U.S. patent application number 15/747424 was filed with the patent office on 2018-08-02 for diagnostic kits.
The applicant listed for this patent is AZOTIC TECHNOLOGIES LTD. Invention is credited to David DENT, Gary DEVINE, Dhaval PATEL.
Application Number | 20180216168 15/747424 |
Document ID | / |
Family ID | 54106731 |
Filed Date | 2018-08-02 |
United States Patent
Application |
20180216168 |
Kind Code |
A1 |
DENT; David ; et
al. |
August 2, 2018 |
DIAGNOSTIC KITS
Abstract
The invention provides a diagnostic kit comprising means to
determine the presence in a sample of preferred strains the
nitrogen-fixing bacteria Gluconacetobacter diazotrophicus (Gd)
which are based upon the finding that such strains comprises unique
nucleic acid sequences and other features, that are detectable,
even in the presence of plant genomic DNA. Methods for using the
kits in agriculture are also described and claimed.
Inventors: |
DENT; David; (Fleet
Hampshire, GB) ; PATEL; Dhaval; (Nottingham, GB)
; DEVINE; Gary; (Nottingham, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AZOTIC TECHNOLOGIES LTD |
Chorley, Lancashire |
|
GB |
|
|
Family ID: |
54106731 |
Appl. No.: |
15/747424 |
Filed: |
July 27, 2016 |
PCT Filed: |
July 27, 2016 |
PCT NO: |
PCT/GB2016/052288 |
371 Date: |
January 24, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C05F 11/08 20130101;
C12N 1/20 20130101; C12Q 1/689 20130101; A01C 1/06 20130101; A01N
63/00 20130101 |
International
Class: |
C12Q 1/689 20060101
C12Q001/689; C05F 11/08 20060101 C05F011/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 28, 2015 |
GB |
1513277.2 |
Claims
1. A diagnostic kit for determining the presence of
plant-colonizing strains of Gluconacetobacter diazotrophicus (Gd),
said kit comprising means to determine the presence in a sample of
at least one nucleic acid sequence selected from SEQ ID NOs
1-10.
2. The diagnostic kit of claim 1 which comprises means for
determining the presence of up to 10 different nucleic acid
sequences of SEQ ID NOs 1-10.
3. (canceled)
4. The diagnostic kit of claim 1 which further comprises means for
determining the presence of at least one nucleic acid sequence
which is characteristic of Gd species.
5. The diagnostic kit of claim 4 wherein the at least one nucleic
acid sequence which is characteristic of a Gd species is a nucleic
acid sequence selected from SEQ ID NOs 11-13.
6. The diagnostic kit of claim 1 which further comprises means for
detecting nucleic acid found in a plant.
7. The diagnostic kit of claim 1 which further comprises means for
determining the presence of SEQ ID NOs 64, 65, 66 or 67 as a
negative control.
8. The diagnostic kit of claim 1 wherein the means for determining
the presence of nucleic acid sequences comprises one or more
amplification primers which target said at least one nucleic acid
sequence.
9. The diagnostic kit of claim 8 which further comprises one or
more additional reagents necessary to carry out a nucleic acid
amplification reaction.
10. The diagnostic kit of claim 9 wherein the nucleic acid
amplification reaction is a polymerase chain reaction (PCR).
11. The diagnostic kit of claim 9 wherein the nucleic acid
amplification reaction is a loop-mediated amplification reaction
(LAMP).
12. The diagnostic kit of claim 10, wherein the amplification
primers are selected from SEQ ID NOs 14 to 39 or 58 to 63.
13. The diagnostic kit of claim 11 wherein the amplification
primers are selected from SEQ ID NOs 40-57.
14. The diagnostic kit of claim 1 which comprises means for
extracting plasmid DNA from a Gd containing sample.
15. (canceled)
16. A method for determining the presence in a sample of a strain
of Gluconacetobacter diazotrophicus (Gd) able to intracellularly
colonise plant cells, said method comprising detecting in said
sample at least one nucleic acid sequence selected from SEQ ID NOs
1-10.
17. The method of claim 16 wherein the presence of at least 5 of
said nucleic acid sequences of SEQ ID NOs 1-10 is detected.
18. (canceled)
19. The method of claim 16 wherein the presence of at least one
nucleic acid sequence which is characteristic of Gd species is
detected, wherein the nucleic acid sequence which is characteristic
of Gd species is selected from SEQ ID NOs 11-13.
20. (canceled)
21. The method of claim 16 wherein the sample is a plant sample and
a nucleic acid sequence found in said plant is also detected.
22. The method of claim 16 wherein the presence of said nucleic
acid sequences are determined by means of a nucleic acid
amplification reaction, wherein the nucleic acid amplification
reaction is a polymerase chain reaction (PCR), a quantitative PCR
(QPCR), or a loop-mediated isothermal amplification (LAMP).
23-27. (canceled)
28. The method of claim 16, which further comprises extracting
plasmid DNA from the sample and detecting the presence of a plasmid
of less than 17566 base pairs.
29. (canceled)
30. The method of claim 16 wherein the sample is a plant sample to
which a strain of Gluconacetobacter diazotrophicus (Gd) has been
applied.
Description
[0001] The present invention relates to a diagnostic kit, able to
identify particular strains of the nitrogen-fixing bacteria
Gluconacetobacter diazotrophicus (Gd) that have good utility in
agriculture, in terms of their ability to colonise plant cells
intracellularly, giving rise to particularly effective nitrogen
fixation, as well as to reagents for use in the kits. Novel strains
that are identified by the kits form the subject of a co-pending
application.
BACKGROUND OF THE INVENTION
[0002] Gluconacetobacter diazotrophicus (Gd) has been well studied
for its nitrogen fixing and plant growth promoting activities as
reviewed in Eskin et al. International Journal of Agronomy
(2014):1-13. Certain strains of Gd however have been shown to be
particularly advantageous in the treatment of plants since they are
able to establish themselves intracellularly within plant cells
along with exhibiting species and tissue independence (Cocking et
al., In vitro Cellular & Developmental Biology Plant (2006) 42
(1). These properties, combined with their ability to travel
throughout a range of plant tissues, make such strains better able
to deliver the benefits to the target crop plants.
[0003] However, a wide range of strains of Gd exist and it has not
yet been possible to provide a means for easily identifying strains
which have these beneficial properties.
[0004] Furthermore, an important aspect of bio-fertiliser has been
to provide an alternative to the chemical fertilisers in a nature
friendly way to agricultural crop plants. However, it would be
helpful to validate the effectiveness of any on-going treatment in
field conditions, so that a farmer is able to determine what levels
of nitrogen fertiliser, if any, are required to be supplied to
enhance growth conditions.
[0005] The applicants have found that specific advantageous strains
of Gd contain a number of unique nucleic acid sequences that are
not found in other Gd species, nor in any other species, including
plant species. This gives rise to the provision of a reliable
diagnostic test, useful in both monitoring of treatments in the
fields and in research, for identifying related beneficial
strains.
SUMMARY OF THE INVENTION
[0006] According to the present invention there is provided a
diagnostic kit comprising means to determine the presence in a
sample of at least one nucleic acid sequence selected from SEQ ID
NOS 1-10 shown in attached Table 1 hereinafter.
[0007] SEQ ID NOs 1-10 represent unique and novel sequences, which
appear in a preferred sub-species or strain of Gd. Strains having
these unique characteristics have been found to be particularly
effective in intracellular colonization of plant cells resulting in
beneficial nitrogen fixing. In particular, it has been found that
use of a stain of the invention leads to yield enhancements in
crops such as cereal crops like maize and wheat. Alternatively,
similar yields can be achieved even with reduction in traditional
nitrogen fertiliser applications.
[0008] Thus the sequences provide a means for identifying these
beneficial strains. In addition, they have been found to be
amenable to detection using primers which do not cross-react with
plant species.
[0009] Furthermore, since the strains can colonise intracellularly
in the host plant cells, they are able to effectively travel
throughout the plant. The kit of the invention can be used to
provide an evaluation of colonisation by Gd post-germination at a
young stage and check its efficiency. This will then allow the
farmers to make an "informed decision" about the existence and
extent of the colonisation and if required how much chemical based
nitrogen fertiliser will be required to be applied. This extra
level of security will provide `assurance` to farmers that their
crops are being well-tended, even when using nature and crop
friendly fertiliser in the form of Gd.
[0010] In a particular embodiment, the kit of the invention
comprises means for determining the presence of more than one of
the nucleic acid sequences of SEQ ID NOs 1-10, for example up to
10, such as up to 5, or up to 3 of said nucleic acid sequences of
SEQ ID NOs 1-10. In this way, a reliable diagnostic kit would be
provided which will ensure that the strain detected is the one
which is similar or substantially similar to the beneficial strain.
Similar strains would be detected even if one or more of the
sequences differs for example as result of mutation.
[0011] Preferred strains appear to contain a plasmid, and so
plasmid detection, for example by isolation using a
commercially-available plasmid isolation kit, may provide further
confirmation of the identification of a beneficial strain. In
particular, any plasmid identified should be less than 27455 bp in
size, for example about 17566 bp. The presence of a plasmid, in
particular of this size, differs from a previously known strain of
Gd, UAP5541, which has been reported as lacking in plasmids (Luis
E. Fuentes-Ramierez et al., FEMS Microbiology Ecology 29 (1999)
117-128). Furthermore, the size of the plasmid is smaller than that
reported previously in respect of PAL5 strains containing a single
plasmid (Giongo et al. Standards in Genomic Sciences, May 2010,
Volume 2, Issue 3, pp 309-317 doi: 10.4056/sigs.972221).
[0012] The plasmid of beneficial strains may also be characterized
in that it is restricted into two fragments by the restriction
enzyme EcoRI, wherein the fragments are about 12 Kb and about 5.6
kb in size respectively. Thus, in certain embodiments, the kit of
the invention may further comprise means for determining the
presence and nature of the plasmid, including the provision of a
restriction enzyme such as EcoR1, to allow for the detection of
fragments of the above-mentioned sizes.
[0013] Furthermore, the plasmid lacks a number of key sequences
which have been previously identified as being present in the
plasmid of PAL5. These sequences are shown as SEQ ID Nos 65, 66, 67
and 68 in the attached sequence listing. Thus the absence of these
particular sequences may provide a further characterizing feature
of the strains. In some embodiments therefore, the kit may comprise
means for detecting these SEQ ID Nos 65, 66, 67 and 68 to provide a
negative control in the sense that the absence of positive results
for these sequences may be used to confirm the presence or identity
of preferred strains, or the presence of the these sequences may be
used to reject less preferred strains in strain selection.
[0014] In a particular embodiment, the diagnostic kit further
comprises means for determining the presence of at least one
nucleic acid sequence which is characteristic of Gd species, for
example 2, or 3 nucleic acid sequences which are characteristic of
Gd species. In this way, the kit would provide confirmation that
some Gd is present in the sample, thus confirming the accuracy of
the test. If the sample is known to contain Gd, a positive result
in this determination would act as a `control` confirming that the
test has been carried out effectively. Suitable specific strains
will be sequences found in Gd species generally but not in other
species, and in particular not in other microbial species or in at
least some plants, in particular plants which may be targeted for
Gd treatments.
[0015] Particular examples of such sequences are shown as SEQ ID
NOS 11-13 in Table 2 hereinafter. In particular, these nucleic acid
sequence which is used to detect Gd species have been found to be
amendable to detection of Gd species present in a range of crops,
without cross-reacting with plant species.
[0016] In a further embodiment, the kit comprises means for
detecting the presence of a plant specific nucleic acid sequence,
such as a chloroplast specific nucleic acid which amplifies
universally from plant DNA. The inclusion of such means will act as
a control when the kit is used in the context of detection of Gd
within a plant species, as this means will produce a detectable
signal, even in the absence of any Gd. If this signal fails, this
would indicate failure in the test rather than necessarily that
there is no Gd present. A particular chloroplast primer set which
is available commercially from Thermo Scientific. Product as Phire
Plant Direct PCR Master Mix, is based on the disclosure by Demesure
B et al (1995) Molecular Ecology 4:129-131.
[0017] The means for detecting the presence of the nucleic acid
sequences may take various forms as would be understood in the
art.
[0018] Where the nucleic acids are genes which are expressed, the
kit may comprise means for detecting the expressed proteins. Such
means may include specific protein tests such as immunochemical
analysis which utilise antibodies specific to the proteins to
immobilise and/or detect the proteins, such as ELISA or RIA
techniques, or immunoelectrophoretic methods such as Western
blotting.
[0019] However, in a particular preferred embodiment, the kit of
the invention comprises means for detecting specific nucleic acids
themselves.
[0020] In particular, nucleic acids may be detected using any of
the available techniques including nucleic acid binding assays and
immunoassays using antibodies raised to haptenised forms of the
nucleic acids. However, in a particular embodiment, the detection
involves nucleic acid amplification reactions, and thus in
particular, the kits will comprise one or more amplification
primers which target the or each nucleic acid sequence being
detected.
[0021] As would be understood by a skilled person, when detection
of a sequence is carried out using an amplification reaction, it
would not be necessary to amplify the entire sequence, but rather
just a characteristic fragment of the entire sequence, for example
a fragment of at least 10, and suitably at least 50 base pairs.
Thus the size of the sequence that may be amplified would could be
in the range of from 10-3070 base pairs, and suitably from 20-2000
base pairs, for example from 50-500 base pairs, such as from
100-300 base pairs. The size of the fragment will depend upon the
nature of the detection reaction being used, but it should be
sufficient to ensure that the product is characteristic of SEQ ID
Nos 1-10 or variants as defined above, and so is not present in
other sequences, in particular plant sequences. Where more than one
sequence is amplified, they may be selected to be of differing
sizes, so that they may be easily differentiated during detection,
using techniques such as separation on the basis of size, or
melting point analysis.
[0022] If required, the kit may further comprise one or more
additional reagents necessary to carry out a nucleic acid
amplification reaction. Thus it may include enzymes such as
polymerases, salts such as magnesium or manganese salts, buffers
and nucleotides as would be understood in the art.
[0023] The kits may, if required, include means for detecting the
products of the amplification. Such means may include dyes or
probes, in particular labelled probes that bind the target sequence
intermediate the primers. Alternatively, the primers themselves may
be labelled to facilitate detection.
[0024] Suitable nucleic amplification reactions include reactions
that utilise thermal cycling such as the polymerase chain reaction
(PCR) and ligase chain reaction (LCR) as well as isothermal
amplification reactions such as nucleic acid sequence based
amplification (NASBA), strand displacement amplification (SDA),
transcription mediated amplification (TMA), loop-mediated
isothermal amplification (LAMP) and rolling circle amplification,
3SR, ramification amplification (as described by Zhang et al.,
Molecular Diagnosis (2001) 6 No 2, p 141-150), recombinase
polymerase amplification (available from TwistDx) and others. In a
particular embodiment, the nucleic acid amplification is a PCR, and
may be a quantitative PCR (QPCR) to provide information regarding
the extent of colonisation.
[0025] In an alternative embodiment, the amplification is LAMP
reaction. LAMP assays utilise at least four and suitably six
primers which are designed to target six different regions of the
target sequence. There will always be two outer primers (F3 and B3)
and two inner primers (FIP and BIP). Optionally, in addition there
are two loop primers (FLoop and BLoop). The use of the Loop primers
usually reduces the amplification time and increases the
specificity of the assay. FIP and BIP primers consist of F2,
complementary to the F2c region of the template sequence, and F1c,
identical to the F1 region of the template. Four main features need
to be considered in order to guarantee a successful LAMP primer
design: the melting temperature of the primers (Tm), given
55-65.degree. C. for F3, FIP, BIP and B3 primers and
.gtoreq.65.degree. C. for FLoop and BLoop; a GC content of 50-60%
in the primer sequences; the absence of secondary structures
formation and stability at the ends of each primers; and finally
the distance between primer regions. Examples of suitable LAMP
primer sets are disclosed hereinafter.
[0026] The presence of the products of amplification reactions may
be determined using any available technology. Thus they may include
techniques where products are separated on a gel on the basis of
size and/or charge and detected such as agarose gel
electrophoresis. Alternatively, they may be detected in situ, using
for example using intercalating dyes or labelled probes or primers.
The detection of amplification products using a wide variety of
signalling and detection systems is known. Many of these systems
can be operated in `real-time`, allowing the progress of
amplification to be monitored as it progresses, allowing for
quantification of the product. Many such systems utilise labels and
in particular fluorescent labels that are associated with elements
such as primers and probes used in the amplification system and
which rely on fluorescent energy transfer (FET) as a basis for
signalling. A particular form of such fluorescent energy transfer
is fluorescent resonance energy transfer or Forster resonance
energy transfer (FRET) for signal generation.
[0027] A major example of such a process used commercially is the
TaqMan.RTM. process, in which a dual-labelled probe, carrying both
a first label comprising a fluorescent energy donor molecule or
reporter and a second label comprising a fluorescent energy
acceptor molecule or quencher, is included in a PCR system. When
bound to the probe, these molecules interact so that the
fluorescent signal from the donor molecule is quenched by the
acceptor. During an amplification reaction however, the probe binds
to the target sequence and is digested as the polymerase extends
primers used in the PCR. Digestion of the probe leads to separation
of the donor and acceptor molecules, so that they no longer
interact. In this way, the quenching effect of the acceptor is
eliminated, thus modifying emissions from the molecule. This change
in emission can be monitored and related to the progress of the
amplification reaction.
[0028] Where more than one nucleic acid sequence is detected, the
kit may comprise components sufficient to carry out multiple
separate amplification reactions, such as individual sets of
primers. Preferably however the kit is set up to carry out a
multiplex reaction, where multiple targets may be detected in a
single reaction. In this case, where the detection is done using
gel electrophoresis, the primers are suitably selected so that each
amplified product has a significantly different size or charge so
that they may be readily separated and identified on an agarose gel
or by melting point analysis using a signalling reagent such as a
DNA intercalating dye.
[0029] Alternatively, where the detection system includes labels,
any labels provided for example on primers or probes, will provide
a different and distinguishable signal from other primer sets, for
example on the basis of the wavelength of the emitted signal and/or
the fact that the product has a different melting point or
annealing temperature, which may be distinguished by carrying out a
melting point analysis of products.
[0030] Suitable amplification primers, in particular for PCR
amplification, together with the approximate size of the products
they generate are selected from those set out in Table 3 below:
TABLE-US-00001 TABLE 3 SEQ SEQ Product ID Forward ID Reverse size A
14 TGAAATTGACGCCCGTTGGA 15 CACGCCGGGAAAGAGGATTC 472 bp B 16
GGCAACGCGGTTTCTACGAA 17 CGTTAGCCGGGGTTGTCAGA 489 bp C 18
TCGTTGCCACTTTCCGAGGG 19 GTCGATTGTGTGCAGCGTCA 268 bp A D 20
CACCGATCTTGTGCGTTTCG 21 CGGCAATGCTCCATACCCAC 522 bp E 22
CACCGGAAAGAGTGGCAGGA 23 AACCGGGTCACTTGCGTCAT 783 bp F 24
AGCCATCGGAGTCACATCGG 25 GGAAACCTCGAAACCCTGCG 1129 bp G 26
TCAGGGCAATCACTAGCCGG 27 TCGAGCAGCCGTTTCATCCA 1118 bp H 28
TGATGCGCTTGTTCGTGACG 29 CGTTCGCCCTTGTCGTCATG 478 bp I 30
GGGCCATCCGTTACCTGCTT 31 TGACACACCCGCTCCGAAAT 1102 bp J 32
GCATTTGCGGTAAGTCATCC 33 GGATCCCGATTTGCAAGCCA 814 bp CA K 34
TGTCGGGTCGGGAACTCAAG 35 CGGGTTCTCGCTGATGACCT 464 bp L 36
TCCCGCCTGCATCTGAAGAC 37 CAGCGATGCCAGCCAATACC 1098 bp M 38
GTTCGTCGCGTCTGATGCAG 39 ACCTGGGCATTGTTGGTGGA 1045 bp
[0031] Primer sets represented by SEQ ID NOS 14-33 have been found
to act as useful strain-specific primers for beneficial strains of
Gd, while primer sets represented by SEQ ID NOS 34-39 act as useful
Gd species-specific primers.
[0032] In a further aspect, the invention provides a method for
determining the presence in a sample of a strain of
Gluconacetobacter diazotrophicus (Gd) able to intracellularly
colonise plant cells, said method comprising detecting in said
sample at least one nucleic acid sequence selected from SEQ ID NOS
1-10.
[0033] Suitably up to 10, for example up to 5 such as about 3 of
said nucleic acid sequences of SEQ ID NOs 1-10 are detected.
[0034] In a particular embodiment, the method further comprises
detecting at least one nucleic acid which is characteristic of Gd
species, as described above, such as a nucleic acid sequence of SEQ
ID NOS 11-13. Again, more than one such species specific nucleic
acid sequence may be detected if required.
[0035] In yet a further embodiment, the method further comprises
detecting a plant specific nucleic acid sequence, which may be
characteristic of the particular Gd colonised plant being examined
or may be universally present in plants, such as a chloroplast
specific nucleic acid sequence, as a control for the reaction.
[0036] Various methods of detection may be used in the method, as
described above, but in particular, the method comprises a nucleic
acid amplification reaction, such as the polymerase chain reaction
(PCR).
[0037] Suitable primers are as described above.
[0038] The sample which may be used in the method of the invention
may be any sample that contains or is suspected of containing a
strain of Gd. This may include cultures or laboratory samples,
which may contain the desired strain of Gd. Alternatively, they may
comprise plant samples, including leaf, stem, or root samples, from
which nucleic acid has been released, for example by causing cell
lysis, for example using mechanical, chemical or sonic means. In
particular, the sample is from a plant to which a strain of
Gluconacetobacter diazotrophicus (Gd) has previously been applied.
In this way, the successful colonisation of the plant by Gd can be
confirmed.
[0039] Typically, such tests will be carried out in a laboratory,
although mobile testing, for example, in field conditions, may be
carried out if suitable equipment, such as mobile PCR machines, are
available, or if detection of targets in particular protein
targets, using techniques such as ELISAs, which may be carried out
on lateral flow devices are employed.
[0040] Novel strains of Gluconacetobacter diazotrophicus (Gd) able
to intracellularly colonise plant cells and identified using a
method described above are described in the applicants copending
application of even date. A particular example of such a strain
IMI504958, deposited at CABI (UK) on 22 May 2015.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The invention will now be particularly described by way of
example with reference to the accompanying figures which are
described as follows:
[0042] FIG. 1A: is a gel showing PCR products from a range of
designed to be strain or species specific, including primer sets
A-H in Table 3 (shown in lanes 4, 5, 6, 7, 8, 9, 12 and 13
respectively). FIG. 1B is a gel showing PCR products from a range
of designed to be strain or species specific, including primer sets
I-M in Table 3 (shown in lanes 4, 5, 9, 12 and 13
respectively).
[0043] FIG. 2: is a gel illustrating PCR products using Primer set
E following inoculation of reactions with 100 ng of (1) OSR var.
Ability, (2) OSR var. Extrovert, (3) rice var. Valencia, (4) wheat
var. Willow, (5) grass var. Aberglyn, (6) grass var. Dickens, (7)
maize, (8) quinoa, (9) Arabidopsis var. Columbia, (10) barley var.
Chapeaux, (11) grass var. Twystar, (12) grass var. J Premier
Wicket, (13) potato, and (14) tomato. Lane (15) contains amplicon
produced from 10 ng genomic DNA from Gd, (16) contains the no
template PCR control, and the molecular weight marker at each end
of the gel is Hyperladder 1 kb plus (Bioline).
[0044] FIG. 3: is a gel illustrating the sensitivity PCR using
Primer set B in reactions containing 100 ng DNA from OSR var.
Ability, co-inoculated with (1) 1 ng, (2) 100 picogram, (3) 10
picogram, (4) 1 picogram, (5) 100 femtogram, (6) 10 femtogram, and
(7) no added genomic DNA from Gd. Lane (8) is the no template
control sample and molecular weight marker at each end of the gel
is Hyperladder 1 kb plus (Bioline).
[0045] FIG. 4A is a graph showing positive amplification of
Gluconacetobacter diazotrophicus by fluorescent LAMP using the
Genie II real-time machine and a primer set embodying the
invention. Positive DNA amplification is detected by a fluorescence
signal. FIG. 4B is an anneal curve for the Gluconacetobacter
diazotrophicus samples, following amplification by LAMP; the
reaction was put through an anneal analysis and the temperature at
which the dsDNA reanneals is detected as a burst of
fluorescence.
[0046] FIGS. 5A-C are graphs showing representative results of QPCR
experiments carried out using primers designed to amplify sequences
according to the method of the invention, when carried out using
serial dilutions of samples containing GD DNA. FIG. 5A shows the
results for primer set designated P5 for SEQ ID NOs 58 and 59. FIG.
5B shows results for a primer set designated P8 for SEQ ID NOs 60
and 61. FIG. 5C shows results for a primer set designated P17 for
SEQ ID NO 62 and 63 as defined hereinafter.
[0047] FIGS. 6A-F are graphs showing representative results of QPCR
experiments carried out using primers designed to amplify sequences
that may be detected using a kit of the invention, when carried out
using serial dilutions of samples containing GD DNA and plant
genomic DNA. FIG. 6A shows the results for primer set designated P5
in the presence of wheat DNA. FIG. 6B shows melt peak graphs of the
products of FIG. 6A for all the samples (i.e. dilutions of Gd in
presence of wheat genome and relevant controls). FIG. 6C shows melt
peak graph of the controls from FIG. 6A where a positive control
comprising Gd DNA only resulted in giving signal, and negative
controls comprising plant DNA only and QPCR negative samples only
(NTC--no transcript control), both did not resulted in giving
signal. FIG. 6D shows results for a primer set designated P17 as
defined hereinafter in the presence of maize DNA. FIG. 6E shows the
melt peak graph of the products of FIG. 6D for all the samples
tested (i.e. dilutions of Gd in presence of Maize genome and
relevant controls). FIG. 6F shows the melt peak graphs of the
controls from FIG. 6D where a positive control comprising Gd DNA
only resulted in giving signal, and negative controls comprising
plant DNA only and QPCR negative samples only (NTC--no transcript
control), both did not resulted in giving signal.
[0048] FIG. 7 shows a resolved 1% agarose gel showing plasmid DNA
extracted from a particular strain of GD (IMI504958).
[0049] FIG. 8 shows resolved 1% agarose gel restriction digestion
product of plasmid DNA with EcoRI from strain of Gd (IMI504958).
The restricted fragments are mentioned as 1) .about.12 Kb and 2)
.about.5.6 Kb when run alongside 1 kb ladder where the nearest
fragment from the ladder is highlighted for the size
comparison.
[0050] FIG. 9 shows an agarose gel obtained from a PCR
amplification to detect Gd from the seedlings of wheat obtained
during a field trial.
[0051] FIG. 10 is a graph showing the chlorophyll index of wheat
treated with Gd in accordance with the invention compared to a
control.
[0052] However, it will be apparent to one skilled in the art that
the specific details are not required in order to practice the
invention. The following descriptions of specific embodiments of
the present invention are presented for purposes of illustration
and description. They are not intended to be exhaustive of or to
limit the invention to the precise forms disclosed. Many
modifications and variations are possible in view of the above
teachings. The embodiments are shown and described in order to best
explain the principles of the invention and its practical
applications, to thereby enable others skilled in the art to best
utilize the invention and various embodiments with various
modifications as are suited to the particular use contemplated.
Example 1
Identification of Unique Sequences
[0053] IMI504958, a Gd strain derived from passaging UAP5541, which
was found to have particularly beneficial plant colonisation
properties was isolated and the full genome sequenced. A comparison
was made against the publically available genome of the type strain
(PAL5; sequenced by JGI, USA [Genbank sequence accession CP001189])
using standard methods.
[0054] Surprisingly, a large number of differences were noted in
the genome, and in particular, a number of genes were identified
which are present in the genome of IMI504958 but not PAL5.
[0055] Many of these genes were annotated with an associated
function. The unique genes with annotations were further checked
for uniqueness across all the genomes sequenced to date using the
NCBI's web-based BLAST tool.
[0056] Analysis of the BLAST result narrowed the list to 20 unique
genes not present in any genome. These unique genes appeared to be
"strain-specific" for IMI504958.
[0057] Also, five sets were found to be unique to the Gd species
and will hereafter be referred to as "species-specific" (i.e.
present in IMI 504853, Pa15 and other Gd strains but in no other
species).
[0058] The applicants used this data to design a diagnostic kit for
IMI504958.
Example 2
PCR Validation
[0059] A set of 25 primer sets were designed based upon the
sequences identified in the analysis of the genome. The specificity
of these 25 primer sets (20 designed to be strain-specific and 5
designed to be species-specific) were first tested by carrying out
a conventional PCR reaction using genomic DNA of IMI504958 and
PAL5. The results with IMI504958 are illustrated in FIGS. 1A-B.
Results showed that 16 strain-specific primer sets delineated
IMI504958 from PAL5 as obtained from three different collections
(ATCC49037, DSM5601 and LMG7603). However, 4 putative
strain-specific primer sets cross-reacted with at least one PAL5
and hence were removed from strain-specific study.
[0060] All 5 species-specific primers reacted as expected.
[0061] Further, testing of strain- and species-specific primers was
done against two other strains of Gd, one originally isolated in
India (IMI 502398) and the other from Mauritius (IMI 502399), as
well as a revived 2001 culture of UAP5541 strain (stored in
glycerol at -80.degree. C.), using the method described above. The
data was in agreement with the 16 strain specific primers and 4
species specific primer sets as one of the species-specific primer
sets produced a higher molecular weight band. This was a surprise
result.
[0062] The sensitivity of detection of all 25 primer sets (20
strain-specific and 5 species-specific) was checked using serial
dilutions of bacterial broth cultures. It was found that 24 of
these sets produced very high levels of detection (requiring 1-10
bacterial cells).
[0063] Further, these 25 primers were then checked for
cross-reactivity with several target plant species and varieties
using DNA extracted in-house. The primers were tested in a PCR
reaction using DNA extracted from plants of the following species:
maize, wheat (var. Willow), quinoa, rice (var. Valencia), barley
(var. Chapeaux), potato, Arabidopsis (var. Columbia), oilseed rape
(vars. Ability and Extrovert) and a range of grasses (vars.
Aberglyn, Dickens, J Premier Wicket, and Twystar). The method for
isolating nucleic acids from plant tissues involved the mechanical
maceration of leaf material followed by a modified CTAB extraction
(Doyle and Doyle, 1987 Phytochem. Bull., 19: 11-15). Briefly,
cellular membranes were disrupted using SDS and CTAB to release
their contents, and cellular proteins were degraded or denatured
using proteinase K and .beta.-mercaptoethanol. The extraction
buffer also contained PVPP to remove plant polyphenols, EDTA to
chelate metal ions, sodium chloride to solubilise nucleic acid
structures, as well as TRIS HCl to stabilise the buffer pH. RNA
molecules were degraded using RNase A treatment. Following the
removal of insoluble cellular debris using chloroform:isoamyl mix
(24:1), deoxynucleic acids were precipitated in ethanol using
sodium acetate, washed using diluted ethanol, and resuspended in
molecular grade water.
[0064] Illustrative results are shown in FIG. 2.
[0065] At the same time, the sensitivity of primers were tested by
co-inoculating PCR reactions containing 100 ng of the above
mentioned plant genomes with six-fold serially diluted genomic DNA
from Gd, starting from 1 ng. It was found that the sensitivity of
the PCR system was generally unaffected by the presence of plant
genome and routine detection was established from a minimum of 1
picogram of Gd DNA.
[0066] Illustrative results are shown in FIG. 3.
[0067] Results suggested that 17 of the 20 strain-specific primer
sets and three of the five species-specific sets either do not
cross react with any plant genomes tested, or cross-react with a
small number but produce a DNA product of a different size and
distinguishable size.
[0068] Of the strain-specific primer sets, only 10 produced results
which were of (1) high specificity, (2) high sensitivity, and (3)
produced no cross-reactions with plant DNA and these are
represented in Table 3 above as primer sets A-J. Similarly only
three of the selected species-specific primer sets were found to be
specific and sensitive enough for use and these are shown as primer
sets K-M in Table 3 respectively. In addition, the size of the
products obtained using these primers is shown in Table 3 and
illustrated in FIGS. 1A and 1B. Thus, methods and kits based upon
these primers are particularly useful in identifying beneficial Gd
in field situations.
Example 3
LAMP Assay
[0069] A series of LAMP primers were designed to amplify regions of
SEQ ID NOS 6, 7 and 9 and are shown in Table 4 below as
follows:
TABLE-US-00002 TABLE 4 SEQ ID NO Sequence Type 40
CTCAGGAAGACCGAATTGATTA F3 41 GCGAAACGTCTGATTGAAC B3 42
CGGATAACCACTGGTGCTCCGACTCGCCTCACTCTACT FIP 43
TCCACGAATCTCACGAAGCACCCCGACCTTATCTCCCAT BIP 44
GCCAGGCGTGTACATATAACTA FL 45 CGGAATACCTAGTTGGAACACT BL 46
TCAAGATCGATGCACCTATTC F3 47 AACAGACAGTTCTGGTAGGA B3 48
CGCATCTCCAGATCGGCAGGTCGTCCAGTCGATCATG FIP 49
ACATCTGTCCACGGCATTGGTGGCTGGCTTATGAGTCT BIP 50 GAGAAGTCCTCTGCTTCGG
FL 51 CGGCGGTTGAGAAGATGT BL 52 GGAAGACATCAACGAAGCA F3 53
TTGACAGTTGCATAGTCCG B3 54 ATACGGCTCGTCATGTCGCGGTGATGGATAATCTCAGCC
FIP 55 CAGTGGCCGAACCTGGAAGCGCTGATATAAGCCTGAAGAT BIP 56
ATTGCACCGCGTTGATG FL 57 GCGTAACGGTCACAAGGA BL
[0070] SEQ ID NOS 40-45 were designed to amplify SEQ ID NO 6 above,
SEQ ID NOS 46-51 were designed to amplify SEQ ID NO 7 above, and
SEQ ID NOS 52-57 were designed to amplify SEQ ID NO 9 above.
[0071] These primers were obtained and tested in a LAMP assay on
samples comprising pure Gd DNA that had been isolated using a
modified CTAB methodology from bacteria grown in liquid
culture.
[0072] In addition, DNAs from a range of plant pathogenic bacteria
and fungi was tested for amplification in LAMP by the primer sets.
These included Bacillus subtilis, Lactobacillus, Fructobacillus,
Pseudomonas spp., Agrobacterium spp., a range of phytoplasmas and
various fungi including species from the Fusarium, Penicillium and
Aspergillus genera.
[0073] Real-time LAMP was carried out on a Genie II instrument
(OptiGene), and 1 .mu.l of sample was added to a 24 .mu.l reaction
mix containing 15 .mu.l Isothermal Master Mix ISO-001 (OptiGene),
200 nM of each external primer (F3 and B3), 2 .mu.M of each
internal primer (FIP and BIP) and 1 .mu.M of loops primer (FLoop
and BLoop). RT-LAMP reaction consisted of 30 minutes of isothermal
amplification at 63.degree. C. To evaluate the annealing
temperature of the products, reactions were then subjected to a
slow annealing step from 95 to 68.degree. C. (0.05.degree. C./s)
with fluorescence monitoring.
[0074] Negative reaction controls, consisting of water, were also
used.
[0075] Of the three sets of primers tested in LAMP, the third
primer set specific for SEQ ID NO 9 gave amplification in nine and
a half minutes with an anneal at 89.2.degree. C. (see FIG. 4A). The
primer set specific for SEQ ID NO 6 amplified the positive control
at around 11 minutes with an anneal of approximately 88.degree. C.,
and the primer set specific for SEQ ID NO 7 was the slowest,
amplifying the positive control at around 23 minutes with an
annealing temperature around 90.degree. C.
[0076] All sets of primers that gave the positive Gd amplification
were specific for the bacterium and did not amplify from DNA of any
of the other bacterial and fungal DNAs they were tested on. They
are therefore all suitable as primer sets to be used for detection
of the Gd bacterium.
Example 4
Detecting Gd on Plant Samples Using LAMP
[0077] To validate the primers on rapidly extracted DNA from
contaminated seed, a series of experiments were set up in which
seed of two plant species, tomato and wheat, were spiked on the
surface with Gd DNA. The samples were then put through the 2-minute
DNA extraction technique in which the samples are placed in plastic
tubes containing steel beads and TE buffer and shaken vigorously
for 2 minutes. Two microliters of the solution was then placed in
the LAMP reaction as described in Example 4 using the primer set
comprising SEQ ID NOs 52 to 57 to test for amplification of the Gd
DNA from these samples.
[0078] The results showed that the Gd DNA is detectable when put
through these assays, against a background of plant DNA.
[0079] In order confirm that any samples that tested negative for
Gd supported LAMP amplification (i.e. they do not contain
inhibitors of LAMP reactions), the cytochrome-oxidase gene (COX)
primers (Tomlinson et al., 2012 Journal of Virological Methods,
191: 148-154.), which amplify DNA from the host plant, were used as
controls for false negatives on all samples.
Example 5
QPCR Determination
[0080] A range of QPCR reactions were carried out on samples
comprising known quantities of DNA from Gd (IMI504958) and also
from a range of crop species including maize, barley and wheat
genomic DNA.
[0081] QPCR reaction mixtures were prepared to a volume of 20 .mu.L
volume per reaction. In the case of Gd DNA alone, these consisted
of 10 .mu.L iTaq.TM. Universal SYBR Green.RTM. Supermix (2.times.)
(Bio-Rad), 1 .mu.L each of forward and reverse primers (final
concentration of 10 .mu.mol), 7 .mu.L SDW (sterile distilled water)
and 1 .mu.L DNA template at the required concentration.
[0082] Primers used in this case were as set out in Table 5.
TABLE-US-00003 TABLE 5 SEQ ID NO Sequence Type 58
AGGAGGCTCTTTCTTTGGAAGC Forward 59 AAGTGCCCCTGTTATCGTACAC Reverse 60
TGGGTCATCGGTTCTGATTTCC Forward 61 TAGTTTGATGTCGGGTGCTGAG Reverse 62
GCGAATACCGGTCTTTTTACGC Forward 63 ATGCAAGCTCCGGATTGAGAG Reverse
[0083] The primer set represented by SEQ ID NOs 58 and 59
(designated P5) was aimed at amplifying a 149 base pair region of
SEQ ID NO 3, the primer set represented by SEQ ID NOS 60 and 61
(designated P8) was designed to amplify a 104 base pair region of
SEQ ID NO 6 above, and the primer pair represented by SEQ ID NO 62
and 63 (designated P17) was designed to amplify a 130 base pair
region of SEQ ID NO 10 above.
[0084] Thermocycling was carried out using a CFX96 Touch.TM.
Real-Time PCR Detection System from Bio-rad. Initial denaturation
was performed at 95.degree. C. for 3 minutes; amplification was
performed using 40 cycles of denaturation at 95.degree. C. for 5
seconds followed by 60.degree. C. for 30 seconds (plate read post
each amplification).
[0085] All of the primer sets amplified Gd DNA with good efficiency
as set out in Table 6, which shows the average Cq values of three
replicates of the amplification, and quantitatively as illustrated
by FIG. 5A-C. The percentage efficiency was calculated using the
formula % E=[10.sup.(-1/slope)]-1.times.100.
TABLE-US-00004 TABLE 6 P5 (SEQ P8 (SEQ P17 (SEQ Log ID NOs ID Nos
ID NOs 62 + Dilutions 58 + 59) 60 + 61) 63) ng/.mu.l -1 19.23 19.68
19.96 12 -2 22.75 22.55 23.39 1.2 -3 26.19 26.10 26.78 0.12 -4
30.23 29.97 30.75 0.012 -5 33.64 32.85 33.96 0.0012 -6 36.15 36.26
37.04 0.00012 -7 NA NA NA Slope -3.4659 -3.3613 -3.4594 %
Efficiency 94.32 98.38 94.57
[0086] The quantitative amplification was carried out in the
presence of genomic plant DNA in order to determine whether there
was any cross reactivity. It was found that whilst there was cross
reactivity with some plants species, the primer pairs P5 showed no
cross-reactivity to wheat and barley genomes, P8 showed no
cross-reactivity to wheat barley and maize and P17 showed no
cross-reactivity to wheat and maize genomes making these
potentially suitable primer sets for detecting Gd in crop
species.
[0087] To ensure that primer efficiency and robustness would be
maintained in the presence of plant genomic DNA, the above QPCR
examples above were repeated but in this case, the composition was
varied in that 6 .mu.L SDW (sterile distilled water) was used
together with 1 .mu.L relevant Gd dilution DNA template and 1 .mu.L
plant genomic DNA template. For instance, Gd DNA (92 ng/.mu.l) was
serially diluted from 10.sup.-1 to 10.sup.-7 with either wheat DNA
(70.6 ng/.mu.l) or maize DNA (111 ng/.mu.l) and amplification
reactions run as described above.
[0088] Representative results are shown in FIG. 6A and FIG. 6C and
in Table 7.
TABLE-US-00005 TABLE 7 Gd + Plant DNA QPCR Std. curve Cq Value from
QPCR run Log P5 (Seq ID P17 (Seq ID ng/.mu.l in dilutions 3)_Wheat
10)_Maize reaction -1 21.47 21.76 9.2 -2 24.56 25.11 9.2 -3 27.69
28.13 0.92 -4 31.38 32.08 0.092 -5 34.89 35.32 0.0092 -6 37.55
37.80 0.00092 -7 N/A N/A 0.000092 AzGd DNA 24.20 25.00 (0.01) Plant
DNA N/A NA NTC NA NA Slope -3.2887 -3.2794 % Efficiency 101.41
101.81
[0089] It appears that the primers will maintain efficiency in the
presence of plant genomes and thus may form the basis of a
detection kit.
[0090] Results were confirmed by carrying out melt analysis post
amplification the denaturation curve (Melt curve) analysis was
performed from 60.degree. C. to 95.degree. C. with 0.5.degree. C.
increment 5 seconds/step followed by plate read after each
increment.
[0091] Representative examples of the results are shown in FIGS.
6(B) and 6(E). Clear melt curves are visible for amplified Gd DNA,
without plant genomic DNA.
Example 6
Plasmid Detection
[0092] Plasmid DNA extraction from Gd (IMI504958) was performed
using Qiagen mini prep kit (Cat. No. 69104). The low copy number
plasmid extraction protocol was followed using 5 ml and 10 ml 48
hour bacterial culture. The extracted plasmid was run on 1% agarose
gel flanked by a 1 kb ladder (FIG. 7) and imaged.
[0093] Alongside the plasmid DNA, genomic DNA of Gd (IMI504958) was
also included on lane-1. The results, shown in FIG. 7 indicate the
presence of a single plasmid of about 17.5 Kb in size, which is
smaller than that reported previously for plasmids found in
PAL5.
[0094] The plasmid DNA was sequenced and a primer was designed
using Primer3 to cover the start and end sites of linear sequence
data (P_End_Fw--CCAAATCTCTGGAACGGGTA (SEQ ID NO 64). Sangar
sequencing was performed using this primer (SEQ ID NO 64) and the
sequenced data was aligned to confirm the plasmid sequence was
complete.
[0095] Since plasmid DNA in its natural form is circular and can
form secondary and tertiary structures, this may impact on the
accuracy of size measurements using agarose gels. To confirm the
results and also validate the sequencing of the plasmid DNA, a
restriction map of plasmid was studied using NEBcutter. The
restriction digestion will linearize the plasmid providing only a
single conformational structure. Also, the restriction enzyme
selection is done after studying the sequence, thus allowing the
plasmid sequence to be validated as well. In case of IMI504958
plasmid DNA, the NEB-cutter showed the restriction enzyme EcoRI to
digest the plasmid DNA at 3864-9461 bp and 9462-3863 bp producing a
DNA fragment of 5598 bp and 11968 bp. Both the size can be studied
using a 1 Kb ladder available in the lab, removing the limitation
of the reference ladder's maximum size detection as well.
[0096] Therefore, restriction digestion was performed on IMI504958
plasmid using double cutter EcoRI as per supplier's protocol. Post
restriction digestion the products were run on 1% agarose gel until
the bands were resolved and imaged. IMI504958 plasmid DNA when
restricted with EcoRI produced two fragments (1. .about.12 Kb and
2. .about.5.6 Kb) of DNA of predicted size (FIG. 8).
[0097] This validated the sequencing data in terms of both size and
sequence. It may further provide an identification test or a
confirmatory test in relation to the kit of the invention.
Example 7
Illustration of Activity of IMI504853
[0098] A field trial was designed to test Gd (IMI504853) as a
bio-fertilizer using wheat (cultivar Mulika). Two plots of Gd
treated and control (untreated) respectively where planted. Post
germination the young wheat seedlings at 10-12 day of growth were
sampled and tested for Gd presence using the primer G (seq ID 26
& 27) representing the DNA seq ID 7. The Gd presence was
detected when PCR was resolved on a 1% agarose gel with respective
negative and positive controls (FIG. 9) confirming that in real
world condition the kit of the invention works well.
[0099] The measurement of chlorophyll content i.e. "greenness"
using a SPAD meter has been shown to correlate with over all plant
health and crop yield. The crop at growth stage 35 and 61 were
checked for its chlorophyll content using SPAD was found to be
statistically significantly (P=0.001) in Gd treated plots when
compared to control plots (FIG. 10). Interestingly, the SPAD showed
a significant increase in the chlorophyll content from the wheat
obtained from plots treated with Gd identifiable using the kit of
the invention compared to untreated controls (FIG. 10). This
indicates that Gd treated plots which have been confirmed to have
the bacterium present using the diagnostics kit results in much
healthier plants and potentially higher yield. The data from wheat
field trial indicates the efficacy of Gd as a bio-fertiliser.
TABLE-US-00006 TABLE 1 SEQ ID 1 Glutathione S-
ATGACAAAATTATACTATTCTCCCGGCGCTTGCTCTTTGGCAGGGCATATTTTGCTCGAAGAGTTGGGAAGAC-
C transferase (EC
ATATGAGTTGAAATTGACGCCCGTTGGAGACGAAGGCACGGGAAGTGAAGAGTTTCTAAAAATAAACCCGCGA-
G 2.5.1.18)
GAAGGGTGCCTGTTTTAATTGATGGTGCGGAAATAATTACCGAAAGCCCCGCAATCTTATTTT-
ATTTATCGAGT
TCATTTTCAGACGGAAATTTCTGGCCAAAATCAGTTTTGGAGCAAGCCCGCTGCTGGGAATGGTTTAACTGG-
TT
ATCGAGCAATGTACACTCGGTTGCCTATGGGCAGGTGTGGCGACCAGGACGGTTCATTGATGATGAGCGTCA-
GT
GGAATAATGTTATTTCAAAAGGGAAAAATAACCTTCATGAATTTAGTGATGTAATAGAAAATAATATCTCCG-
GG
AAAACGTGGTGTGTGGGTGAATCGTATTCATGCGTTGATCCGTATTTGTTTGTTTTTTATTCTTGGGGGAAA-
GC
CATCGGATTGGATATGGAATCCTCTTTCCCGGCGTGGTCGCGTCATGCAGCGCGGATGCTGGAGCGGTTGGC-
CG TTCAAAACGCTTTACGGCAAGAAGGTTTGATCTCGTAA SEQ ID 2 O-
TTGGATGCCTCTCGTTTTCCTTGCGGAGTCATCATGACCATTCCTCTCTTTCGTCCGCAATTCACACCGCAGA-
T methyl-
TCAACGTGCGCTTGATCGCCTTTATTCCGAGACACTCTCGCAAGATCCAGCGATACGCCAATTGG-
CGCAAGCCA transferase
AAGGACTGACACATGACGGGCAACGCGGTTTCTACGAAGCCATGAAAGATGCCAGACTACCCGTTACGCCAGA-
G (EC 2.1.1.-)
TTCGGCGCCCTGCTCTATATTCTGGCACGCAGCACCAGAGCCCAACATATCATCGAATTCGGCACGTCCTTCG-
G
TGTTTCAACATTATTTCTCGCAGCGGCTTTACGCGACAATGGCGGGGGCCGACTGGTGACCTCGGAACTTAT-
CT
CAGACAAAGCAGAAAGGGCTTCCGCCAATCTGCGGGAGGCAGGACTGGCAGACCTCGTAGACATTCGCATCG-
GA
GATGCCCGCGCCACGTTATCGCGTGATCTTCCTGAGTCGATCGATCTGATCCTGCTGGATGCGACTAAAGGA-
CT
CTACCTCGATCTCTTACTCCTGCTGGAACCTGCATTACGAAAAGGTGGCCTGGTGATCAGCGATCGCGCCGA-
TC
TCGATGGTGACGACGGCGGTCGCGCAGCAGCCTACCTTACCTATCTGACAACCCCGGCTAACGGATATCGCA-
TC GCCGGCATCACTACACAGGCGTTGGGACAAACCTTCGCTCACGATGTGGCGGTGCGCACCTGA
SEQ ID 3 Transcriptional
GTGGGAATAGCCACGCTCTACCAATACTTTGAGAACAAGGAGAGCGTTGTCGCGGCACTTAGTCGTCGGGTAC-
G regulator, TetR
GGAAACACTGCTCCATGATGTTGCGTCATTACTCGAAACCGCTTGTTCGTTGCCACTTTCCGAGGGTGTGCGC-
T family
GTCTGGTCGTCGCTGCCGTGAAGGCGGACAAGAGCCGTCCATCGCTTACGGTCCGGCTTGATCGGT-
TGGAGGAG
GCTCTTTCTTTGGAAGCGGATCATCTGCTGGTAGCGGCTGAGCTTTGCACGGTTGTTGCGTCGTTCCTCAAG-
TG
CCAGGGAATTATTCAGGAGAACACTGCAAAAATTCTGGCAGATGATCTGTGTACGATAACAGGGGCACTTAT-
TG
ACGCTGCACACAATCGACAGATACCTATCGATGACTTGCTAATTGACCGTATTACGCGAAGGCTGGTCGCGA-
TT ATTCAGAGCGCGCTTTAA SEQ ID 4 RNA polymerase
GTGGGACAGCCGGACAAATATTTCGAGCTTTTCGCGATACATCGCACCGATCTTGTGCGTTTCGCCAGAGGTA-
T ECF-type sigma
CATGAGAGATGATAGTCTGGCGGAAGATGTTGTACAGGATGCTTTTCTGCGGCTGACTACTGTAACAGTGGCA-
C factor::RNA
AGGACCGCGTTCTTTCGGATCCTCTGAATTACGTTTACCGCATTATTCGGAATCTGGCCTTTGACCGTTATCG-
A polymerase ECF-
CGACGGCAATTCGAGGCCGGATTGTTTGACCATGGGGTAGATAGTTCTTCCGAAACAATCGAAGCGGATGCCC-
C type sigma
TACACCGGAAGGTGAGGCTTCAGGGAAATCCGACATGCGGGCAATGCGCGCCGCTATGGCGGAACTGCCAGAA-
C factor
GGACGTGCGTCGCATTGGAAATGCATCTGTTCGACGGACGAAAGCTACGGGAAATAGCGGCTCATT-
TAGGTGTT
TCTATTGGGATGGCCCACTCCCTTGTCGCAGTGGGTATGGAGCATTGCCGCAAACGTCTTTCCACACCTGAA-
AC CTGA SEQ ID 5 FecR family
GTGAGCGAAGACTTCAATCCAACAACGGCGGTTGAGTGGAAAATAGCCCTTTCAGAAGAGCCTGACGATTCTG-
T protein,
GCTCAGAGAACGCTTTGAAGCATGGCTTGCTGCCGCAGAGGATCACCGGAAAGAGTGGCAGGAA-
CTTACGCAGG COG:Fe2+-
GACTTGAGAATTTCCGCCAGATTGGCCCGCTTTATCGTGAGAAATGGGTGCCTTCATCAAGTG-
GGGCACAAAAT dicitrate
ACTGCGTCAAAACAAGGTAGGCTCAAAGGAAGACCTGCTAATTTTGTTAGGTTTTCAGTTGCT-
GCATTTGCGGC sensor, membrane
TGCTGCTGCCGTTACATTGGTATGGTCCTCTGACCTTCTGCTTCGATTACAGGCCGATTACGCCACAGGCTCG-
G component;
CAGAAACGCGAACAGTCAGTCTCCCCGATGGTAGTGAATTGACCCTCGCGCCGCGAAGTGCGGTAAAAATGTC-
T
TACTCTGTAGAGAAACGGGATATTCGTCTTTTAAAGGGAGAGGCGCTCTTCACGGTTCGACATGATATGGCG-
CG
ACCTTTTGAGGTCCACACAGACAAATTCACCGTAACGGACATCGGAACTATTTTTGACGTCAGAATGTCTCA-
GG
GCGAGGAAGAAGTCTCTGTCCGGGAAGGAGAGGTCCGGGTGCAGGATGTTTCCGGTGGATTTCATAATCTCG-
AT
GCCGGAACGTGGGAGCGGATTAGAACTGTAGGCAATGGAGTGAGCGTCACTCATGGGAGCGGCTCTCCGGAA-
GA
TGTGGGCGCATGGTCAGCGGGGCAAATTATTGCCAAGGAAAACAGCGTGTCCAGCGTCGTGGAAAGGCTTAG-
GC
CCTACTACCGGGGGGTTGTTGTCCTTTATGGTTCTTCCTTTGGGGAGAAGTCACTCACTGGTGTTTATGACG-
CA
AGTGACCCGGTTGGCGCATTTCGGGCGATCGCAGCCGCGCATCATGCTCAGATGCATCAGGTTTCGCCATGG-
CT GACAATATTGGCCGCACCGTAG SEQ ID 6 Ferrichrome-iron
ATGAAGGGTGCGGTTGCATTGCATTCGCAATTGTGGCGGCTCATACGAATGGGAACGGATAAGGTGATGACGA-
T receptor
TGATGATAGAATGAAGCGGTGTGGGCGGCAGGTGGCGTGGCTTATCGCGCTGGGTAGTACGACG-
TTTCTGAATG
CCGCTGTGACGAAAAGCTATGGTGCAGAACCTTCCCAAAGTGCTCGGGCCGTCAGATCATTTTCCATTCCGG-
CC
CAATCTCTTGAAGATGGTCTCGCAAGGTTCGGACAGCAAAGTGGGTGGCAGGTTTCTGTTGACGGAAATCTT-
GC
AAAATCTCTGACAACGCACGGTGTTAGCGGTACGATGACATCTGCTCAGGCCCTCAATGCGATCCTGTCCGG-
GA
CTGGCCTGACATACACGATCAGGGGTGGCCGAACCGTCGTGCTGACGAAAGCAGTAGCCAACATCACGCTTG-
GT
CCGGTCCGTGTCGGAGGAACCCTCGCGCGTCAGGATCCAACAGGGCCGGGTGTCGGCTACTTCGCCGAAAAC-
AC
AATGGTTGGTACAAAGACGGATACGCCCATCACGGAAATACCGAACTCAGTCTACGTCGTGACCAAGCAGTT-
GA
TGACCGATCAGCAGCCGCAGAATATCCTACAGGCTTTGCGTTACACTCCCGGCATCTACTCTGAAGCCGGAG-
GA
ACGACAAATCGCGGATCTGCCCAGAATGACAACATGGGCATTTATCAGCGTGGATTTCTCTCGAGCCAGTTC-
GT
GGATGGGTTGATGACGAATTCGTATGCCGCCGCCGAGCCAAGCTTTCTGGACCGTATCGAGGCGCTCAACGG-
TC
CAGCATCGGTGATGTATGGCCAGACGACACCCGGAGGAATGGTCGGTATGAGCCTGAAGAAACCCACCGAAA-
CG
CCGCTGCATCAGGTTTCGCTAGGCTTCGGAAGCTGGGGACGGTACGAGGCAACGTTCGATGTCAGCGATAAG-
AT
CACGCAGTCCGGTAATCTGCGCTATCGTATTGCAGCCATCGGAGTCACATCGGGCACTCAGGAAGACCGAAT-
TG
ATTATCATCGGGTGGGTGTACTTCCTTCAATCACGTGGGATATCGATCCCAAGACTCGCCTCACTCTACTTG-
GT
AGTTATATGTACACGCCTGGCTCAGGGAGCACCAGTGGTTATCCGGTCCTCGGGACTCTTATTCACAATTCG-
GA
AATTCCACGAATCTCACGAAGCACATTTATCGGAATACCTAGTTGGAACACTATGGGAGATAAGGTCGGGAT-
GT
TCGAATATCAATTTAGTCATAAATTTAATAAATTTATTGAGTTCAATCAGACGTTTCGCGTAGAGAATTCCA-
AC
GTTCATGAGTCAAATATCACCGATGTAACACCTGTAGATGTTGAAGGAAAATGGACATATTTTTATCCTTGG-
AA
ACAAAATTATGAAAACACAACTGAGGTACTTGATACTCGCTTAGGGGGGCGGTTTCTAACTGGTCCTGTACA-
AC
ATACATGGGTCATCGGTTCTGATTTCCGCAATTATGACTATCATTATACTGAGCTCATCGACGACGGTGCGA-
CA
ATCGTTGTGCCCACTCAGCACCCGACATCAAACTATTCCCCATGTATAAGTTTAACCTCCGCGAAGTGTGAC-
GC
CTTCGCGGGAATAAACCCAGACTATAACTCGTTTCAGGAGGGCGTGTATTTTCAGGACCAGATAAAATGGCA-
GC
GCCTGTCCGTTCTCTTGGGTGGACGCCAAGATTGGGTTAATTCATCTAATAAAAATTACAGTGTAACGAACT-
TT
TATGGAAACGTCAGCACCCGCGTTAATAACACTGCTCCACACCCTCAATCGGCCTTCACCTGGAGAGCTGGT-
AT
AATCTATAATTTTGACTTTGGGCTTGCCCCGTACTTCAGTTACGCAACATCCTTTGTGCCACAAGGAGGTAC-
GG
ATTGGCAGGGTAAGATTTTCGCGCCTTTGAGCGGAAAGCAACTCGAAGCCGGGTTGAAGTATAAAGTTCCAA-
AC
GAAGATATCCTCATAACGGCATCAGCATTCCGAATTGATGAAGACCACTATCTTATCAGTGATCTTGTTCAC-
AC
GGGCTTTAGCAGTGACGCGGGAACGGTACGCTCGCAGGGTTTCGAGGTTTCCGCCAGTGCGAACATTACCAA-
AA
ACCTCAAACTTGTCGCCTCTTACACATATGAGGATGTGCGGTTCAGAAAGAACAATTTGGCCGTAAATTCGG-
TC
GATCCCGTCACGCTAACATATGGAGCAAAGGTAAGCGAGAATGGAAAATTCGTTCCTCGAGTTCCTCGGAAT-
AT
GTTTAATATGTTTCTTGATTACACCTTCCACGACGCCCCATTGAAGGGTCTCGGCTTTAATGGAGGAATTCG-
CT
ACACCGGTTTTACCTATGCGGACTATGTGGAGTCTTACAAAACGCCGGCGTATTATCTGTTTGACATTGGCG-
CA
CACTATGATTTTGAGGAAATAATCCCTTCTCTCAAAGGTCTGCGTGCCCAGTTGGCAATCTCAAATTTGGCC-
AA
TAAATATTATATTACTTCGTGCAATACCGCCATATGTACTCTCGGTTATGCTCGAAAGTTTTACGGTAACGT-
GA CGTATAGCTGGTGA SEQ ID 7 Reverse
GTGACGCCCGAATTGCTCCTCTCCAAGGTGCGGCTGCTGCGGTCGCCCAATGACGACGGCGCGTTCTTCGACC-
T transcriptase
AGTCGGCAGTGTTCTTAATTGGTCCTGGGAGGAAAGAGACGAACGTCAATTCGCCCGCTTCAAGCAGCGCGCG-
G family protein
GCATCCCTGAGTTCGATGGCGTCGCCCTTCCACAGGGTTTGGTTGCAGCTGGCTTCTTCTCGAACATCGTGCT-
G
CTTGATTTCGATCGGATCGTCATCGGACAGATTGGGAGAGAAGTTACAAACGGAGTGTGGCTCCGGGACGCC-
TG
CCGGTACGTCGACGACATTAGACTGACCATAACAACTGCACCAGGTATTGACCCAAGAGAAGCTCAGGCGCG-
TG
TAATGGCGTGGCTTGGGCAACTCCTCACGGGGAGCTGTCCGGGCTTGGAATTCTCCCCGGAGAAGACGTCAA-
CT
GCGTCGGTTGGAGGCGAGCAGATGCCGCTGGTTCGCCAATCCCGAAAGATGGAGCGCATCCAGACCGCGATT-
TC
CGGCGGCTTCGATGCCAGTGGTGGCGAGGAGGTGATCCACGCGATCGAAGCCCTCGTCCGATCCCAGCTAAC-
GA
TCAACAGCGTCGAAGAGTCGCCTACCCCTCCCGGCTTGAGAGCGGTACCCGATGTCAAAGACGAGACAGTCG-
GT
CGTTTCGCTGCTGGTCGGTTCAGAAAAACCTTTCGTTCATTGAGGCCACTACTCGATGATCGACCTTACATG-
GA
GATTGCTGAATTCGGGGAGGAGACGTTCCGGCGCACCCGACTTTCGCAATCGGAGCTTGACGAGGAAGCACG-
CG
CATTTGCGCTAATCCTAGTCGAACGGTGGATACTCGATCCTTCGAATGTGCGGCTGCTGCGCGTCGCACTCG-
AC
CTCTGGCCGTCCCGCCAACTCCTCAAGGAAGTACTGAAACTCTTTGAGCCCTATCTTGTCGGGAAGATCAGG-
GC
AATCACTAGCCGGCAAGTTGCATACTACTGCCTCGCCGAGATATTTCGAGCAGGGGCGACCGAGACGGGCTT-
CA
TTGACGATCCAGAGTGCCTTCCCGCTGCCGTCGATCTCGCCGGTTATAGATCTCTGCTTCTGGAGGCCGCAG-
TA
CGAGTGGCCCGGGGCGAAGCCGAACGTGTCCCGTGGTATCTCGCGCAACAAGCACTGCTTTACATTGCGGTC-
CA
CGATCCCCGGGCTATCCAAGATCGAGGAATTTCAAAGACCAATCGATCCTATTGGCGCCTCGTCTCATTTCT-
GA
AAGGCGAACGCGACGTCTCTTCAGATCGCGAATTCGCAGTAGCCGCGGTGGTGAGCCGCAGGTCGTTCCTTT-
CG
AATGATCAGGCCGTGGATCTCGTCGGTCGGATGCTCACGCCAGAGCGGTTCGCCGAGGTGGCCGCGCGCGAC-
AT
AGAATTCGCCCGCGATCTCTTTCGCGCCGTCGACCGACACCTCACCGTTCCGGCAGGCATTGCCGAGGACTT-
GG
GGGTCGCCGAATGGTCCATGTCAGAGGAAATGAGCTCTCTGCAAAGCTATATCCAAGGCAAAGGGCCTCTGA-
AT
CCGCTACGCAATGAGATCGGCGTACTCAGTTTTGCAGAGAAATTCATCTCCCATCTCCAAGAAGGAAATTTG-
CC
GGAAGTCGTGACGCCGTCGACGACGCAGATAGCGGTACAGCAAGTGGGCAAATATGTCCGCGTCGAACGGGT-
GA
TCTTCAGATCGGCCCAGACAACGCCGACTTACCGGTCTATTTATACTGCTCCCAGATGGGCGCCGGAATCTC-
AA
CGCTGGCGCTTTCAGCTCGGTTATTTACTTCGCTTCATTCTTACTGCCAGAATAGACTTCAGCCTTCCAGTT-
AG
GCCGCCATCGTGGAAGGAAGGTAAACACATCTATCGGCCTACCAGAAGTCACTGGTTTCAGCGGCAATACGG-
CT
TCTATAATGGGCATGAGGCCTTCGGGGACGATTGGCTACCCATTTCGCAGTTCACTCAGGATCTTCTCTTCG-
AT
CTGCTCACCTGGCCCGGCTGCCGCACAAGTAGCCCGGATGTCGATCAGTTGTCCCTGGATGAAACGGCTGCT-
CG
AATCCGCGCAGCTCTCGTAGAAGCCACCGCTGCGATTGGCCCGGCTACAGGAACCCTGATGCTCAAGATCGA-
TG
CACCTATTCCAGGTACCACATCGAAGGGGCGCCCGCTTCGGGCCTGCGTCGTCCAGTCGATCATGCCCGAAG-
CA
GAGGACTTCTCGGCTGCCGATCTGGAGATGCGCTCGCCGGCCCTTCGACGAAAGCACCGCAAACATCTGTCC-
AC
GGCATTGGCGGCGGTTGAGAAGATGTTGGATCTTCGCGAGACTCATAAGCCAGCCAGCAAGCGTCTCGACTG-
GC
TCATCCTACCAGAACTGTCTGTTCACCCGGATGACGTTGCCACCCACCTCGTGCCGTTCGCGCGAGCGTTCA-
AG
ACCGCGATCCTGGTCGGCATGGCCTACGAACAAGTCGTCACGGGAGAGCCGCTGATCAACTCGGCCCTCTGG-
AT
TATCCCGAGGATGGTGCGGGGCATGGGCCTACAGACGGTGATCAGACGGCAGGGAAAACAGCACCTCTCTCC-
GA
TGGAACAGAAGTACGTCAAACCGGTCGAACTGATCACCGGATTCCGCCCGTGCCAGTGGCTGGTGGGGTACG-
AA
TGGTCGAACAATCCGGCCAAAGACGCACTTTGGCTCACCGCGTCCATCTGCTACGATGCAACAGACCTGAAG-
CT
GGCGAGCGATCTTCGTGATCGCTCAGACGTGTTTGCGATCCCAGCCCTGAATCTCGACGTCGGCACCTTCGA-
TC
AGATGGCGCAGGCGCTGCATTATCATATGTTCCAACTCGTGCTGATCGCGAACAACGGAGCTTATGGGGGCA-
GC
AATGCTCACGTTCCCAAGGGGGAGGCCTATCAACGCCAAGTGTTCCATACCCATGGCCAGCCCCAGGCTACA-
AT
TTCCTTTTTCGAGATCGACGATATCGAGGGCATGAAGCAGAGACACAAGCTCGGCGCTGGGAAGGAAGGCGG-
GT GGAAATATCCACCTGCCGGCTGTCAAGTCTGA SEQ ID 8 DNA topology
TTGATGCGCTTGTTCGTGACGGGGCCAACTGGCAGTGGAAAATCAACGCTGGCTGCAAAGTTGGCTCAAAGGG-
C modulation
AGCTATACCACTGTTCCCGCTCGATGAAATTCATTGGGTTCGCCATCTCTCCGGGGATTGGCGGCGCGATCCT-
G protein
TTGAACGCCTGTCTATGCTCGGAGAGATTGTACAGCTCGATGCCTGGGTCATCGAAGGCGTGCAG-
TTCAAATGG
ACTGATATAGCGATAGAACGAGCAGACTGGATCGTCGTCCTCGATCCACCACGTTGGCGGAACATCGCTCGT-
AT
CCTGCGCCGTTTCGTCAATCGCCGATGCTTTTCTGGGGCGGGGCACCGTGGAACGGTAAAGGCTCTATTGGA-
GG
AGATGCGTTGGTCAGCCGACTACTATGGTCATGAACGCGGTATGCTGTTCGAGAAGATTGGACAATCGCCAG-
AC
AAGCTCATCGTCGTACATGACGACAAGGGCGAACGCGCTTTGACCGAGGCTGTATTCGCGACTGCGTGA
SEQ ID 9 Cycloisomaltooli
ATGGCATATTGGATCAGGCTCTCGCTGGCCGTGTGGCCGCCCGATCAGCAACGTTGTAGCGAAGGCCGCGTAA-
T gosaccharide
GCGCCGCTATCTTTTCACAACCATTCTCTCGCTCTTACCGTCCCTTGCGGCGGCGGCATCCCTCCAAGGTCCG-
A glucano-
TTGTTTCGCATGTGCGGGATGATCGGGCTTTCTACCAGGCAGGCAATGTCGCGATGATTTCCGT-
GGAACTGACC transferase
CCACTAGCCGCTTGGACGGGAGGCCATGTGGATCTAGCGATATGTTCGCGTGGGCAAGTCGTGGGCACGATTC-
A precursor (EC
GAGCCAAGCGGTCACCAGCATGGTGGCTGGGGCGGACCAGACACTTCACTATCCCGTCACCGTTCCCAGTCTC-
C 2.4.1.-)
ATGCTCATGGGTATCAGTTGGCTATCGCGGCCCTGAACAATGGGGACAGCGGGACAGCGTCCTG-
TACCGGGACA
GGCAGCACTTCCACGTCGCCGGCCGATGTGGCGTCAGGCGGCATCAACGTGGCCGCGAATGCCTGGGAAGAC-
AT
CAACGAAGCATGGGTCGACGCGCCGACGCTCGGCAACGTCTCCGCGGCCCGGGTGATGGATAATCTCAGCCA-
GT
ATCACATCAACGCGGTGCAATTTTACGACGTGCTGTGGCGACATGACGAGCCGTATTCATCCGCCCCGCAGT-
GG
CCGAACCTGGAAGGCGTAACGGTCACAAGGACCAATCTTCAGGCTTATATCAGCGCGGCGCATAGCCGCGGC-
AT
GGTGGCGCTCGCCTATAATCTCTGGAACGGAGCCTGGGCGGACTATGCAACTGTCAATCCGAAGGTCACGGC-
GG
CAATGGGGCTCTATGCTTCGTCCGGACAGAAACACCAACTGACCAACGGCGGGGGCTGGCTGTCCTGGGGGT-
GG
TCGACCGACCATATTGCTGAAATGAACCCGTTCAATGGCGACTGGGCCAGATGGCTAACCAGCCAGATCCAG-
AA
GACCATGTGGAATTTAGGATTCGACGCCGCGCATCTGGATACGTTGGGTGACCCTGGTGGTCAGCAATATGA-
CG
GCGAGGGCCATCCGTTACCTGCTTTAGGAACGATTCTGGCAGACTTTGCGAATAATGTACAGGCTCAGACCG-
GG
GCACCAACTGACATCAACGCCGTTTCGGGTTGGAATACCACCGACCTTTACCTACGCGGTACGGGACCCAAC-
CT
GTATATCGAACCCCATCCCGAATTCGGAAACACGCCGGGCTACGATGATTCCCGAAGCTTATGGGACATCAA-
AC
AGAAATATACGTCGCGCCCGCTGATGACGGCGTTTTATCCGCAGCAGGTCCAGAGCGGTTCGCTGAGCACGT-
CC
TTTGCCGTCAAGGGTGAGAGTGTGAAGGTTTGCGACCCGACGTTAAAATCCGGATGCATAGCCAATAACCTC-
GG
CATTGAGTTGTTGCTCGGCCAGATTGCGCTCAATGGAGGCTCCAATATTACTCTTGGTGATTTTGATCATCT-
GA
TACCGGGGCCATATTTCCCCCGTCCGACCCTTAAGATCGACGGTCCATTGCAGCAATATCTGGCGGATTACT-
AC
AACTGGTGGGTCGGAATGCGCGATCTGCTGCGTGTCGGCGTCATCTCATCCAATGAGAGGGAGTCCATCCGG-
AA
TGGAAACGGAGCCAGTATCGGCCAACCTTATGCCCAACCGGGAACCGTCTACTATCATCCCCTGATACGCGC-
TG
GCATCGCTGGTGAATTGGCGCTCACAAACATGATCGGGTTGCATTATAATCGGATTGACGACCCTGACGGCA-
AA
AACAATCCGACCCCGGTGAACAACCTGTCGATCGAGATGGAATTCTGGGAAAGAAGCACACCGGGGGCATTG-
TA
CTATAGCGCGCCCGACATCAACCACGGCTTCCCACAGCCCCTCACCTATAGGCTGAACGGAAACGGTAGCGT-
GA
TGTTTACGCTACCGACTCTCAAGACGGTGGCGCTTGTTTGGCTGGAAGGCACCAATTTCACCACTACGACCG-
AT
TACACGATCGGTACGGCGCAGGATGTGAGGGGTGGCACAGCAAACTTCTGGACGAACGGCAGCGGAGAGGAT-
GC
TACCGGATATCGTGGCTGCTGTGGTCGCTCCGCACGCTGGGACAGCATCGATTTCGGAGCGGGTGTGTCAAC-
GC
TAACGATGGTAACCCGAAGCCAACTCGGCGGACTGGTCGAATTTCGCCTGGATGCACCTGATGGACCAGTCA-
TC
GCCCGTAATTATGTTCCTGCGTCTAGCGCCACGACAACAACCACTCAATTACGCAGGCCAGTATTCGGGACA-
CA TACCGTCTTCGCTAAAATTCCTGGTCGCGAGATTACGCTGATATCCTGGAAGCCATAA SEQ
ID 10 Methyl-
ATGATGGCTAACGACAATACCACTGAGGTGGTTGGTGCATTTGCGGTAAGTCATCCCAACTTGGCGCAAGGTT-
T transferase
TACTTTTAGTAACAGCAGTCAACTAGATACGATTGCTTCTACTATTCATAAAAGCGGTTTGGAGACTTATGAA-
G (EC 2.1.1.-)
CTCCGACAACTAATATAATTATCGAACTGATCAGGAGTTCGTCTGGTCTTATTTTAGATGTGGGAGCGAATAC-
C
GGTCTTTTTACGCTAGTCGCCGCAGCAGCCAACCCCCTGATCCGCGTCTGCTCTTTTGAGCCGCTTGCGAGT-
AT
TCGTGAACTTCTCAAGAGCAATATTGCTCTCAATCCGGAGCTTGCATCACGTATCGCTGTCGAGCCTGTCGG-
GT
TATCGAATGAACGGGGCACTTTCACTTTTTACGAAACGATCAACAATCGTGGCTTTGTCACGACGAGTTCAT-
CG
CTTGAAAAAGCACATGCAGAGCGAATCGGCGATTTGTACGTCGAGCGCACTATCGAGACCCGGACACTTGAT-
GA
ATTCGGAGAAACGCTCGGGAATGCGAGCGTTCCGTTCGTCAAAATTGACGTTGAGGGACATGAGCATGCCGT-
TA
TCTCCGGTGGCCGCCACTTTATCGCCAAGCACCGCCCTTTTCTTACTCTCGAAGTCCTGAGAGAGGCTAACA-
CT
TTGAGTCTGGACCAGTTGGTGACCGAGTCCAACTACCTTGCCCTGGCAATGGCACCCGACGAATTGCGGCAG-
TG
CGAGCGTTTACGGTTTCATGACGACGCCTGGAATCATCTTTTGGTCCCCGCCGAAAAAGCGGAACGGCTATT-
TT CGCTCTGCCGCCGACTTGGCTTGCAAATCGGGATCCGCTGA
TABLE-US-00007 TABLE 2 SEQ ID 11 Transcriptional
ATGTCGAATTCCGAGCGCCCAATGCGCGATTTGTCGGACCTGGCAAAAAACCGACAAATAGAGCCGATGGTTA-
T repressor;
CAGGCTACGAGAAGTAGTGGATCGGACCGGAGGCGCGAAAGCTGTGGCCGCACGCACGGACATCCCTCTCAGC-
A asserted pathway
CACTTTCAGGTTACCTGTCGGGTCGGGAACTCAAGCTTTCCGTCGCGCGCAAGATCACGGAAGCCTGCGGTGT-
C PF01381<21br>
AGTCTTGACTGGCTTGCGGCAGGAGAGGACGGACCTGCGGCCCGGGAATTCGGCAATGCCCGGCAGGCGGGTC-
C Peptidase S24-
CGAGTCGGTCGAGTTTCTGAATTACGACGTCATTCTCTCCGCCCACCAGGGCGTCGACGGGGATAGTTCTTAT-
A like::PF00717
TCGAAACGAGAATATCGATACCGCGGGATTTTCTCCCTTTGTCCATTCAGTCCAATACGGACAACATTTCGGC-
C
GTCACGGCGAAATGCGACAGCATGAATCCGATCATAGACGATGGAGACATTCTTTTAATAAGAACGGATGTG-
CA
TACGCTCACAAGTGGCAGCATCTATGCCCTGCGGGTAGAAAACACCCTTCTGGTCAGGCGTCTGATCCTCAA-
GA
CCAACGGCAACGTCCAGGTCATCAGCGAGAACCCGCGTTACCCGACCGAGGAACTGAACGCCGAGGACGTTC-
GC
AGGATGGTCCAGGACGACGGCTTTCCGGCCAGGATCATCGGCCGGGTCATCTGGCGCGCCGGTAGCCTGATT-
CC ATAG SEQ ID 12 outer membrane
ATGCGCATCGTCCTCTTGCCCTGCCTCGTCGCGACCTCAATAAGTATGTTGGCGGTTTCCGCATCCTATGCTT-
G heme receptor
GGCGGACAATAGCCCGTCGCCCCCCAGGACGAACAAACAGGCCAAATCGCGGCCGTTACATGCGCAGGGGACG-
C
GCAAAGCGGGCAGCGCCATCACCAGCCAGGATGAAGCGGTGGCTGTCGTGGGAACACGTGAGACATCGCATG-
GG
ATGGAGCAGAGCGTTACCCGTGCGACGATGGACAAGTTCGTGGCGGGGACCAGTCCCCTGCAGATTCTGTCG-
GC
CACGACACCGGGTGTCAATTTTGCCTCGGACGACCCGTTCGGCCTGGATACATGGGCGAACACATTTTATAT-
TC
GCGGCTATTCCCAAAGCCAGTTGGGCATCACCCTGGACGGTATCCCGCTGGGCGATGCCCAGTTCATCAATT-
CC
AACGGCCTCGATATCAATCAGGCGATCATCCAGAACAATATCGGTCGCGTCGACATGTCGCAGGGTGGCGGT-
GC
GCTCGATGTCATGTCCGTCACCAACCTGGGTGGCGCGCTGCAATATTATTCACTCGATCCGCGCGACAAGGC-
TG
GTGGAGACATTTCACAGACGTTCGGCAGCAACCAGACCTATCGCACGTACGTCAGCGCCCAGAGCGGCAAGC-
TC
AATCCCAGCGGGACGAAGTTCTATGCGTCGTACGCGCGCACCGATGCCGGGAAATGGAAAGGCGCCGGGGAC-
CA
GTTCGAACAGCAGGCGAATTTCAAGATCGTACAGCCGCTCGGGCGTTACGGAAAACTGTCCGGATTCTTCAA-
TT
ATTCCGAATTCGACCAGTATAATTACAGCGATTTGAGCCTGGAAATCATCCAGAAGCTCGGCCGGAACGTGG-
AT
TATTTCTATCCGAACTACAAAGCCGCGTATCAGGCTGCCGAGGGGATCTATCCCGCAGGCTATGCCAAGGTC-
GG
AGATGCCATGGACGTCTCCTATTACGATGGTGGCCAGGACCAGCGGAATTATCTTTCCGGCATCACGTCCAC-
GA
TCGACCTGACGTCCCGCCTGCATCTGAAGACGGTGCTGTACGACCAGCAATCGGCGGGGGACTACGAATGGA-
CC
AACCCCTATGTGTCGTCGCCCTCGGGCGCGCCCATGATCCAGCAGGTCGGGCACACATCGATGACGCGCGTG-
GG
CGGGATTGGCGCGGTGCAGTACCAGATCGCCAATCATTCGCTTGAAACCGGCGTCTGGTACGAAAACAACGG-
AT
ATAGCTGGGCGCAACGGTACTACAACCAGCCGCTTCTGGGGGAGGGTACGCCCCGAAGCGCCACCGGACCGT-
AC
AACGATCCGTTCGCCACCGCATACGCCATGACCTTCAATACCAACAGTTTTCAATATTACCTGGAAGATTCC-
TA
CCGTATCTTGAAGACGCTGCGGGTGCACGCGGGCTTCAAATCCATGCTGACGACGACGTCGGGCGGCGCATC-
CT
ATAACAATCCCGTCTATACGGGCCAGGACACCCTGCCCAGTGGCAGCCTGACCACCGCCAGCGCCTTCCTGC-
CG
CATGTCAGCATCAACTGGAATTTCCTGCCCCGGAACGAACTGTTTTTCGACTTCGCGGAGAACATGCGCGCA-
TT
CACCTATAATACATGGCAGAGCGGGAATGCATGGGGAGTCAATGAGATGCCCCAGAACCTGAAGCCCGAGAC-
CA
CCTTCAATTACGAGGTCGGTTATCGATATAATTCCCGCTTCGTCACGGGCCTCGTCAATCTGTATCATATCG-
AT
TACAGGAACCGGCTGGCCACCATCACCACCGGCAGCCTGGTGAACGCCCACAATACCTATATCAACGTGGGG-
AA
CATGGCGATCTGGGGTGCCGATGCCGGCGTGACGGTGCGCCCGCTGCCGGGCCTCGAGATCTTCAACAGCGC-
CA
GCTACAACAAATCCACCTATGGGCAGGATGTATCCAGCGGCGGGGTAAATTATCCCATCAGCGGCAAGCAGG-
AG
GCCGGCTATCCGCAATGGATGTACAAGGCCAACGTCTCGTACAGGTATGGCAACGCGAAGGTCAACTTCAAC-
GT
CAACTATATGGGAAAGCGATACATCTCGTACATGAACGACGCCGCCGTGAACGGGTATTGGCTGGCATCGCT-
GT
CGGCGACGTATATCTTCAAAACCATTCCCCATCTCTCTCAGCTTGAATTCAATTTCGGCGTCTACAACCTGT-
TC
AACCAGGAATATATCGGCGGCATCGGCGGGTTCTCACTGTCCGGTGACACGCAGCAACTCTTTGCCGGCGCG-
CC ACGCCAGTTCTTCGGTACGCTGCACGCACGGTTCTAG SEQ ID 13 Levansucrase
GTGACGGCGCGGTCGTGGTTGCTCTGCAATCTGAAGAGTTTCCTTCAGGAGGATGGAATGGCGCATGTACGCC-
G
AAAAGTAGCCACGCTGAATATGGCGTTGGCCGGGTCCCTGCTCATGGTGCTGGGCGCGCAAAGTGCGCTGGC-
GC
AAGGGAATTTCAGCCGGCAGGAAGCCGCGCGCATGGCGCACCGTCCGGGTGTGATGCCTCGTGGCGGCCCGC-
TC
TTCCCCGGGCGGTCGCTGGCCGGGGTGCCGGGCTTCCCGCTGCCCAGCATTCATACGCAGCAGGCGTATGAC-
CC
GCAGTCGGACTTTACCGCCCGCTGGACACGTGCCGACGCATTGCAGATCAAGGCGCATTCGGATGCGACGGT-
CG
CGGCCGGGCAGAATTCCCTGCCGGCGCAACTGACCATGCCGAACATCCCGGCGGACTTCCCGGTGATCAATC-
CG
GATGTCTGGGTCTGGGATACCTGGACCCTGATCGACAAGCACGCCGATCAGTTCAGCTATAACGGCTGGGAA-
GT
CATTTTCTGCCTGACGGCCGACCCCAATGCCGGATACGGTTTCGACGACCGCCACGTGCATGCCCGCATCGG-
CT
TCTTCTATCGTCGCGCGGGTATTCCCGCCAGCCGGCGGCCGGTGAATGGCGGCTGGACCTATGGCGGCCATC-
TC
TTCCCCGACGGAGCCAGCGCGCAGGTCTACGCCGGCCAGACCTACACGAACCAGGCGGAATGGTCCGGTTCG-
TC
GCGTCTGATGCAGATACATGGCAATACCGTATCGGTCTTCTATACCGACGTGGCGTTCAACCGTGACGCCAA-
CG
CCAACAACATCACCCCGCCGCAGGCCATCATCACCCAGACCCTGGGGCGGATCCACGCCGACTTCAACCATG-
TC
TGGTTCACGGGCTTCACCGCCCACACGCCGCTGCTGCAGCCCGACGGCGTGCTGTATCAGAACGGTGCGCAG-
AA
CGAATTCTTCAATTTCCGCGATCCGTTCACCTTCGAGGACCCGAAGCATCCCGGCGTGAACTACATGGTGTT-
CG
AGGGCAATACCGCGGGCCAGCGTGGCGTCGCCAACTGCACCGAGGCCGATCTGGGCTTCCGCCCGAACGATC-
CC
AATGCGGAAACCCTGCAGGAAGTCCTGGATAGCGGGGCCTATTACCAGAAGGCCAATATCGGCCTGGCCATC-
GC
CACGGATTCGACCCTGTCGAAATGGAAGTTCCTGTCGCCGCTGATTTCGGCCAACTGCGTCAATGACCAGAC-
CG
AACGGCCGCAGGTGTACCTCCATAACGGAAAATACTATATCTTCACCATCAGCCACCGCACGACCTTCGCGG-
CC
GGTGTCGATGGACCGGACGGCGTCTACGGCTTCGTGGGTGACGGCATCCGCAGTGACTTCCAGCCGATGAAC-
TA
TGGCAGCGGCCTGACGATGGGCAATCCGACCGACCTCAACACGGCGGCAGGCACGGATTTCGATCCCAGCCC-
GG
ACCAGAACCCGCGGGCCTTCCAGTCCTATTCGCACTACGTCATGCCGGGGGGACTGGTTGAATCGTTCATCG-
AC
ACGGTGGAAAACCGTCGCGGGGGTACCCTGGCGCCCACGGTCCGGGTGCGCATCGCCCAGAACGCGTCCGCG-
GT
CGACCTGCGGTACGGCAATGGCGGCCTGGGCGGCTATGGCGATATTCCGGCCAACCGCGCGGACGTGAACAT-
CG
CCGGCTTCATCCAGGATCTGTTCGGCCAGCCCACGTCGGGTCTGGCGGCGCAGGCGTCCACCAACAATGCCC-
AG GTGCTGGCGCAGGTTCGCCAATTCCTGAACCAGTAA
Sequence CWU 1
1
681630DNAAcetobacterGlutathione S-transferase (EC 2.5.1.18)
1atgacaaaat tatactattc tcccggcgct tgctctttgg cagggcatat tttgctcgaa
60gagttgggaa gaccatatga gttgaaattg acgcccgttg gagacgaagg cacgggaagt
120gaagagtttc taaaaataaa cccgcgagga agggtgcctg ttttaattga
tggtgcggaa 180ataattaccg aaagccccgc aatcttattt tatttatcga
gttcattttc agacggaaat 240ttctggccaa aatcagtttt ggagcaagcc
cgctgctggg aatggtttaa ctggttatcg 300agcaatgtac actcggttgc
ctatgggcag gtgtggcgac caggacggtt cattgatgat 360gagcgtcagt
ggaataatgt tatttcaaaa gggaaaaata accttcatga atttagtgat
420gtaatagaaa ataatatctc cgggaaaacg tggtgtgtgg gtgaatcgta
ttcatgcgtt 480gatccgtatt tgtttgtttt ttattcttgg gggaaagcca
tcggattgga tatggaatcc 540tctttcccgg cgtggtcgcg tcatgcagcg
cggatgctgg agcggttggc cgttcaaaac 600gctttacggc aagaaggttt
gatctcgtaa 6302729DNAAcetobacterO-methyltransferase (EC 2.1.1.-)
2ttggatgcct ctcgttttcc ttgcggagtc atcatgacca ttcctctctt tcgtccgcaa
60ttcacaccgc agattcaacg tgcgcttgat cgcctttatt ccgagacact ctcgcaagat
120ccagcgatac gccaattggc gcaagccaaa ggactgacac atgacgggca
acgcggtttc 180tacgaagcca tgaaagatgc cagactaccc gttacgccag
agttcggcgc cctgctctat 240attctggcac gcagcaccag agcccaacat
atcatcgaat tcggcacgtc cttcggtgtt 300tcaacattat ttctcgcagc
ggctttacgc gacaatggcg ggggccgact ggtgacctcg 360gaacttatct
cagacaaagc agaaagggct tccgccaatc tgcgggaggc aggactggca
420gacctcgtag acattcgcat cggagatgcc cgcgccacgt tatcgcgtga
tcttcctgag 480tcgatcgatc tgatcctgct ggatgcgact aaaggactct
acctcgatct cttactcctg 540ctggaacctg cattacgaaa aggtggcctg
gtgatcagcg atcgcgccga tctcgatggt 600gacgacggcg gtcgcgcagc
agcctacctt acctatctga caaccccggc taacggatat 660cgcatcgccg
gcatcactac acaggcgttg ggacaaacct tcgctcacga tgtggcggtg 720cgcacctga
7293462DNAAcetobacterTranscriptional regulator, TetR family
3gtgggaatag ccacgctcta ccaatacttt gagaacaagg agagcgttgt cgcggcactt
60agtcgtcggg tacgggaaac actgctccat gatgttgcgt cattactcga aaccgcttgt
120tcgttgccac tttccgaggg tgtgcgctgt ctggtcgtcg ctgccgtgaa
ggcggacaag 180agccgtccat cgcttacggt ccggcttgat cggttggagg
aggctctttc tttggaagcg 240gatcatctgc tggtagcggc tgagctttgc
acggttgttg cgtcgttcct caagtgccag 300ggaattattc aggagaacac
tgcaaaaatt ctggcagatg atctgtgtac gataacaggg 360gcacttattg
acgctgcaca caatcgacag atacctatcg atgacttgct aattgaccgt
420attacgcgaa ggctggtcgc gattattcag agcgcgcttt aa
4624522DNAAcetobacterRNA polymerase ECF-type sigma factor :: RNA
polymerase ECF-type sigma factor 4gtgggacagc cggacaaata tttcgagctt
ttcgcgatac atcgcaccga tcttgtgcgt 60ttcgccagag gtatcatgag agatgatagt
ctggcggaag atgttgtaca ggatgctttt 120ctgcggctga ctactgtaac
agtggcacag gaccgcgttc tttcggatcc tctgaattac 180gtttaccgca
ttattcggaa tctggccttt gaccgttatc gacgacggca attcgaggcc
240ggattgtttg accatggggt agatagttct tccgaaacaa tcgaagcgga
tgcccctaca 300ccggaaggtg aggcttcagg gaaatccgac atgcgggcaa
tgcgcgccgc tatggcggaa 360ctgccagaac ggacgtgcgt cgcattggaa
atgcatctgt tcgacggacg aaagctacgg 420gaaatagcgg ctcatttagg
tgtttctatt gggatggccc actcccttgt cgcagtgggt 480atggagcatt
gccgcaaacg tctttccaca cctgaaacct ga 5225984DNAAcetobacterFecR
family protein, COGFe2+-dicitrate sensor, membrane component;
5gtgagcgaag acttcaatcc aacaacggcg gttgagtgga aaatagccct ttcagaagag
60cctgacgatt ctgtgctcag agaacgcttt gaagcatggc ttgctgccgc agaggatcac
120cggaaagagt ggcaggaact tacgcaggga cttgagaatt tccgccagat
tggcccgctt 180tatcgtgaga aatgggtgcc ttcatcaagt ggggcacaaa
atactgcgtc aaaacaaggt 240aggctcaaag gaagacctgc taattttgtt
aggttttcag ttgctgcatt tgcggctgct 300gctgccgtta cattggtatg
gtcctctgac cttctgcttc gattacaggc cgattacgcc 360acaggctcgg
cagaaacgcg aacagtcagt ctccccgatg gtagtgaatt gaccctcgcg
420ccgcgaagtg cggtaaaaat gtcttactct gtagagaaac gggatattcg
tcttttaaag 480ggagaggcgc tcttcacggt tcgacatgat atggcgcgac
cttttgaggt ccacacagac 540aaattcaccg taacggacat cggaactatt
tttgacgtca gaatgtctca gggcgaggaa 600gaagtctctg tccgggaagg
agaggtccgg gtgcaggatg tttccggtgg atttcataat 660ctcgatgccg
gaacgtggga gcggattaga actgtaggca atggagtgag cgtcactcat
720gggagcggct ctccggaaga tgtgggcgca tggtcagcgg ggcaaattat
tgccaaggaa 780aacagcgtgt ccagcgtcgt ggaaaggctt aggccctact
accggggggt tgttgtcctt 840tatggttctt cctttgggga gaagtcactc
actggtgttt atgacgcaag tgacccggtt 900ggcgcatttc gggcgatcgc
agccgcgcat catgctcaga tgcatcaggt ttcgccatgg 960ctgacaatat
tggccgcacc gtag 98462604DNAAcetobacterFerrichrome-iron receptor
6atgaagggtg cggttgcatt gcattcgcaa ttgtggcggc tcatacgaat gggaacggat
60aaggtgatga cgattgatga tagaatgaag cggtgtgggc ggcaggtggc gtggcttatc
120gcgctgggta gtacgacgtt tctgaatgcc gctgtgacga aaagctatgg
tgcagaacct 180tcccaaagtg ctcgggccgt cagatcattt tccattccgg
cccaatctct tgaagatggt 240ctcgcaaggt tcggacagca aagtgggtgg
caggtttctg ttgacggaaa tcttgcaaaa 300tctctgacaa cgcacggtgt
tagcggtacg atgacatctg ctcaggccct caatgcgatc 360ctgtccggga
ctggcctgac atacacgatc aggggtggcc gaaccgtcgt gctgacgaaa
420gcagtagcca acatcacgct tggtccggtc cgtgtcggag gaaccctcgc
gcgtcaggat 480ccaacagggc cgggtgtcgg ctacttcgcc gaaaacacaa
tggttggtac aaagacggat 540acgcccatca cggaaatacc gaactcagtc
tacgtcgtga ccaagcagtt gatgaccgat 600cagcagccgc agaatatcct
acaggctttg cgttacactc ccggcatcta ctctgaagcc 660ggaggaacga
caaatcgcgg atctgcccag aatgacaaca tgggcattta tcagcgtgga
720tttctctcga gccagttcgt ggatgggttg atgacgaatt cgtatgccgc
cgccgagcca 780agctttctgg accgtatcga ggcgctcaac ggtccagcat
cggtgatgta tggccagacg 840acacccggag gaatggtcgg tatgagcctg
aagaaaccca ccgaaacgcc gctgcatcag 900gtttcgctag gcttcggaag
ctggggacgg tacgaggcaa cgttcgatgt cagcgataag 960atcacgcagt
ccggtaatct gcgctatcgt attgcagcca tcggagtcac atcgggcact
1020caggaagacc gaattgatta tcatcgggtg ggtgtacttc cttcaatcac
gtgggatatc 1080gatcccaaga ctcgcctcac tctacttggt agttatatgt
acacgcctgg ctcagggagc 1140accagtggtt atccggtcct cgggactctt
attcacaatt cggaaattcc acgaatctca 1200cgaagcacat ttatcggaat
acctagttgg aacactatgg gagataaggt cgggatgttc 1260gaatatcaat
ttagtcataa atttaataaa tttattgagt tcaatcagac gtttcgcgta
1320gagaattcca acgttcatga gtcaaatatc accgatgtaa cacctgtaga
tgttgaagga 1380aaatggacat atttttatcc ttggaaacaa aattatgaaa
acacaactga ggtacttgat 1440actcgcttag gggggcggtt tctaactggt
cctgtacaac atacatgggt catcggttct 1500gatttccgca attatgacta
tcattatact gagctcatcg acgacggtgc gacaatcgtt 1560gtgcccactc
agcacccgac atcaaactat tccccatgta taagtttaac ctccgcgaag
1620tgtgacgcct tcgcgggaat aaacccagac tataactcgt ttcaggaggg
cgtgtatttt 1680caggaccaga taaaatggca gcgcctgtcc gttctcttgg
gtggacgcca agattgggtt 1740aattcatcta ataaaaatta cagtgtaacg
aacttttatg gaaacgtcag cacccgcgtt 1800aataacactg ctccacaccc
tcaatcggcc ttcacctgga gagctggtat aatctataat 1860tttgactttg
ggcttgcccc gtacttcagt tacgcaacat cctttgtgcc acaaggaggt
1920acggattggc agggtaagat tttcgcgcct ttgagcggaa agcaactcga
agccgggttg 1980aagtataaag ttccaaacga agatatcctc ataacggcat
cagcattccg aattgatgaa 2040gaccactatc ttatcagtga tcttgttcac
acgggcttta gcagtgacgc gggaacggta 2100cgctcgcagg gtttcgaggt
ttccgccagt gcgaacatta ccaaaaacct caaacttgtc 2160gcctcttaca
catatgagga tgtgcggttc agaaagaaca atttggccgt aaattcggtc
2220gatcccgtca cgctaacata tggagcaaag gtaagcgaga atggaaaatt
cgttcctcga 2280gttcctcgga atatgtttaa tatgtttctt gattacacct
tccacgacgc cccattgaag 2340ggtctcggct ttaatggagg aattcgctac
accggtttta cctatgcgga ctatgtggag 2400tcttacaaaa cgccggcgta
ttatctgttt gacattggcg cacactatga ttttgaggaa 2460ataatccctt
ctctcaaagg tctgcgtgcc cagttggcaa tctcaaattt ggccaataaa
2520tattatatta cttcgtgcaa taccgccata tgtactctcg gttatgctcg
aaagttttac 2580ggtaacgtga cgtatagctg gtga
260473066DNAAcetobacterReverse transcriptase family protein
7gtgacgcccg aattgctcct ctccaaggtg cggctgctgc ggtcgcccaa tgacgacggc
60gcgttcttcg acctagtcgg cagtgttctt aattggtcct gggaggaaag agacgaacgt
120caattcgccc gcttcaagca gcgcgcgggc atccctgagt tcgatggcgt
cgcccttcca 180cagggtttgg ttgcagctgg cttcttctcg aacatcgtgc
tgcttgattt cgatcggatc 240gtcatcggac agattgggag agaagttaca
aacggagtgt ggctccggga cgcctgccgg 300tacgtcgacg acattagact
gaccataaca actgcaccag gtattgaccc aagagaagct 360caggcgcgtg
taatggcgtg gcttgggcaa ctcctcacgg ggagctgtcc gggcttggaa
420ttctccccgg agaagacgtc aactgcgtcg gttggaggcg agcagatgcc
gctggttcgc 480caatcccgaa agatggagcg catccagacc gcgatttccg
gcggcttcga tgccagtggt 540ggcgaggagg tgatccacgc gatcgaagcc
ctcgtccgat cccagctaac gatcaacagc 600gtcgaagagt cgcctacccc
tcccggcttg agagcggtac ccgatgtcaa agacgagaca 660gtcggtcgtt
tcgctgctgg tcggttcaga aaaacctttc gttcattgag gccactactc
720gatgatcgac cttacatgga gattgctgaa ttcggggagg agacgttccg
gcgcacccga 780ctttcgcaat cggagcttga cgaggaagca cgcgcatttg
cgctaatcct agtcgaacgg 840tggatactcg atccttcgaa tgtgcggctg
ctgcgcgtcg cactcgacct ctggccgtcc 900cgccaactcc tcaaggaagt
actgaaactc tttgagccct atcttgtcgg gaagatcagg 960gcaatcacta
gccggcaagt tgcatactac tgcctcgccg agatatttcg agcaggggcg
1020accgagacgg gcttcattga cgatccagag tgccttcccg ctgccgtcga
tctcgccggt 1080tatagatctc tgcttctgga ggccgcagta cgagtggccc
ggggcgaagc cgaacgtgtc 1140ccgtggtatc tcgcgcaaca agcactgctt
tacattgcgg tccacgatcc ccgggctatc 1200caagatcgag gaatttcaaa
gaccaatcga tcctattggc gcctcgtctc atttctgaaa 1260ggcgaacgcg
acgtctcttc agatcgcgaa ttcgcagtag ccgcggtggt gagccgcagg
1320tcgttccttt cgaatgatca ggccgtggat ctcgtcggtc ggatgctcac
gccagagcgg 1380ttcgccgagg tggccgcgcg cgacatagaa ttcgcccgcg
atctctttcg cgccgtcgac 1440cgacacctca ccgttccggc aggcattgcc
gaggacttgg gggtcgccga atggtccatg 1500tcagaggaaa tgagctctct
gcaaagctat atccaaggca aagggcctct gaatccgcta 1560cgcaatgaga
tcggcgtact cagttttgca gagaaattca tctcccatct ccaagaagga
1620aatttgccgg aagtcgtgac gccgtcgacg acgcagatag cggtacagca
agtgggcaaa 1680tatgtccgcg tcgaacgggt gatcttcaga tcggcccaga
caacgccgac ttaccggtct 1740atttatactg ctcccagatg ggcgccggaa
tctcaacgct ggcgctttca gctcggttat 1800ttacttcgct tcattcttac
tgccagaata gacttcagcc ttccagttag gccgccatcg 1860tggaaggaag
gtaaacacat ctatcggcct accagaagtc actggtttca gcggcaatac
1920ggcttctata atgggcatga ggccttcggg gacgattggc tacccatttc
gcagttcact 1980caggatcttc tcttcgatct gctcacctgg cccggctgcc
gcacaagtag cccggatgtc 2040gatcagttgt ccctggatga aacggctgct
cgaatccgcg cagctctcgt agaagccacc 2100gctgcgattg gcccggctac
aggaaccctg atgctcaaga tcgatgcacc tattccaggt 2160accacatcga
aggggcgccc gcttcgggcc tgcgtcgtcc agtcgatcat gcccgaagca
2220gaggacttct cggctgccga tctggagatg cgctcgccgg cccttcgacg
aaagcaccgc 2280aaacatctgt ccacggcatt ggcggcggtt gagaagatgt
tggatcttcg cgagactcat 2340aagccagcca gcaagcgtct cgactggctc
atcctaccag aactgtctgt tcacccggat 2400gacgttgcca cccacctcgt
gccgttcgcg cgagcgttca agaccgcgat cctggtcggc 2460atggcctacg
aacaagtcgt cacgggagag ccgctgatca actcggccct ctggattatc
2520ccgaggatgg tgcggggcat gggcctacag acggtgatca gacggcaggg
aaaacagcac 2580ctctctccga tggaacagaa gtacgtcaaa ccggtcgaac
tgatcaccgg attccgcccg 2640tgccagtggc tggtggggta cgaatggtcg
aacaatccgg ccaaagacgc actttggctc 2700accgcgtcca tctgctacga
tgcaacagac ctgaagctgg cgagcgatct tcgtgatcgc 2760tcagacgtgt
ttgcgatccc agccctgaat ctcgacgtcg gcaccttcga tcagatggcg
2820caggcgctgc attatcatat gttccaactc gtgctgatcg cgaacaacgg
agcttatggg 2880ggcagcaatg ctcacgttcc caagggggag gcctatcaac
gccaagtgtt ccatacccat 2940ggccagcccc aggctacaat ttcctttttc
gagatcgacg atatcgaggg catgaagcag 3000agacacaagc tcggcgctgg
gaaggaaggc gggtggaaat atccacctgc cggctgtcaa 3060gtctga
30668513DNAAcetobacterDNA topology modulation protein 8ttgatgcgct
tgttcgtgac ggggccaact ggcagtggaa aatcaacgct ggctgcaaag 60ttggctcaaa
gggcagctat accactgttc ccgctcgatg aaattcattg ggttcgccat
120ctctccgggg attggcggcg cgatcctgtt gaacgcctgt ctatgctcgg
agagattgta 180cagctcgatg cctgggtcat cgaaggcgtg cagttcaaat
ggactgatat agcgatagaa 240cgagcagact ggatcgtcgt cctcgatcca
ccacgttggc ggaacatcgc tcgtatcctg 300cgccgtttcg tcaatcgccg
atgcttttct ggggcggggc accgtggaac ggtaaaggct 360ctattggagg
agatgcgttg gtcagccgac tactatggtc atgaacgcgg tatgctgttc
420gagaagattg gacaatcgcc agacaagctc atcgtcgtac atgacgacaa
gggcgaacgc 480gctttgaccg aggctgtatt cgcgactgcg tga
51392352DNAAcetobacterCycloisomaltooligosaccharide
glucanotransferase precursor (EC 2.4.1.-) 9atggcatatt ggatcaggct
ctcgctggcc gtgtggccgc ccgatcagca acgttgtagc 60gaaggccgcg taatgcgccg
ctatcttttc acaaccattc tctcgctctt accgtccctt 120gcggcggcgg
catccctcca aggtccgatt gtttcgcatg tgcgggatga tcgggctttc
180taccaggcag gcaatgtcgc gatgatttcc gtggaactga ccccactagc
cgcttggacg 240ggaggccatg tggatctagc gatatgttcg cgtgggcaag
tcgtgggcac gattcagagc 300caagcggtca ccagcatggt ggctggggcg
gaccagacac ttcactatcc cgtcaccgtt 360cccagtctcc atgctcatgg
gtatcagttg gctatcgcgg ccctgaacaa tggggacagc 420gggacagcgt
cctgtaccgg gacaggcagc acttccacgt cgccggccga tgtggcgtca
480ggcggcatca acgtggccgc gaatgcctgg gaagacatca acgaagcatg
ggtcgacgcg 540ccgacgctcg gcaacgtctc cgcggcccgg gtgatggata
atctcagcca gtatcacatc 600aacgcggtgc aattttacga cgtgctgtgg
cgacatgacg agccgtattc atccgccccg 660cagtggccga acctggaagg
cgtaacggtc acaaggacca atcttcaggc ttatatcagc 720gcggcgcata
gccgcggcat ggtggcgctc gcctataatc tctggaacgg agcctgggcg
780gactatgcaa ctgtcaatcc gaaggtcacg gcggcaatgg ggctctatgc
ttcgtccgga 840cagaaacacc aactgaccaa cggcgggggc tggctgtcct
gggggtggtc gaccgaccat 900attgctgaaa tgaacccgtt caatggcgac
tgggccagat ggctaaccag ccagatccag 960aagaccatgt ggaatttagg
attcgacgcc gcgcatctgg atacgttggg tgaccctggt 1020ggtcagcaat
atgacggcga gggccatccg ttacctgctt taggaacgat tctggcagac
1080tttgcgaata atgtacaggc tcagaccggg gcaccaactg acatcaacgc
cgtttcgggt 1140tggaatacca ccgaccttta cctacgcggt acgggaccca
acctgtatat cgaaccccat 1200cccgaattcg gaaacacgcc gggctacgat
gattcccgaa gcttatggga catcaaacag 1260aaatatacgt cgcgcccgct
gatgacggcg ttttatccgc agcaggtcca gagcggttcg 1320ctgagcacgt
cctttgccgt caagggtgag agtgtgaagg tttgcgaccc gacgttaaaa
1380tccggatgca tagccaataa cctcggcatt gagttgttgc tcggccagat
tgcgctcaat 1440ggaggctcca atattactct tggtgatttt gatcatctga
taccggggcc atatttcccc 1500cgtccgaccc ttaagatcga cggtccattg
cagcaatatc tggcggatta ctacaactgg 1560tgggtcggaa tgcgcgatct
gctgcgtgtc ggcgtcatct catccaatga gagggagtcc 1620atccggaatg
gaaacggagc cagtatcggc caaccttatg cccaaccggg aaccgtctac
1680tatcatcccc tgatacgcgc tggcatcgct ggtgaattgg cgctcacaaa
catgatcggg 1740ttgcattata atcggattga cgaccctgac ggcaaaaaca
atccgacccc ggtgaacaac 1800ctgtcgatcg agatggaatt ctgggaaaga
agcacaccgg gggcattgta ctatagcgcg 1860cccgacatca accacggctt
cccacagccc ctcacctata ggctgaacgg aaacggtagc 1920gtgatgttta
cgctaccgac tctcaagacg gtggcgcttg tttggctgga aggcaccaat
1980ttcaccacta cgaccgatta cacgatcggt acggcgcagg atgtgagggg
tggcacagca 2040aacttctgga cgaacggcag cggagaggat gctaccggat
atcgtggctg ctgtggtcgc 2100tccgcacgct gggacagcat cgatttcgga
gcgggtgtgt caacgctaac gatggtaacc 2160cgaagccaac tcggcggact
ggtcgaattt cgcctggatg cacctgatgg accagtcatc 2220gcccgtaatt
atgttcctgc gtctagcgcc acgacaacaa ccactcaatt acgcaggcca
2280gtattcggga cacataccgt cttcgctaaa attcctggtc gcgagattac
gctgatatcc 2340tggaagccat aa
235210855DNAAcetobacterMethyltransferase (EC 2.1.1.-) 10atgatggcta
acgacaatac cactgaggtg gttggtgcat ttgcggtaag tcatcccaac 60ttggcgcaag
gttttacttt tagtaacagc agtcaactag atacgattgc ttctactatt
120cataaaagcg gtttggagac ttatgaagct ccgacaacta atataattat
cgaactgatc 180aggagttcgt ctggtcttat tttagatgtg ggagcgaata
ccggtctttt tacgctagtc 240gccgcagcag ccaaccccct gatccgcgtc
tgctcttttg agccgcttgc gagtattcgt 300gaacttctca agagcaatat
tgctctcaat ccggagcttg catcacgtat cgctgtcgag 360cctgtcgggt
tatcgaatga acggggcact ttcacttttt acgaaacgat caacaatcgt
420ggctttgtca cgacgagttc atcgcttgaa aaagcacatg cagagcgaat
cggcgatttg 480tacgtcgagc gcactatcga gacccggaca cttgatgaat
tcggagaaac gctcgggaat 540gcgagcgttc cgttcgtcaa aattgacgtt
gagggacatg agcatgccgt tatctccggt 600ggccgccact ttatcgccaa
gcaccgccct tttcttactc tcgaagtcct gagagaggct 660aacactttga
gtctggacca gttggtgacc gagtccaact accttgccct ggcaatggca
720cccgacgaat tgcggcagtg cgagcgttta cggtttcatg acgacgcctg
gaatcatctt 780ttggtccccg ccgaaaaagc ggaacggcta ttttcgctct
gccgccgact tggcttgcaa 840atcgggatcc gctga 85511744DNAAcetobacter
11atgtcgaatt ccgagcgccc aatgcgcgat ttgtcggacc tggcaaaaaa ccgacaaata
60gagccgatgg ttatcaggct acgagaagta gtggatcgga ccggaggcgc gaaagctgtg
120gccgcacgca cggacatccc tctcagcaca ctttcaggtt acctgtcggg
tcgggaactc 180aagctttccg tcgcgcgcaa gatcacggaa gcctgcggtg
tcagtcttga ctggcttgcg 240gcaggagagg acggacctgc ggcccgggaa
ttcggcaatg cccggcaggc gggtcccgag 300tcggtcgagt ttctgaatta
cgacgtcatt ctctccgccc accagggcgt cgacggggat 360agttcttata
tcgaaacgag aatatcgata ccgcgggatt ttctcccttt gtccattcag
420tccaatacgg acaacatttc ggccgtcacg gcgaaatgcg acagcatgaa
tccgatcata 480gacgatggag acattctttt aataagaacg gatgtgcata
cgctcacaag tggcagcatc 540tatgccctgc gggtagaaaa cacccttctg
gtcaggcgtc tgatcctcaa gaccaacggc 600aacgtccagg tcatcagcga
gaacccgcgt tacccgaccg aggaactgaa cgccgaggac 660gttcgcagga
tggtccagga cgacggcttt ccggccagga tcatcggccg ggtcatctgg
720cgcgccggta gcctgattcc atag 744122331DNAAcetobacterouter membrane
heme receptor 12atgcgcatcg tcctcttgcc ctgcctcgtc gcgacctcaa
taagtatgtt ggcggtttcc 60gcatcctatg cttgggcgga caatagcccg tcgcccccca
ggacgaacaa acaggccaaa 120tcgcggccgt tacatgcgca ggggacgcgc
aaagcgggca gcgccatcac cagccaggat 180gaagcggtgg ctgtcgtggg
aacacgtgag acatcgcatg ggatggagca gagcgttacc 240cgtgcgacga
tggacaagtt cgtggcgggg accagtcccc tgcagattct gtcggccacg
300acaccgggtg tcaattttgc ctcggacgac ccgttcggcc tggatacatg
ggcgaacaca 360ttttatattc gcggctattc ccaaagccag ttgggcatca
ccctggacgg tatcccgctg 420ggcgatgccc agttcatcaa ttccaacggc
ctcgatatca atcaggcgat
catccagaac 480aatatcggtc gcgtcgacat gtcgcagggt ggcggtgcgc
tcgatgtcat gtccgtcacc 540aacctgggtg gcgcgctgca atattattca
ctcgatccgc gcgacaaggc tggtggagac 600atttcacaga cgttcggcag
caaccagacc tatcgcacgt acgtcagcgc ccagagcggc 660aagctcaatc
ccagcgggac gaagttctat gcgtcgtacg cgcgcaccga tgccgggaaa
720tggaaaggcg ccggggacca gttcgaacag caggcgaatt tcaagatcgt
acagccgctc 780gggcgttacg gaaaactgtc cggattcttc aattattccg
aattcgacca gtataattac 840agcgatttga gcctggaaat catccagaag
ctcggccgga acgtggatta tttctatccg 900aactacaaag ccgcgtatca
ggctgccgag gggatctatc ccgcaggcta tgccaaggtc 960ggagatgcca
tggacgtctc ctattacgat ggtggccagg accagcggaa ttatctttcc
1020ggcatcacgt ccacgatcga cctgacgtcc cgcctgcatc tgaagacggt
gctgtacgac 1080cagcaatcgg cgggggacta cgaatggacc aacccctatg
tgtcgtcgcc ctcgggcgcg 1140cccatgatcc agcaggtcgg gcacacatcg
atgacgcgcg tgggcgggat tggcgcggtg 1200cagtaccaga tcgccaatca
ttcgcttgaa accggcgtct ggtacgaaaa caacggatat 1260agctgggcgc
aacggtacta caaccagccg cttctggggg agggtacgcc ccgaagcgcc
1320accggaccgt acaacgatcc gttcgccacc gcatacgcca tgaccttcaa
taccaacagt 1380tttcaatatt acctggaaga ttcctaccgt atcttgaaga
cgctgcgggt gcacgcgggc 1440ttcaaatcca tgctgacgac gacgtcgggc
ggcgcatcct ataacaatcc cgtctatacg 1500ggccaggaca ccctgcccag
tggcagcctg accaccgcca gcgccttcct gccgcatgtc 1560agcatcaact
ggaatttcct gccccggaac gaactgtttt tcgacttcgc ggagaacatg
1620cgcgcattca cctataatac atggcagagc gggaatgcat ggggagtcaa
tgagatgccc 1680cagaacctga agcccgagac caccttcaat tacgaggtcg
gttatcgata taattcccgc 1740ttcgtcacgg gcctcgtcaa tctgtatcat
atcgattaca ggaaccggct ggccaccatc 1800accaccggca gcctggtgaa
cgcccacaat acctatatca acgtggggaa catggcgatc 1860tggggtgccg
atgccggcgt gacggtgcgc ccgctgccgg gcctcgagat cttcaacagc
1920gccagctaca acaaatccac ctatgggcag gatgtatcca gcggcggggt
aaattatccc 1980atcagcggca agcaggaggc cggctatccg caatggatgt
acaaggccaa cgtctcgtac 2040aggtatggca acgcgaaggt caacttcaac
gtcaactata tgggaaagcg atacatctcg 2100tacatgaacg acgccgccgt
gaacgggtat tggctggcat cgctgtcggc gacgtatatc 2160ttcaaaacca
ttccccatct ctctcagctt gaattcaatt tcggcgtcta caacctgttc
2220aaccaggaat atatcggcgg catcggcggg ttctcactgt ccggtgacac
gcagcaactc 2280tttgccggcg cgccacgcca gttcttcggt acgctgcacg
cacggttcta g 2331131812DNAAcetobacterLevansucrase 13gtgacggcgc
ggtcgtggtt gctctgcaat ctgaagagtt tccttcagga ggatggaatg 60gcgcatgtac
gccgaaaagt agccacgctg aatatggcgt tggccgggtc cctgctcatg
120gtgctgggcg cgcaaagtgc gctggcgcaa gggaatttca gccggcagga
agccgcgcgc 180atggcgcacc gtccgggtgt gatgcctcgt ggcggcccgc
tcttccccgg gcggtcgctg 240gccggggtgc cgggcttccc gctgcccagc
attcatacgc agcaggcgta tgacccgcag 300tcggacttta ccgcccgctg
gacacgtgcc gacgcattgc agatcaaggc gcattcggat 360gcgacggtcg
cggccgggca gaattccctg ccggcgcaac tgaccatgcc gaacatcccg
420gcggacttcc cggtgatcaa tccggatgtc tgggtctggg atacctggac
cctgatcgac 480aagcacgccg atcagttcag ctataacggc tgggaagtca
ttttctgcct gacggccgac 540cccaatgccg gatacggttt cgacgaccgc
cacgtgcatg cccgcatcgg cttcttctat 600cgtcgcgcgg gtattcccgc
cagccggcgg ccggtgaatg gcggctggac ctatggcggc 660catctcttcc
ccgacggagc cagcgcgcag gtctacgccg gccagaccta cacgaaccag
720gcggaatggt ccggttcgtc gcgtctgatg cagatacatg gcaataccgt
atcggtcttc 780tataccgacg tggcgttcaa ccgtgacgcc aacgccaaca
acatcacccc gccgcaggcc 840atcatcaccc agaccctggg gcggatccac
gccgacttca accatgtctg gttcacgggc 900ttcaccgccc acacgccgct
gctgcagccc gacggcgtgc tgtatcagaa cggtgcgcag 960aacgaattct
tcaatttccg cgatccgttc accttcgagg acccgaagca tcccggcgtg
1020aactacatgg tgttcgaggg caataccgcg ggccagcgtg gcgtcgccaa
ctgcaccgag 1080gccgatctgg gcttccgccc gaacgatccc aatgcggaaa
ccctgcagga agtcctggat 1140agcggggcct attaccagaa ggccaatatc
ggcctggcca tcgccacgga ttcgaccctg 1200tcgaaatgga agttcctgtc
gccgctgatt tcggccaact gcgtcaatga ccagaccgaa 1260cggccgcagg
tgtacctcca taacggaaaa tactatatct tcaccatcag ccaccgcacg
1320accttcgcgg ccggtgtcga tggaccggac ggcgtctacg gcttcgtggg
tgacggcatc 1380cgcagtgact tccagccgat gaactatggc agcggcctga
cgatgggcaa tccgaccgac 1440ctcaacacgg cggcaggcac ggatttcgat
cccagcccgg accagaaccc gcgggccttc 1500cagtcctatt cgcactacgt
catgccgggg ggactggttg aatcgttcat cgacacggtg 1560gaaaaccgtc
gcgggggtac cctggcgccc acggtccggg tgcgcatcgc ccagaacgcg
1620tccgcggtcg acctgcggta cggcaatggc ggcctgggcg gctatggcga
tattccggcc 1680aaccgcgcgg acgtgaacat cgccggcttc atccaggatc
tgttcggcca gcccacgtcg 1740ggtctggcgg cgcaggcgtc caccaacaat
gcccaggtgc tggcgcaggt tcgccaattc 1800ctgaaccagt aa
18121420DNAAcetobacterPrimer 14tgaaattgac gcccgttgga
201520DNAAcetobacterPrimer 15cacgccggga aagaggattc
201620DNAAcetobacterPrimer 16ggcaacgcgg tttctacgaa
201720DNAAcetobacterPrimer 17cgttagccgg ggttgtcaga
201820DNAAcetobacterPrimer 18tcgttgccac tttccgaggg
201921DNAAcetobacterPrimer 19gtcgattgtg tgcagcgtca a
212020DNAAcetobacterPrimer 20caccgatctt gtgcgtttcg
202120DNAAcetobacterPrimer 21cggcaatgct ccatacccac
202220DNAAcetobacterPrimer 22caccggaaag agtggcagga
202320DNAAcetobacterPrimer 23aaccgggtca cttgcgtcat
202420DNAAcetobacterPrimer 24agccatcgga gtcacatcgg
202520DNAAcetobacterPrimer 25ggaaacctcg aaaccctgcg
202620DNAAcetobacterPrimer 26tcagggcaat cactagccgg
202720DNAAcetobacterPrimer 27tcgagcagcc gtttcatcca
202820DNAAcetobacterPrimer 28tgatgcgctt gttcgtgacg
202920DNAAcetobacterPrimer 29cgttcgccct tgtcgtcatg
203020DNAAcetobacterPrimer 30gggccatccg ttacctgctt
203120DNAAcetobacterPrimer 31tgacacaccc gctccgaaat
203222DNAAcetobacterPrimer 32gcatttgcgg taagtcatcc ca
223320DNAAcetobacterPrimer 33ggatcccgat ttgcaagcca
203420DNAAcetobacterPrimer 34tgtcgggtcg ggaactcaag
203520DNAAcetobacterPrimer 35cgggttctcg ctgatgacct
203620DNAAcetobacterPrimer 36tcccgcctgc atctgaagac
203720DNAAcetobacterPrimer 37cagcgatgcc agccaatacc
203820DNAAcetobacterPrimer 38gttcgtcgcg tctgatgcag
203920DNAAcetobacterPrimer 39acctgggcat tgttggtgga
204022DNAAcetobacterPrimer 40ctcaggaaga ccgaattgat ta
224119DNAAcetobacterPrimer 41gcgaaacgtc tgattgaac
194238DNAAcetobacterPrimer 42cggataacca ctggtgctcc gactcgcctc
actctact 384339DNAAcetobacterPrimer 43tccacgaatc tcacgaagca
ccccgacctt atctcccat 394422DNAAcetobacterPrimer 44gccaggcgtg
tacatataac ta 224522DNAAcetobacterPrimer 45cggaatacct agttggaaca ct
224621DNAAcetobacterPrimer 46tcaagatcga tgcacctatt c
214720DNAAcetobacterPrimer 47aacagacagt tctggtagga
204837DNAAcetobacterPrimer 48cgcatctcca gatcggcagg tcgtccagtc
gatcatg 374938DNAAcetobacterPrimer 49acatctgtcc acggcattgg
tggctggctt atgagtct 385019DNAAcetobacterPrimer 50gagaagtcct
ctgcttcgg 195118DNAAcetobacterPrimer 51cggcggttga gaagatgt
185219DNAAcetobacterPrimer 52ggaagacatc aacgaagca
195319DNAAcetobacterPrimer 53ttgacagttg catagtccg
195439DNAAcetobacterPrimer 54atacggctcg tcatgtcgcg gtgatggata
atctcagcc 395540DNAAcetobacterPrimer 55cagtggccga acctggaagc
gctgatataa gcctgaagat 405617DNAAcetobacterPrimer 56attgcaccgc
gttgatg 175718DNAAcetobacterPrimer 57gcgtaacggt cacaagga
185822DNAAcetobacterPrimer 58aggaggctct ttctttggaa gc
225922DNAAcetobacterPrimer 59aagtgcccct gttatcgtac ac
226022DNAAcetobacterPrimer 60tgggtcatcg gttctgattt cc
226122DNAAcetobacterPrimer 61tagtttgatg tcgggtgctg ag
226222DNAAcetobacterPrimer 62gcgaataccg gtctttttac gc
226321DNAAcetobacterPrimer 63atgcaagctc cggattgaga g
216420DNAAcetobacterPrimer 64ccaaatctct ggaacgggta
2065420DNAAcetobacterhypothetical protein coding sequence
65atgcggatca gcggcatatc ttcgattctc gataatgcaa cggcccagtc ctcatcgggt
60gccacgacac agaagagcgg gattttgcaa tcatcgtctc tcgatttcag caatatttcc
120gcgtcgaatc tgcaagacgt gaatgcggaa ctctataatc aggggaagat
atcgctccgt 180cagagcggtg acttgtcctt gttggatggg tgggcactca
agggcgtcaa taatggtcaa 240cttcggcagt catcgaccgg aaccctcaac
gcctattcct tgcttgatac catgattaat 300tatcaggaaa cgaacggtat
cggcgatgta aaagatacgg tcgcatccct caaggcattg 360cgcagtacac
ttgaggaata tgataccaac aatcagaata aaacggcgat cacggcctaa
42066864DNAAcetobacterGDIA_RS17945 (7136..7999)- hypothetical
protein 66atgtacgttt cagtggcaaa ggctggtacc atcgttgata gcctgtcgac
gagggagaag 60agcaaagagg gaggcggcac gctcctgacg tccgatcagc cagattccgc
tgaaacaggc 120cacgccgaga ccgaatctgt ttcgattacg ctgtctcagg
cggccgtcga cgctttgaac 180gggactgcct cgcagaacac tccggcttcc
atgcaactcg ccatgtcccg catggaccag 240attatccggt ccggcaacag
cgcggcaaaa gccgatgccg gggcgcgtgt cgccaatttg 300caggcgcaga
tgcggcagct catggagacc aaggatctca tgtcgccaaa agcgctggcg
360gccgccctgg cgctgatggc ccgcgaactg gccgccgccg tcagcgagta
tgtgcaatgc 420ggcggatcgg ctgccaacgc ggccattggt accgtcgtac
tatccgcgtc ggccgacaca 480tcggctgatc cgtccactgc cgccccggca
acatctgtaa cggcggatca acccgttgcg 540tcggttcccc aggacgcgac
acaggaacag agtgcccaag gccagaccgc tcccgggcag 600agcggtcagc
agacccagga aggaaacgcc gtcgatcatt cgagcgatga gacggaacag
660accgcaggtg cggcaaatac ccagcctaaa gaaagcgggg acgacctgtt
tgccaaggcc 720gtgaagagcc tggcggagga aatgaagagc atcctcaacg
aactgaagaa caagaagaaa 780aaggacgatg cgtccgaaga ccatgatctt
caatcgacac agggttctct cgacagaatc 840gatcagatga ttggaggcat ataa
864671392DNAAcetobacterGDIA_RS17965 (13228..14619)- HlyD family
type I secretion periplasmic adaptor subunit 67tcatggctcc
ctcataccgt cagtcatgag cggaatgaga ttgttaagaa gaaattgcat 60aatggttcgt
gtacccactt gaatatctgc tgtaatgggc attcctggcc gggggtgaaa
120cgatgaaggt acgccgtgca accgataatc ttcgatatgc agccgtgcgc
gataatagga 180ctgtgccgca gcggcaggat cgttgctcgc tcgctgcccg
atcccgccgt ttgatacggc 240gtcctgtgac gtattaccat cgaacgtgtc
tgcgctgatt tctcgtacca gcgcctcagc 300accgccatac tgagagtatg
gaaatgccgc gaacttgata attgccttgt tgccgggttg 360cacaaaaccc
gcatctttgc tgcttaaatg cacctcgact tcaagaccgc tatcgactgg
420aacaagccgc atcagctctg ccccggttgt cagaacggac ccaggtgaca
atctggcgat 480cgtcaggacg actgcatcct cttgcgccgt caagatggtc
atgcttttgc ggagcttcgc 540cttttgatag tcggcatccg tggttgagag
cttgtgttgt gcctcgctta aatcgcgata 600gatgtccgct ctccatgttt
caatatattc ttggcgttcg gcgacgagcg cctgcaactt 660ggcttttgcc
gatgctgcgt catgctgggc aaggacctcg gagcgctgaa cttccatcat
720atcgttctga gcgccaagag tagaaaggcg gctgcccacc tgttcctcct
gcaagcgagt 780tcgcatcctg tgtacgttgg tcgcgacttc gagccgttta
gcgtagatgg tagcgttggc 840cagcgcgcct tggagatcgg ccgcctggga
agccagttgt tcttgatagt tctcgatttt 900ggccgcaaac tcagccttac
gacgcaggaa tgtcgccgct tgctgtattg acgctgggtc 960gtcggggtcc
gacaggtagt ccctaccctg cgcttccgca gaaaggcgtg cgacttccgc
1020agtataactt tgggtttgtg acttcaaatt tgcgatatca gcgtcgttaa
ccgtagggtc 1080aaggcgggat aatacttggc ccttatgaac aaggtcacct
tcccgcacgt ccacactacg 1140aataatcgac gtttcgaacg gctgtacaac
gagaggtctc tgaatagata ctattttccc 1200ttcggtggac accacccgag
tcagcggaaa gaccatcatg aatacgatac tactcgcggt 1260cagcgcacca
attatccaag atatatagcg ggctgaaggc gtggccggca tgttgacaag
1320cgcggcagtc ggcgaatgaa actcaagcaa cgccggaggc aaatcggcat
agtcaaagga 1380gtcagtctcc ac 139268849DNAAcetobacterGDIA_RS18030
(25282..26130) - hypothetical protein 68tcaagatttt ttaccttccc
gctcgattgt gacggaagcg gcgagttcct tgatcgcggc 60gacgctggca tcggctcgtt
cctcagcccg gatggcgcgg gcttccgctg cctgcacgcg 120ggattccgca
cgggcttcgg ccgcgtcacg ggacttttca gcgttctcca gtgccgtggc
180agcggtgctg gcccgacgct ccgcgatgtc ctgggctttc tccgccgcct
gggcacgggc 240ctgggcctcg gacacggcct gccgcagggc gtcgatctcc
ccggtctgcc gctcggccat 300ggcacgcgcc gcctgatggg cctgacgttc
agcggtcagt tcgaccgtca gccggtccac 360ctcccgctgc aaggcctcca
gccggcgtgt cagcccttca gcctcggccg tagcgccagc 420cagggcttcc
gccaattcca tcgcctcccc cctggcatcg gccagctccg cacccgcctg
480agcctggata tcggcgatct gctggtccgc cgcttcccgc gccagcgcca
ccttctggtc 540ggccgccgcc tggatggcct gctggacgga ttgccctttc
tggcgctccg catcgacaaa 600gccggtcagc atcgccagga agccctgctt
catccgttcg gccgcgttgt ccatctcggc 660cgagacgaga tccccggctt
cgctccgggg ctcctccttc ggtgcttcga ccggccgccc 720gcgctgactg
tctttccagg cgttgagcag cggcaggatg gcgttcggac tgccgccccc
780cagctcgtcc cgcactttgc gcacactggg cttctcgccc cgcccgacca
gcgcttcggc 840ggcctgcat 849
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