing (AR) domain; a mutated amtB gene that results
in the lack of expression of said amtB gene; and combinations
thereof. [0801] 319. The method of clause 300, wherein the
composition comprises: a purified population of bacteria that
comprise bacteria with a mutated nifL gene that has been altered to
comprise a heterologous promoter inserted into said nifL gene and a
mutated glnE gene that results in a truncated GlnE protein lacking
an adenylyl-removing (AR) domain. [0802] 320. The method of clause
300, wherein the composition comprises: a purified population of
bacteria that comprise bacteria with a mutated nifL gene that has
been altered to comprise a heterologous promoter inserted into said
nifL gene and a mutated glnE gene that results in a truncated GlnE
protein lacking an adenylyl-removing (AR) domain and a mutated am/B
gene that results in the lack of expression of said amtB gene.
[0803] 321. The method of clause 300, wherein the composition
comprises: a purified population of bacteria that comprise bacteria
with a nifL modification that expresses a Nil protein at less than
about 50% of a bacteria lacking the nifL modification. [0804] 322.
The method of clause 300, wherein the composition comprises: a
purified population of bacteria that comprise bacteria with a nifL
modification that expresses a Nil protein at less than about 50% of
a bacteria lacking the nifL modification, wherein the modification
is a disruption, knockout, or truncation. [0805] 323. The method of
clause 300, wherein the composition comprises: a purified
population of bacteria that comprise bacteria with a raft,
modification that expresses a Nil protein at less than about 50% of
a bacteria lacking the nifL modification, and wherein the bacteria
further comprise a promoter operably linked to a nifA sequence.
[0806] 324. The method of clause 300, wherein the composition
comprises: a purified population of bacteria that comprise bacteria
with a nifL modification that expresses a Nil protein at less than
about 50% of a bacteria lacking the nifL modification, and wherein
the bacteria lack a nifL homolog. [0807] 325. The method of clause
300, wherein the composition comprises: a purified population of
bacteria that comprise bacteria with a glnE modification that
expresses a GlnE protein at less than about 50% of a bacteria
lacking the glnE modification. [0808] 326. The method of clause
300, wherein the composition comprises: a purified population of
bacteria that comprise bacteria with a glnE modification that
expresses a GlnE protein at less than about 50% of a bacteria
lacking the glnE modification, wherein the modification is a
disruption, knockout, or a truncation. [0809] 327. The method of
clause 300, wherein the composition comprises: a purified
population of bacteria that comprise bacteria with a glnE
modification that expresses a GlnE protein at less than about 50%
of a bacteria lacking the glnE modification, and wherein the GlnE
protein sequence lacks an adenylyl removing (AR) domain. [0810]
328. The method of clause 300, wherein the composition comprises: a
purified population of bacteria that comprise bacteria with a glnE
modification that expresses a GlnE protein at less than about 50%
of a bacteria lacking the glnE modification, and wherein the GlnE
protein sequence lacks adenylyl removing (AR) activity. [0811] 329.
The method of clause 300, wherein the purified population of
bacteria, in planta, provide at least 1% of fixed nitrogen to the
plant. [0812] 330. The method of clause 300, wherein the purified
population of bacteria, in planta, provide at least 5% of fixed
nitrogen to the plant. [0813] 331. The method of clause 300,
wherein the purified population of bacteria, in planta, provide at
least 10% of fixed nitrogen to the plant. [0814] 332. The method of
clause 300, wherein the purified population of bacteria are capable
of fixing atmospheric nitrogen in non-nitrogen-limiting conditions.
[0815] 333. The method of clause 300, wherein the purified
population of bacteria, in planta, excrete nitrogen-containing
products of nitrogen fixation. [0816] 334. The method of clause
300, wherein the purified population of bacteria, in planta,
provide at least 1% of fixed nitrogen to the plant, and wherein
said fixed nitrogen is measured through dilution of enriched
fertilizer by atmospheric N2 gas in plant tissue. [0817] 335. The
method of clause 300, wherein the purified population of bacteria
colonize a root of said plant and are present in an amount of at
least about 1.0.times.10.sup.4 bacterial cells per gram of fresh
weight of plant root tissue. [0818] 336. The method of clause 300,
wherein the plant comprises the seed, stalk, flower, fruit, leaves,
or rhizome. [0819] 337. The method of clause 300, wherein the
composition comprises: an agriculturally acceptable carrier. [0820]
338. The method of clause 300, wherein the composition comprising
the purified population of bacteria is administered into furrows in
which seeds of said plant are planted. [0821] 339. The method of
clause 300, wherein the composition comprising the purified
population of bacteria is formulated as a liquid in-furrow
composition comprising bacteria at a concentration of about
1.times.10.sup.7 to about 1.times.10.sup.10 du per milliliter.
[0822] 340. The method of clause 300, wherein the composition
comprising the purified population of bacteria is administered onto
a seed of said plant. [0823] 341. The method of clause 300, wherein
the composition comprising the purified population of bacteria is
formulated as a seed coating and is administered onto a seed of
said plant. [0824] 342. The method of clause 300, wherein the
composition comprising the purified population of bacteria is
formulated as a seed coating and is administered onto a seed of
said plant, at a concentration of about 1.times.10.sup.5 to about
1.times.10.sup.7 cfu per seed. [0825] 343. The method of clause
300, wherein the plant is non-leguminous. [0826] 344. The method of
clause 300, wherein the plant is a cereal crop. [0827] 345. The
method of clause 300, wherein the plant is selected from the group
consisting of: corn, rice, wheat, barley, sorghum, millet, oat,
rye, and triticale. [0828] 346. The method of clause 300, wherein
the plant is corn. [0829] 347. The method of clause 300, wherein
the plant is a legume. [0830] 348. The method of clause 300,
wherein the plant is a grain crop. [0831] 349. The method of clause
300, wherein the purified population of bacteria comprise bacteria
selected from: Rahnella aquatilis, Klebsiella variicola,
Achromobacter spiritinus, Achromobacter marplatensis,
Microbacterium murale, Kluyvera intermedia, Kosakonia
pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia
sacchari, and combinations thereof. [0832] 350. The method of
clause 300, wherein the purified population of bacteria comprise a
bacteria of genus Rahnella. [0833] 351. The method of clause 300,
wherein the purified population of bacteria comprise a bacteria of
species Rahnella aquatilis. [0834] 352. The method of clause 300,
wherein the purified population of bacteria comprise a bacteria
deposited as PTA-122293. [0835] 353. The method of clause 300,
wherein the purified population of bacteria comprise a bacteria
deposited as NCMA 201701003. [0836] 354. The method of clause 300,
wherein the purified population of bacteria comprise a bacteria of
genus Kosakonia. [0837] 355. The method of clause 300, wherein the
purified population of bacteria comprise a bacteria of species
Kosakonia sacchari. [0838] 356. The method of clause 300, wherein
the purified population of bacteria comprise a bacteria deposited
as PTA-122294. [0839] 357. The method of clause 300, wherein the
purified population of bacteria comprise a bacteria deposited as
NCMA 201701001. [0840] 358. The method of clause 300, wherein the
purified population of bacteria comprise a bacteria deposited as
NCMA 201701002. [0841] 359. The method of clause 300, wherein the
purified population of bacteria comprise a bacteria deposited as
NCMA 201708004. [0842] 360. The method of clause 300, wherein the
purified population of bacteria comprise a bacteria deposited as
NCMA 201708003. [0843] 361. The method of clause 300, wherein the
purified population of bacteria comprise a bacteria deposited as
NCMA 201708002. [0844] 362. The method of clause 300, wherein the
purified population of bacteria comprise a bacteria of genus
Klebsiella [0845] 363. The method of clause 300, wherein the
purified population of bacteria comprise a bacteria of species
Klebsiella variicola. [0846] 364. The method of clause 300, wherein
the purified population of bacteria comprise a bacteria deposited
as NCMA 201708001. [0847] 365. The method of clause 300, wherein
the purified population of bacteria comprise a bacteria deposited
as NCMA 201712001. [0848] 366. The method of clause 300, wherein
the purified population of bacteria comprise a bacteria deposited
as NCMA 201712002. [0849] 367. An isolated bacteria, comprising:
[0850] i. a 16S nucleic acid sequence that is at least about 97%
identical to a nucleic acid sequence selected from SEQ ID NOs: 85,
96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269,
277-283; [0851] ii. a nucleic acid sequence that is at least about
90% identical to a nucleic acid sequence selected from SEQ ID NOs:
86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166,
168-176, 263-268, 270-274, 275, 276, 284-295; and/or [0852] iii. a
nucleic acid sequence that is at least about 90% identical to a
nucleic acid sequence selected from SEQ ID NOs: 177-260, 296-303.
[0853] 368. The isolated bacteria of clause 367, comprising: a 1.65
nucleic acid sequence that is at least about 97% identical to a
nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121,
122, 123, 124, 136, 149, 157, 167, 261, 262, 269, and 277-283.
[0854] 369. The isolated bacteria of clause 367, comprising: a 16S
nucleic acid sequence that is at least about 99% identical to a
nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121,
122, 123, 124, 136, 149, 157, 167, 261, 262, 269, and 277-283.
[0855] 370. The isolated bacteria of clause 367, comprising: a 16S
nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121,
122, 123, 124, 136, 149, 157, 167, 261, 262, 269, and 277-283.
[0856] 371. The isolated bacteria of clause 367, comprising: a
nucleic acid sequence that is at least about 90% identical to a
nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110,
112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268,
270-274, 275, 276, and 284-295. [0857] 372. The isolated bacteria
of clause 367, comprising: a nucleic acid sequence that is at least
about 95% identical to a nucleic acid sequence selected from SEQ ID
NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166,
168-176, 263-268, 270-274, 275, 276, and 284-295. [0858] 373. The
isolated bacteria of clause 367, comprising: a nucleic acid
sequence that is at least about 99% identical to a nucleic acid
sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135,
137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and
284-295. [0859] 374. The isolated bacteria of clause 367,
comprising: a nucleic acid sequence selected from SEQ ID NOs:
86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166,
168-176, 263-268, 270-274, 275, 276, and 284-295. [0860] 375. The
isolated bacteria of clause 367, comprising: a nucleic acid
sequence that is at least about 90% identical to a nucleic acid
sequence selected from SEQ ID NOs: 177-260 and 296-303. [0861] 376.
The isolated bacteria of clause 367, comprising: a nucleic acid
sequence that is at least about 95% identical to a nucleic acid
sequence selected from SEQ ID NOs: 177-260 and 296-303. [0862] 377.
The isolated bacteria of clause 367, comprising: a nucleic acid
sequence that is at least about 99% identical to a nucleic acid
sequence selected from SEQ ID NOs: 177-260 and 296-303.
[0863] 378. The isolated bacteria of clause 367, comprising: a
nucleic acid sequence selected from SEQ ID NOs: 177-260 and
296-303. [0864] 379. The isolated bacteria of clause 367,
comprising: at least one genetic variation introduced into a gene
selected from the group consisting of nifA, nifL, ntrB, ntrC,
polynucleotide encoding glutamine synthetase, glnA, glnB, glnK,
drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ,
nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifW,
nifZ, nifM, nifF, nifB, nifQ, and a gene associated with
biosynthesis of a nitrogenase enzyme. [0865] 380. The isolated
bacteria of clause 367, comprising: at least one genetic variation
introduced into a gene selected from the group consisting of nifA,
nifL, ntrB, polynucleotide encoding glutamine synthetase, glnA,
glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD,
glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV,
nifW, nifZ, nifM, nifF, nifB, nifQ, and a gene associated with
biosynthesis of a nitrogenase enzyme, wherein the genetic variation
(A) is a knock-out mutation; (B) alters or abolishes a regulatory
sequence of a target gene; (C) comprises the insertion of a
heterologous regulatory sequence; or (D) a domain deletion. [0866]
381. The isolated bacteria of clause 367, comprising: at least one
mutation that results in one or more of: increased expression or
activity of NifA or glutaminase; decreased expression or activity
of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, or AmtB;
decreased adenylyl-removing activity of GlnE; or decreased
uridylyl-removing activity of GlnD. [0867] 382. The isolated
bacteria of clause 367, comprising: a mutated nifL gene that has
been altered to comprise a heterologous promoter inserted into said
nifL gene. [0868] 383. The isolated bacteria of clause 367,
comprising: a mutated glnE gene that results in a truncated GlnE
protein lacking an adenylyl-removing (AR) domain. [0869] 384. The
isolated bacteria of clause 367, comprising: a mutated amtB gene
that results in the lack of expression of said amtB gene. [0870]
385. The isolated bacteria of clause 367, comprising: at least one
of the following genetic alterations: a mutated nifL gene that has
been altered to comprise a heterologous promoter inserted into said
MIL gene; a mutated glnE gene that results in a truncated GlnE
protein lacking an adenylyl-removing (AR) domain; a mutated amtB
gene that results in the lack of expression of said amtB gene; and
combinations thereof. [0871] 386. The isolated bacteria of clause
367, comprising: a mutated nifL gene that has been altered to
comprise a heterologous promoter inserted into said nifL gene and a
mutated glnE gene that results in a truncated GlnE protein lacking
an adenylyl-removing (AR) domain. [0872] 387. The isolated bacteria
of clause 367, comprising: a mutated nifL gene that has been
altered to comprise a heterologous promoter inserted into said nifL
gene and a mutated glnE gene that results in a truncated GlnE
protein lacking an adenylyl-removing (AR) domain and a mutated amtB
gene that results in the lack of expression of said amtB gene.
[0873] 388. The isolated bacteria of clause 367, comprising: a nifL
modification that expresses a NifL protein at less than about 50%
of a bacteria lacking the nifL modification. [0874] 389. The
isolated bacteria of clause 367, comprising: a MIL modification
that expresses a nifL protein at less than about 50% of a bacteria
lacking the nifL modification, wherein the modification is a
disruption, knockout, or truncation. [0875] 390. The isolated
bacteria of clause 367, comprising: a nifL modification that
expresses a NifL protein at less than about 50% of a bacteria
lacking the nifL modification, and wherein the bacteria further
comprise a promoter operably linked to a nifA sequence. [0876] 391.
The isolated bacteria of clause 367, comprising: a nifL
modification that expresses a NifL protein at less than about 50%
of a bacteria lacking the nifL modification, and wherein the
bacteria lack a nifL homolog. [0877] 392. The isolated bacteria of
clause 367, comprising: a glnE modification that expresses a GlnE
protein at less than about 50% of a bacteria lacking the glnE
modification. [0878] 393. The isolated bacteria of clause 367,
comprising: a glnE modification that expresses a GlnE protein at
less than about 50% of a bacteria lacking the glnE modification,
wherein the modification is a disruption, knockout, or a
truncation. [0879] 394. The isolated bacteria of clause 367,
comprising: a glnE modification that expresses a GlnE protein at
less than about 50% of a bacteria lacking the glnE modification,
and wherein the GlnE protein sequence lacks an adenylyl removing
(AR) domain. [0880] 395. The isolated bacteria of clause 367,
comprising: a glnE modification that expresses a GlnE protein at
less than about 50% of a bacteria lacking the glnE modification,
and wherein the GlnE protein sequence lacks adenylyl removing (AR)
activity. [0881] 396. The isolated bacteria of clause 367, wherein
the bacteria, in planta, provide at least 1% of fixed nitrogen to a
plant exposed to said bacteria. [0882] 397. The isolated bacteria
of clause 367, wherein the bacteria, in planta, provide at least 5%
of fixed nitrogen to a plant exposed to said bacteria. [0883] 398.
The isolated bacteria of clause 367, wherein the bacteria, in
planta, provide at least 10% of fixed nitrogen to a plant exposed
to said bacteria. [0884] 399. The isolated bacteria of clause 367,
wherein the bacteria is capable of fixing atmospheric nitrogen in
non-nitrogen-limiting conditions. [0885] 400. The isolated bacteria
of clause 367, wherein the bacteria, in planta, excretes
nitrogen-containing products of nitrogen fixation. [0886] 401. The
isolated bacteria of clause 367, wherein the bacteria, in planta,
provide at least 1% of fixed nitrogen to a plant exposed to said
bacteria, and wherein said fixed nitrogen is measured through
dilution of enriched fertilizer by atmospheric N.sub.2 gas in plant
tissue. [0887] 402. The isolated bacteria of clause 367, wherein
the bacteria colonize a root of a plant exposed to said bacteria to
a concentration of at least about 1.0.times.10.sup.4 bacterial
cells per gram of fresh weight of plant root tissue. [0888] 403.
The isolated bacteria of clause 367, formulated into an
agricultural composition. [0889] 404. The isolated bacteria of
clause 367, formulated into an in-furrow composition. [0890] 405.
The isolated bacteria of clause 367, formulated as a liquid
in-furrow composition that comprises bacteria at a concentration of
about 1.times.10.sup.7 to about 1.times.10.sup.10 cfu per
milliliter. [0891] 406. The isolated bacteria of clause 367,
formulated as a seed treatment or seed coating. [0892] 407. The
isolated bacteria of clause 367, formulated as a seed treatment or
seed coating that comprises bacteria at a concentration of about
1.times.10.sup.5 to about 1.times.10.sup.7 cfu per seed. [0893]
408. The isolated bacteria of clause 367, wherein the bacteria is
in contact with a plant and increases nitrogen fixation in the
plant. [0894] 409. The isolated bacteria of clause 367, disposed on
a non-leguminous plant. [0895] 410. The isolated bacteria of clause
367, disposed on a cereal crop. [0896] 411. The isolated bacteria
of clause 367, disposed on a plant selected from the group
consisting of: corn, rice, wheat, barley, sorghum, millet, oat,
rye, and triticale. [0897] 412. The isolated bacteria of clause
367, disposed on corn. [0898] 413. The isolated bacteria of clause
367, disposed on a legume. [0899] 414. The isolated bacteria of
clause 367, disposed on a grain crop. [0900] 415. The isolated
bacteria of clause 367, selected from: Rahnella aquatilis,
Klebsiella variicola, Achromobacter spiritinus, Achromobacter
marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia
pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia
sacchari, and combinations thereof. [0901] 416. The isolated
bacteria of clause 367, wherein the bacteria is of the genus
Rahnella. [0902] 417. The isolated bacteria of clause 367, wherein
the bacteria is of the species Rahnella aquatilis. [0903] 418. The
isolated bacteria of clause 367, deposited as PTA-122293. [0904]
419. The isolated bacteria of clause 367, deposited as NCMA
201701003. [0905] 420. The isolated bacteria of clause 367, wherein
the bacteria is of the genus Kosakonia. [0906] 421. The isolated
bacteria of clause 367, wherein the bacteria is of the species
Kosakonia sacchari. [0907] 422. The isolated bacteria of clause
367, deposited as PTA-122294. [0908] 423. The isolated bacteria of
clause 367, deposited as NCMA 201701001. [0909] 424. The isolated
bacteria of clause 367, deposited as NCMA 201701002. [0910] 425.
The isolated bacteria of clause 367, deposited as NCMA 201708004.
[0911] 426. The isolated bacteria of clause 367, deposited as NCMA
201708003. [0912] 427. The isolated bacteria of clause 367,
deposited as NCMA 201708002. [0913] 428. The isolated bacteria of
clause 367, wherein the bacteria is of the genus Klebsiella. [0914]
429. The isolated bacteria of clause 367, wherein the bacteria is
of the species Klebsiella variicola. [0915] 430. The isolated
bacteria of clause 367, deposited as NCMA 201708001. [0916] 431.
The isolated bacteria of clause 367, deposited as NCMA 201712001.
[0917] 432. The isolated bacteria of clause 367, deposited as NCMA
201712002. [0918] 433. A composition comprising any one or more
bacteria of clauses 415 to 432. [0919] 434. A method of detecting a
non-native junction sequence, comprising: amplifying a nucleotide
sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to SEQ ED NOs: 372-405. [0920] 435. The method
according to clause 434, wherein said amplifying is by conducting a
polymerase chain reaction. [0921] 436. The method according to
clause 434, wherein said amplifying is by conducting a quantitative
polymerase chain reaction. [0922] 437. A method of detecting a
non-native junction sequence, comprising: amplifying a nucleotide
sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to at least a 10 contiguous base pair fragment
contained in SEQ ID NOs: 372-405, said contiguous base pair
fragment being comprised of nucleotides at the intersection of: an
upstream sequence comprising SEQ ID NOs: 304-337 and downstream
sequence comprising SEQ ID NOs: 338-371. [0923] 438. The method
according to clause 437, wherein said amplifying is by conducting a
polymerase chain reaction. [0924] 439. The method according to
clause 437, wherein said amplifying is by conducting a quantitative
polymerase chain reaction. [0925] 440. A non-native junction
sequence, comprising: a nucleotide sequence that shares at least
about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:
372-405. [0926] 441. A non-native junction sequence, comprising: a
nucleotide sequence that shares at least about 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
1.00% sequence identity to at least a 10 contiguous base pair
fragment contained in SEQ ID NOs: 372-405, said contiguous base
pair fragment being comprised of nucleotides at the intersection
of: an upstream sequence comprising SEQ ID NOs: 304-337 and
downstream sequence comprising SEQ ID NOs: 338-371. [0927] 442. A
bacterial composition, comprising: at least one remodeled bacterial
strain that fixes atmospheric nitrogen, the at least one remodeled
bacterial strain comprising exogenously added DNA wherein said
exogenously added DNA shares at least 80% identity to a
corresponding native bacterial strain. [0928] 443. A method of
maintaining soil nitrogen levels, comprising: [0929] planting, in
soil of a field, a crop inoculated by a remodeled bacterium that
fixes atmospheric nitrogen; and [0930] harvesting said crop,
wherein no more than 90% of a nitrogen dose required for producing
said crop is administered to said soil of said field between
planting and harvesting. [0931] 444. A method of delivering a
probiotic supplement to a crop plant, comprising: [0932] coating a
crop seed with a seed coating, seed treatment, or seed dressing,
wherein said seed coating, seed dressing, or seed treatment
comprising living representatives of said probiotic; and [0933]
applying in soil of a field, said crop seeds. [0934] 445. A method
of increasing the amount of atmospheric derived nitrogen in a
non-leguminous plant, comprising: [0935] exposing said
non-leguminous plant to remodeled non-intergeneric microbes, said
remodeled non-intergeneric microbes comprising at least one genetic
variation introduced into a nitrogen fixation genetic regulatory
network or at least one genetic variation introduced into a
nitrogen assimilation genetic regulatory network. [0936] 446. A
method of increasing an amount of atmospheric derived nitrogen in a
corn plant, comprising: [0937] exposing said corn plant to
remodeled non-intergeneric microbes comprising remodeled genetic
variations within at least two genes selected from the group
consisting of nifL, glnB, glnE, and amtB. [0938] 447. A method of
increasing an amount of atmospheric derived nitrogen in a corn
plant, comprising: [0939] exposing said corn plant to remodeled
non-intergeneric microbes comprising at least one genetic variation
introduced into a nitrogen fixation genetic regulatory network and
at least one genetic variation introduced into a nitrogen
assimilation genetic regulatory network, wherein said remodeled
non-intergeneric microbes, in planta, produce at least 5% of fixed
nitrogen in said corn plant as measured by dilution of 15N in crops
grown in fields treated with fertilizer containing 1.2% 15N. [0940]
448. The method of any of the previous clauses, wherein the
bacterium produces fixed N of at least about 1.times.10.sup.-17
mmol N per bacterial cell per hour when in the presence of
exogenous nitrogen. [0941] 449. The bacterium of any of the
previous clauses, wherein the bacterium produces fixed N of at
least about 1.times.10.sup.-17 mmol N per bacterial cell per hour
when in the presence of exogenous nitrogen. [0942] 450. The
bacterial population of any of the previous clauses, wherein the
bacterium produces fixed N of at least about 1.times.10.sup.-17
mmol N per bacterial cell per hour when in the presence of
exogenous nitrogen. [0943] 451. The isolated bacteria of any of the
previous clauses, wherein the bacterium produces fixed N of at
least about 1
.times.10.sup.-17 mmol N per bacterial cell per hour when in the
presence of exogenous nitrogen. [0944] 452. The non-intergeneric
bacterial population of any of the previous clauses, wherein the
bacterium produces fixed N of at least about 1.times.10.sup.-17
mmol N per bacterial cell per hour when in the presence of
exogenous nitrogen. [0945] 453. The non-intergeneric bacterium of
any of the previous clauses, wherein the bacterium produces fixed N
of at least about 1.times.10.sup.-17 mmol N per bacterial cell per
hour when in the presence of exogenous nitrogen. [0946] 454. The
method of any of the previous clauses, wherein the plurality of
bacteria, in planta, produce 5% or more of the fixed nitrogen in
the plant. [0947] 455. The method of any of the previous clauses,
wherein the plurality of non-intergeneric bacteria, in planta,
produce 5% or more of the fixed nitrogen in the plant. [0948] 456.
The bacterium of any of the previous clauses, wherein the plurality
of bacteria, in plank; produce 5% or more of the fixed nitrogen in
the plant. [0949] 457. The bacterial population of any of the
previous clauses, wherein the plurality of bacteria, in planta,
produce 5% or more of the fixed nitrogen in the plant. [0950] 458.
The isolated bacteria of any of the previous clauses, wherein the
plurality of bacteria, in planta, produce 5% or more of the fixed
nitrogen in the plant. [0951] 459. The non-intergeneric bacterial
population of any of the previous clauses, wherein the plurality of
non-intergeneric bacteria, in planta, produce 5% or more of the
fixed nitrogen in the plant. [0952] 460. The non-intergeneric
bacterium of any of the previous clauses, wherein the plurality of
non-intergeneric bacteria, in planta, produce 5% or more of the
fixed nitrogen in the plant. [0953] 461. The method of any of the
previous clauses, wherein the plurality of bacteria, in planta,
produce 10% or more of the fixed nitrogen in the plant. [0954] 462.
The method of any of the previous clauses, wherein the plurality of
non-intergeneric bacteria, in planta, produce 10% or more of the
fixed nitrogen in the plant. [0955] 463. The bacterium of any of
the previous clauses, wherein the plurality of bacteria, in planta,
produce 10% or more of the fixed nitrogen in the plant. [0956] 464.
The bacterial population of any of the previous clauses, wherein
the plurality of bacteria, in planta, produce 10% or more of the
fixed nitrogen in the plant. [0957] 465. The isolated bacteria of
any of the previous clauses, wherein the plurality of bacteria, in
planta, produce 10% or more of the fixed nitrogen in the plant.
[0958] 466. The non-intergeneric bacterial population of any of the
previous clauses, wherein the plurality of non-intergeneric
bacteria, in planta, produce 10% or more of the fixed nitrogen in
the plant. [0959] 467. The non-intergeneric bacterium of any of the
previous clauses, wherein the plurality of non-intergeneric
bacteria, in planta, produce 10% or more of the fixed nitrogen in
the plant. [0960] 468. The method of any of the previous clauses,
wherein the plurality of bacteria, in planta, produce 15% or more
of the fixed nitrogen in the plant. [0961] 469. The method of any
of the previous clauses, wherein the plurality of non-intergeneric
bacteria, in planta, produce 15% or more of the fixed nitrogen in
the plant. [0962] 470. The bacterium of any of the previous
clauses, wherein the plurality of bacteria, in planta, produce 15%
or more of the fixed nitrogen in the plant. [0963] 471. The
bacterial population of any of the previous clauses, wherein the
plurality of bacteria, in planta, produce 15% or more of the fixed
nitrogen in the plant. [0964] 472. The isolated bacteria of any of
the previous clauses, wherein the plurality of bacteria, in planta,
produce 15% or more of the fixed nitrogen in the plant. [0965] 473.
The non-intergeneric bacterial population of any of the previous
clauses, wherein the plurality of non-intergeneric bacteria, in
planta, produce 15% or more of the fixed nitrogen in the plant.
[0966] 474. The non-intergeneric bacterium of any of the previous
clauses, wherein the plurality of non-intergeneric bacteria, in
planta, produce 15% or more of the fixed nitrogen in the plant.
[0967] 475. The method of any of the previous clauses, wherein the
plurality of bacteria, in planta, produce 20% or more of the fixed
nitrogen in the plant. [0968] 476. The method of any of the
previous clauses, wherein the plurality of non-intergeneric
bacteria, in planta, produce 20% or more of the fixed nitrogen in
the plant. [0969] 477. The bacterium of any of the previous
clauses, wherein the plurality of bacteria, in planta, produce 20%
or more of the fixed nitrogen in the plant. [0970] 478. The
bacterial population of any of the previous clauses, wherein the
plurality of bacteria, in planta, produce 20% or more of the fixed
nitrogen in the plant. [0971] 479. The isolated bacteria of any of
the previous clauses, wherein the plurality of bacteria, in planta,
produce 20% or more of the fixed nitrogen in the plant. [0972] 480.
The non-intergeneric bacterial population of any of the previous
clauses, wherein the plurality of non-intergeneric bacteria, in
planta, produce 20% or more of the fixed nitrogen in the plant.
[0973] 481. The non-intergeneric bacterium of any of the previous
clauses, wherein the plurality of non-intergeneric bacteria, in
planta, produce 20% or more of the fixed nitrogen in the plant.
[0974] 482. The method of any of the previous clauses, wherein the
product of (i) the average colonization ability per unit of plant
root tissue and (ii) produced fixed N per bacterial cell per hour,
is at least about 2.0.times.10.sup.-7 mmol N per gram of fresh
weight of plant root tissue per hour. [0975] 483. The bacterium of
any of the previous clauses, wherein the product of (i) the average
colonization ability per unit of plant root tissue and (ii)
produced fixed N per bacterial cell per hour, is at least about
2.0.times.10.sup.-7 mmol N per gram of fresh weight of plant root
tissue per hour. [0976] 484. The bacterial population of any of the
previous clauses, wherein the product of (i) the average
colonization ability per unit of plant root tissue and (ii)
produced fixed N per bacterial cell per hour, is at least about
2.0.times.10.sup.-7 mmol N per grain of fresh weight of plant root
tissue per hour. [0977] 485. The isolated bacteria of any of the
previous clauses, wherein the product of (i) the average
colonization ability per unit of plant root tissue and (ii)
produced fixed N per bacterial cell per hour, is at least about
2.0.times.10.sup.-7 mmol N per gram of fresh weight of plant root
tissue per hour. [0978] 486. The non-intergeneric bacterial
population of any of the previous clauses, wherein the product of
(i) the average colonization ability per unit of plant root tissue
and (ii) produced fixed N per bacterial cell per hour, is at least
about 2.0.times.10.sup.-7 mmol N per gram of fresh weight of plant
root tissue per hour. [0979] 487. The non-intergeneric bacterium of
any of the previous clauses, wherein the product of (i) the average
colonization ability per unit of plant root tissue and (ii)
produced fixed N per bacterial cell per hour, is at least about
2.0.times.10.sup.-7 mmol N per gram of fresh weight of plant root
tissue per hour. [0980] 488. The method of any of the previous
clauses, wherein the product of (i) the average colonization
ability per unit of plant root tissue and (ii) produced fixed N per
bacterial cell per hour, is at least about 2.0.times.10.sup.-6 mmol
N per gram of fresh weight of plant root tissue per hour. [0981]
489. The bacterium of any of the previous clauses, wherein the
product of (i) the average colonization ability per unit of plant
root tissue and (ii) produced fixed N per bacterial cell per hour,
is at least about 2.0.times.10.sup.-6 mmol N per gram of fresh
weight of plant root tissue per hour. [0982] 490. The bacterial
population of any of the previous clauses, wherein the product of
(i) the average colonization ability per unit of plant root tissue
and (ii) produced fixed N per bacterial cell per hour, is at least
about 2.0.times.10.sup.-6 mmol N per gram of fresh weight of plant
root tissue per hour. [0983] 491. The isolated bacteria of any of
the previous clauses, wherein the product of (i) the average
colonization ability per unit of plant root tissue and (ii)
produced fixed N per bacterial cell per hour, is at least about
2.0.times.10.sup.-6 mmol N per gram of fresh weight of plant root
tissue per hour. [0984] 492. The non-intergeneric bacterial
population of any of the previous clauses, wherein the product of
(i) the average colonization ability per unit of plant root tissue
and (ii) produced fixed N per bacterial cell per hour, is at least
about 2.0.times.10.sup.16 mmol N per gram of fresh weight of plant
root tissue per hour. [0985] 493. The non-intergeneric bacterium of
any of the previous clauses, wherein the product of (i) the average
colonization ability per unit of plant root tissue and (ii)
produced fixed N per bacterial cell per hour, is at least about
2.0.times.10.sup.0.6 mmol N per grain of fresh weight of plant root
tissue per hour. [0986] 494. The method of any of the previous
clauses, wherein the plant has been remodeled or bred for efficient
nitrogen use. [0987] 495. The bacterial composition of any of the
previous clauses, wherein said at least one remodeled bacterial
strain comprises at least one variation in a gene or intergenic
region within 10,000 bp of a gene selected from the group
consisting of: nifA, ntrB, ntrC, glnA, glnB, glnK, draT, amtB,
glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS,
nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ. [0988] 496. The
method of any of the previous clauses, wherein said remodeled
bacterium comprises a bacterial composition of any of the preceding
clauses. [0989] 497. The method of any of the previous clauses,
wherein said remodeled bacterium consists of a bacterial
composition of any of the preceding clauses. [0990] 498. The method
of any of the previous clauses, wherein the remodeled bacterial
strain is a remodeled Gram-positive bacterial strain. [0991] 499.
The method of any of the previous clauses, wherein the remodeled
Gram-positive bacterial strain has an altered expression level of a
regulator of a Nif cluster. [0992] 500. The method of any of the
previous clauses, wherein the remodeled Gram-positive bacterial
strain expresses a decreased amount of a negative regulator of a
Nif cluster. [0993] 501. The method of any of the previous clauses,
wherein the remodeled bacterial strain expresses a decreased amount
of GlnR. [0994] 502. The method of any of the previous clauses,
wherein the genome of the remodeled bacterial strain encodes a
polypeptide with at least 75% identity to a sequence from the Zehr
lab NifH database. [0995] 503. The method of any of the previous
clauses, wherein the genome of the remodeled bacterial strain
encodes a polypeptide with at least 85% identity to a sequence from
the Zehr lab NifH database. [0996] 504. The method of any of the
previous clauses, wherein the genome of the remodeled bacterial
strain encodes a polypeptide with at least 75% identity to a
sequence from the Buckley lab NifH database. [0997] 505. The method
of any of the previous clauses, wherein the genome of the remodeled
bacterial strain encodes a polypeptide with at least 85% identity
to a sequence from the Buckley lab NifH database. [0998] 506. The
method of any of the previous clauses, wherein said remodeled
non-intergeneric microbes comprise at least one genetic variation
introduced into said nitrogen fixation genetic regulatory network.
[0999] 507. The method of any of the previous clauses, wherein said
remodeled non-intergeneric microbes comprise at least one genetic
variation introduced into said nitrogen assimilation genetic
regulatory network. [1000] 508. The method of any of the previous
clauses, wherein said remodeled non-intergeneric microbes comprise
at least one genetic variation introduced into said nitrogen
fixation genetic regulatory network and at least one genetic
variation introduced into said nitrogen assimilation genetic
regulatory network. [1001] 509. The method of any of the previous
clauses, wherein said remodeled non-intergeneric microbes are
applied into furrows in which seeds of said non-leguminous plant
are planted. [1002] 510. The method of any of the previous clauses,
wherein said remodeled non-intergeneric microbes are coated onto a
seed of said non-leguminous plant. [1003] 511. The method of any of
the previous clauses, wherein said remodeled non-intergeneric
microbes colonize at least a root of said non-leguminous plant such
that said remodeled non-intergeneric microbes are present in said
non-leguminous plant in an amount of at least 10.sup.5 colony
forming units per gram fresh weight of tissue. [1004] 512. The
method of any of the previous clauses, wherein said remodeled
non-intergeneric microbes are capable of fixing atmospheric
nitrogen in non-nitrogen-limiting conditions. [1005] 513. The
method of any of the previous clauses, wherein said remodeled
non-intergeneric microbes, in planta, excrete nitrogen-containing
products of nitrogen fixation. 514. The method of any of the
previous clauses, wherein said remodeled non-intergeneric microbes,
in planta, produce at least 1% of fixed nitrogen in said
non-leguminous plant. [1006] 515. The method of any of the previous
clauses, wherein said fixed nitrogen in said non-leguminous plant
produced by said remodeled non-intergeneric microbes is measured by
dilution of 15N in crops grown in fields treated with fertilizer
containing 1.2% 15N. [1007] 516. The method of any of the previous
clauses, wherein said remodeled non-intergeneric microbes, in
planta, produce 5% or more of the fixed nitrogen in said
non-leguminous plant. [1008] 517. The method of any of the previous
clauses, wherein said non-intergeneric microbes are remodeled using
at least one type of engineering selected from the group consisting
of directed mutagenesis, random mutagenesis, and directed
evolution. [1009] 518. The method of any of the previous clauses,
wherein said remodeled non-intergeneric microbes, in planta,
excrete nitrogen-containing products of nitrogen fixation. [1010]
519. The method of any of the previous clauses, wherein said
remodeled non-intergeneric microbes are applied into furrows in
which seeds of said corn plant are planted. [1011] 520. The method
of any of the previous clauses, wherein said remodeled
non-intergeneric microbes are coated onto a seed of said corn
plant. [1012] 521. The method of any of the previous clauses,
wherein said remodeled non-intergeneric microbes comprise at least
one genetic variation introduced into a nitrogen fixation genetic
regulatory network and at least one genetic variation introduced
into a nitrogen assimilation gentic regulatory network.
[1013] 522. The method of any of the previous clauses, wherein said
non-intergeneric microbes comprise at least one genetic variation
introduced into a nitrogen fixation genetic regulatory network and
at least one genetic variation introduced into a nitrogen
assimilation gentic regulatory network. [1014] 523. The method of
any of the previous clauses, wherein said genetically engineered
non-intergeneric microbes comprise at least one genetic variation
introduced into a nitrogen fixation genetic regulatory network and
at least one genetic variation introduced into a nitrogen
assimilation gentic regulatory network. [1015] 524. The method of
any of the previous clauses, wherein said remodeled
non-intergeneric bacteria comprise at least one genetic variation
introduced into a nitrogen fixation genetic regulatory network and
at least one genetic variation introduced into a nitrogen
assimilation gentic regulatory network. [1016] 525. The method of
any of the previous clauses, wherein said non-intergeneric bacteria
comprise at least one genetic variation introduced into a nitrogen
fixation genetic regulatory network and at least one genetic
variation introduced into a nitrogen assimilation gentic regulatory
network. [1017] 526. The method of any of the previous clauses,
wherein said genetically engineered non-intergeneric bacteria
comprise at least one genetic variation introduced into a nitrogen
fixation genetic regulatory network and at least one genetic
variation introduced into a nitrogen assimilation gentic regulatory
network. [1018] 527. The bacterium of any of the previous clauses,
wherein said bacterium comprise at least one genetic variation
introduced into a nitrogen fixation genetic regulatory network and
at least one genetic variation introduced into a nitrogen
assimilation gentic regulatory network. [1019] 528. The bacterial
population of any of the previous clauses, wherein bacteria within
said bacterial population comprise at least one genetic variation
introduced into a nitrogen fixation genetic regulatory network and
at least one genetic variation introduced into a nitrogen
assimilation gentic regulatory network. [1020] 529. The isolated
bacteria of any of the previous clauses, wherein said isolated
bacteria comprise at least one genetic variation introduced into a
nitrogen fixation genetic regulatory network and at least one
genetic variation introduced into a nitrogen assimilation gentic
regulatory network. [1021] 530. The non-intergeneric bacterial
population of any of the previous clauses, wherein non-intergeneric
bacteria within said non-intergeneric bacterial population comprise
at least one genetic variation introduced into a nitrogen fixation
genetic regulatory network and at least one genetic variation
introduced into a nitrogen assimilation gentic regulatory network.
[1022] 531. The non-intergeneric bacterium of any of the previous
clauses, wherein said non-intergeneric bacterium comprises at least
one genetic variation introduced into a nitrogen fixation genetic
regulatory network and at least one genetic variation introduced
into a nitrogen assimilation gentic regulatory network. [1023] 532.
A composition comprising any one or more bacteria of any of the
previous clauses.
[1024] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190039964A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20190039964A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References