U.S. patent application number 14/063640 was filed with the patent office on 2014-08-07 for phage &phgr;mru polynucleotides and polypeptides and uses thereof.
This patent application is currently assigned to Pastoral Greenhouse Gas Research Limited. The applicant listed for this patent is Pastoral Greenhouse Gas Research Limited. Invention is credited to Eric Heinz Altermann, Graeme Trevor Attwood, Debjit Dey, Petrus Hendricus Janssen, William John Kelly, Zhanhao Kong, Sinead Christine Leahy, Dong Li, Christina Diane Moon, Robert Starr Ronimus, Carrie Sang, Linley Rose Schofield, Catherine Mary Totill.
Application Number | 20140220636 14/063640 |
Document ID | / |
Family ID | 40511642 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140220636 |
Kind Code |
A1 |
Altermann; Eric Heinz ; et
al. |
August 7, 2014 |
PHAGE &phgr;MRU POLYNUCLEOTIDES AND POLYPEPTIDES AND USES
THEREOF
Abstract
The invention encompasses phage .phi.mru including phage
induction, phage particles, and the phage genome. Also encompassed
are phage polypeptides, as well as polynucleotides which encode
these polypeptides, expression vectors comprising these
polynucleotides, and host cells comprising these vectors. The
invention further encompasses compositions and methods for
detecting, targeting, permeabilising, and inhibiting microbial
cells, especially methanogen cells, using the disclosed phage,
polypeptides, polynucleotides, expression vectors, or host
cells.
Inventors: |
Altermann; Eric Heinz;
(Palmerston North, NZ) ; Attwood; Graeme Trevor;
(Ashhurst, NZ) ; Leahy; Sinead Christine;
(Palmerston North, NZ) ; Kelly; William John;
(Ashhurst, NZ) ; Ronimus; Robert Starr;
(Palmerston North, NZ) ; Li; Dong; (Palmerston
North, NZ) ; Kong; Zhanhao; (Shanghai, CN) ;
Schofield; Linley Rose; (Palmerston North, NZ) ; Dey;
Debjit; (Palmerston North, NZ) ; Totill; Catherine
Mary; (Palmerston North, NZ) ; Sang; Carrie;
(Palmerston North, NZ) ; Moon; Christina Diane;
(Palmerston North, NZ) ; Janssen; Petrus Hendricus;
(Palmerston North, NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pastoral Greenhouse Gas Research Limited |
Wellington |
|
NZ |
|
|
Assignee: |
Pastoral Greenhouse Gas Research
Limited
Wellington
NZ
|
Family ID: |
40511642 |
Appl. No.: |
14/063640 |
Filed: |
October 25, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12678936 |
Mar 24, 2010 |
8592556 |
|
|
PCT/NZ08/00248 |
Sep 25, 2008 |
|
|
|
14063640 |
|
|
|
|
60975104 |
Sep 25, 2007 |
|
|
|
60989840 |
Nov 22, 2007 |
|
|
|
60989841 |
Nov 22, 2007 |
|
|
|
Current U.S.
Class: |
435/69.7 ;
435/252.3; 435/252.31; 435/252.33; 435/252.34; 435/252.35;
435/252.8; 435/254.11; 435/254.2; 435/254.21; 435/320.1; 530/350;
536/23.72 |
Current CPC
Class: |
A61P 31/04 20180101;
A61K 35/13 20130101; C07K 2319/00 20130101; C07K 14/005 20130101;
C12N 9/641 20130101; A61P 1/00 20180101; A61P 37/04 20180101; C12N
2795/10021 20130101; C12N 1/20 20130101; A61P 31/00 20180101; C12N
7/00 20130101; C12N 2795/10022 20130101 |
Class at
Publication: |
435/69.7 ;
530/350; 536/23.72; 435/320.1; 435/252.3; 435/254.2; 435/254.11;
435/252.34; 435/252.33; 435/252.31; 435/252.35; 435/254.21;
435/252.8 |
International
Class: |
C07K 14/005 20060101
C07K014/005; C12N 1/20 20060101 C12N001/20 |
Claims
1-57. (canceled)
58. An isolated polypeptide or peptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-116,
117, 118, 119, 120-136, and 138-172.
59. An isolated polypeptide or peptide which comprises: a) an amino
acid sequence sharing at least 90% identity with SEQ ID NO:117; b)
an amino acid sequence sharing at least 95% identity with SEQ ID
NO:118; c) an amino acid sequence sharing at least 90% identity
with SEQ ID NO:119; d) an amino acid sequence sharing at least 90%
identity with an amino acid sequence selected from the group
consisting of SEQ ID NO:1-31, 33-104, 106-112, 114-116, 120-136,
and 138-172; e) an amino acid sharing at least 95% identity with an
amino acid sequence selected from the group consisting of SEQ ID
NO:105 and 113; or f) an amino acid sharing at least 98% identity
with amino acid sequence SEQ ID NO:32.
60. An isolated polypeptide or peptide which comprises: a) at least
15 amino acids of SEQ ID NO:117 or 118; b) at least 15 amino acids
of SEQ ID NO:119; c) at least 15 amino acids of an amino acid
sequence selected from the group consisting of SEQ ID NO:1-31,
34-73, 75-104, 106-112, 114-116, 120-132, 134-136, and 138-172; d)
at least 17 amino acids SEQ ID NO:105; e) at least 19 amino acids
of SEQ ID NO:74; or f) at least 22 amino acids of an amino acid
sequence selected from the group consisting of SEQ ID NO:33, 113,
and 133.
61. An isolated polynucleotide which comprises: a) a nucleotide
sequence which encodes the amino acid sequence SEQ ID NO: 1-116,
117, 118, 119, 120-136, and 138-172; b) a nucleotide sequence
selected from the group consisting of SEQ ID NO: 173-341, 342-510,
511, 512, and 513; or e) a nucleotide sequence complementary to (a)
or (b).
62. An isolated polynucleotide which comprises: a) a nucleotide
sequence which encodes an amino acid sequence which shares at least
90% identity with SEQ ID NO:117; b) a nucleotide sequence which
encodes an amino acid sequence which shares at least 95% identity
with SEQ ID NO:118; c) a nucleotide sequence which encodes an amino
acid sequence which shares at least 90% identity with SEQ ID
NO:119; d) a nucleotide sequence which encodes an amino acid
sequence which shares at least 90% identity with an amino acid
sequence selected from the group consisting of SEQ ID NO:1-31,
33-104, 106-112, 114-116, 120-136, and 138-172; e) a nucleotide
sequence which encodes an amino acid sharing at least 95% identity
with an amino acid sequence selected from the group consisting of
SEQ ID NO:105 and 113; f) a nucleotide sequence which encodes an
amino acid sharing at least 98% identity with amino acid sequence
SEQ ID NO:32; or g) a nucleotide sequence complementary to any one
of (a) to (f).
63. An isolated polynucleotide which comprises: a) a nucleotide
sequence which encodes at least 15 amino acids of SEQ ID NO:117 or
118; b) a nucleotide sequence which encodes at least 15 amino acids
of SEQ ID NO:119; c) a nucleotide sequence which encodes at least
15 amino acids of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-31, 34-73, 75-104, 106-112, 114-116,
120-132, 134-136, 138-172; d) a nucleotide sequence which encodes
an amino acid sequence which comprises at least 17 amino acids SEQ
ID NO:105; e) a nucleotide sequence which encodes an amino acid
sequence which comprises at least 19 amino acids of SEQ ID NO:74;
f) a nucleotide sequence which encodes an amino acid sequence which
comprises at least 22 amino acids of an amino acid sequence
selected from the group consisting of SEQ ID NO:33, 113, and 133;
or g) a nucleotide sequence complementary to any one of (a) to
(f).
64. A vector which comprises the isolated polynucleotide of any one
of claims 61 to 63.
65. A host cell which is genetically modified to express the
polypeptide or peptide of any one of claims 58 to 60.
66. A host cell which is genetically modified to comprise the
polynucleotide of any one of claims 61 to 63.
67. A host cell which comprises the vector of claim 64.
68. A conjugate molecule or fusion molecule which comprises the
polypeptide or peptide of any one of claims 58 to 60.
69. A conjugate molecule or fusion molecule which comprises the
polynucleotide of any one of claims 61 to 63.
70. A method of permeabilising a microbial cell comprising: a)
optionally, producing or isolating the polypeptide or peptide of
any one of claims 58 to 60; and b) contacting the cell with the
polypeptide or peptide.
71. The method of claim 70, wherein the cell is a methanogen.
72. The method of claim 71, wherein the cell is Methanobrevibacter
ruminantium.
73. The method of claim 72, wherein the cell is Methanobrevibacter
ruminantium strain M1.sup.T (DSM1093).
74. A method of permeabilising a microbial cell comprising: a)
optionally, producing or isolating the conjugate molecule or fusion
molecule of claim 68; and b) contacting the cell with the conjugate
molecule or the fusion molecule.
75. The method of claim 74, wherein the cell is a methanogen.
76. The method of claim 75, wherein the cell is Methanobrevibacter
ruminantium.
77. The method of claim 76, wherein the cell is Methanobrevibacter
ruminantium strain MIT (DSM1093).
Description
RELATED APPLICATIONS
[0001] This is a continuation application under 35 U.S.C. .sctn.120
of U.S. patent application Ser. No. 12/678,936, entitled "Phage
.phi.mru Polynucleotides And Polypeptides And Uses Thereof," which
has a filing date of Mar. 24, 2010, which is a 35 U.S.C. .sctn.371
national phase application of International Application No.
PCT/NZ2008/000248, filed Sep. 25, 2008, which claims the benefit of
U.S. Provisional Application No. 60/975,104, filed Sep. 25, 2007,
U.S. Provisional Application No. 60/989,840, filed Nov. 22, 2007,
and U.S. Provisional Application No. 60/989,841, filed Nov. 22,
2007, the contents of all of which are hereby incorporated by
reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods for
delivering inhibitory molecules into microbial cells, in
particular, methanogen cells. Specifically, the invention relates
to newly identified phage .phi.mru, including phage induction,
phage particles, and the phage genome, and also phage polypeptides,
as well as polynucleotides which encode these polypeptides. The
invention also relates to expression vectors and host cells for
producing these polypeptides. The invention further relates to
methods for detecting, targeting, and inhibiting microbial cells,
especially methanogen cells, using the disclosed phage,
polypeptides, polynucleotides, expression vectors, and host
cells.
BACKGROUND OF THE INVENTION
[0003] In New Zealand, agricultural activity accounts for the
majority of greenhouse gas emissions. Therefore, reducing
agricultural emissions of greenhouse gases is important for meeting
New Zealand's obligations under the Kyoto Protocol. The Protocol
requires reduction of greenhouse gases to 1990 levels by the end of
the first commitment period (2008-2012). To this end, agricultural
sector groups and the New Zealand government established the
Pastoral Greenhouse Gas Research Consortium (PGGRC) to identify
means for reducing New Zealand's agricultural greenhouse gas
emissions.
[0004] An important part of the PGGRC's activities has been
research into reducing methane emissions from New Zealand's grazing
ruminants. Mitigating methane emissions from ruminants is of
commercial interest for two reasons. First, failure to meet
commitments under the Kyoto Protocol will force the government to
purchase carbon credits. This is currently estimated to cost $350
million. Second, methane production results in the loss of 8-12% of
the gross energy produced in the rumen. This energy could be used,
instead, to improve ruminant productivity.
[0005] Methane is produced in the rumen by microbes called
methanogens which are part of the phylum Euryarchaeota within the
kingdom Archaea. Most methanogens grow on CO.sub.2 and H.sub.2 as
their sole energy source, but some can use acetate or methyl
compounds for growth. Several different genera of methanogenic
archaea exist in the rumen, but species of the genus
Methanobrevibacter, especially M. ruminantium, and M. smithii are
thought to be the predominant methanogens in New Zealand ruminants.
M. ruminantium is currently the subject of a genome sequencing
project funded by the PGGRC. The project is the first genome
sequencing of a rumen methanogen and it aims to build a better
understanding of the biology of Methanobrevibacter to discover
targets for inhibition of methane formation.
[0006] Reducing methane production in the rumen requires the
inhibition of methanogens or the inactivation of their
methanogenesis pathway. A means of inhibiting methane production is
to deliver specific inhibitory molecules into methanogen cells.
This may be achieved, for example, by use of agents, such as
bacteriophage, which specifically target methanogens. Several phage
have been characterised for non-rumen methanogens but there have
been no published accounts of phage able to infect or lyse rumen
methanogens. Therefore, it would be highly advantageous to identify
phage that have the ability to infect methanogen cells and/or
deliver inhibitors.
SUMMARY OF THE INVENTION
[0007] The invention features an isolated phage .phi.mru, including
a phage particle and/or phage genome, produced in whole or in part,
as well as isolated polynucleotides and polypeptides of the phage
as described in detail herein.
[0008] The invention also features an isolated polypeptide
comprising at least one phage amino acid sequence selected from the
group consisting of SEQ ID NO:1-69. In a particular aspect, the
polypeptide comprises the amino acid sequence selected from the
group consisting of SEQ ID NO:2-5 and 62-68. In a further aspect,
the polypeptide comprises the amino acid sequence of SEQ ID NO:63.
In another aspect, the polypeptide is a fragment, for example,
comprising at least one amino acid sequence extending from residues
32-186 of SEQ ID NO:63.
[0009] The invention additionally features an isolated
polynucleotide comprising a coding sequence for at least one phage
polypeptide. In one aspect, the polynucleotide comprises a coding
sequence for at least one amino acid sequence selected from the
group consisting of SEQ ID NO:1-69. In a particular aspect, the
polynucleotide comprises a coding sequence for a sequence selected
from the group consisting of SEQ ID NO:2-5 and 62-68. In a further
aspect, the polynucleotide comprises a coding sequence for SEQ ID
NO:63. In another aspect, the polynucleotide comprises a fragment
of a coding sequence, for example, least one amino acid sequence
extending from residues 32-186 of SEQ ID NO:63.
[0010] In an additional aspect, the invention features an isolated
polynucleotide comprising a phage nucleic acid sequence selected
from the group consisting of SEQ ID NO:74-142. In a particular
aspect, the polynucleotide comprises a nucleic acid sequence
selected from the group consisting of SEQ ID NO:75-78 and 135-141,
or is particularly, SEQ ID NO:136. In another aspect, the
polynucleotide is a fragment or an oligonucleotide comprising, for
example, the nucleic acid sequence extending from nucleotides
94-558 of SEQ ID NO:136. In addition, the invention encompasses an
isolated polynucleotide, or fragment thereof, which hybridizes to
any one of the nucleic acid sequences of SEQ ID NO:74-142. The
invention further encompasses an isolated polynucleotide comprising
the complement, reverse complement, reverse sequence, or fragments
thereof, of any one of the nucleic acid sequences.
[0011] The invention features an expression vector comprising a
polynucleotide comprising a coding sequence for at least one phage
polypeptide. In one aspect, the expression vector comprises a
coding sequence for at least one amino acid sequence selected from
the group consisting of SEQ ID NO:1-69. In a particular aspect, the
expression vector comprises a coding sequence for at least one
amino acid sequence of SEQ ID NO:2-5 and 62-68. In a further
aspect, the expression vector comprises a coding sequence for at
least one amino acid sequence of SEQ ID NO:63. In another aspect,
the expression vector comprises a coding sequence for at least one
amino acid sequence extending from residues 32-186 of SEQ ID
NO:63.
[0012] As a specific aspect, the invention features an expression
vector which produces phage .phi.mru, in whole or in part, as
described in detail herein. In particular, the expression vector
may produce phage particles, a phage genome, or modified phage,
including any alterations, derivatives, variants, or fragments
thereof.
[0013] The invention also features a host cell, for example, a
microbial host cell, comprising at least one expression vector.
[0014] The invention specifically features an antibody raised to a
polypeptide or polynucleotide as disclosed herein. In certain
aspects, the antibody is directed to at least one polypeptide
sequence selected from the group consisting of SEQ ID NO:1-69, or a
modified sequence thereof. In alternate aspects, the antibody is
raised to at least a fragment of a polynucleotide selected from the
group consisting of SEQ ID NO:74-142, or a complement, or modified
sequence thereof. In another aspect, the antibody includes one or
more fusions or conjugates with at least one cell inhibitor, for
example, anti-methanogenesis compounds (e.g., bromoethanesulphonic
acid), antibodies and antibody fragments, lytic enzymes, peptide
nucleic acids, antimicrobial peptides, and other antibiotics as
described in detail herein.
[0015] The invention additionally features modified phage
polypeptides, e.g., for at least one of SEQ ID NO:1-69, including
biologically active alterations, fragments, variants, and
derivatives, described herein. The invention additionally features
modified antibodies, e.g., directed to at least one of SEQ ID
NO:1-69, including biologically active alterations, fragments,
variants, and derivatives, described herein. Also featured are
polynucleotides encoding these modified polypeptides, as well as
alterations, fragments, variants, and derivatives of the disclosed
polynucleotides, expression vectors comprising these
polynucleotides, and host cells comprising these vectors. In
specific aspects, the compositions and methods of the invention
employ these modified polynucleotides or polypeptides, or
corresponding expression vectors or host cells.
[0016] In addition, the invention features phage polypeptides,
e.g., at least one of SEQ ID NO:1-69 or modified sequences thereof,
which include fusions or conjugates with at least one cell
inhibitor, for example, anti-methanogenesis compounds (e.g.,
bromoethanesulphonic acid), antibodies and antibody fragments,
lytic enzymes, peptide nucleic acids, antimicrobial peptides, and
other antibiotics as described in detail herein.
[0017] The invention features a composition comprising an isolated
polypeptide, e.g., at least one of SEQ ID NO:1-69, or a modified
sequence thereof. The invention additionally features a composition
comprising an antibody, e.g., directed to at least one of SEQ ID
NO:1-69, or a modified sequence thereof. Also featured is a
composition comprising an isolated polynucleotide, e.g., at least
one of SEQ ID NO:74-142, or a complement or modified sequence
thereof. Further featured is a composition that includes an
expression vector, or host cell comprising an expression vector, in
accordance with the invention. The composition can include any one
of the biologically active alterations, fragments, variants, and
derivatives described herein. The compositions can include at least
one cell inhibitor (e.g., as a fusion or conjugate), and can be
formulated, for example, as pharmaceutical compositions or as food
supplements, in particular, ruminant feed components.
[0018] The invention also features a composition of the invention
as part of a kit for targeting and/or inhibiting microbial cells,
especially methanogen cells, in accordance with the disclosed
methods. The kits comprise: a) at least one composition as set out
herein; and b) optionally, instructions for use, for example, in
targeting cells or inhibiting cell growth or replication for
methanogens or other microbes.
[0019] The invention features a method for producing a phage, the
method comprising: a) culturing an expression vector or host cell
comprising an expression vector, which comprises at least part of
the phage genome under conditions suitable for the production of
the phage; and b) recovering the phage from the culture. In
particular aspects, the phage comprises at least one polypeptide
selected from the group consisting of SEQ ID NO:1-69, or modified
sequences thereof. In further aspects, the phage comprises at least
one polynucleotide selected from the group consisting of SEQ ID
NO:74-142, or modified sequences thereof.
[0020] The invention also features a method for producing a phage
polypeptide, the method comprising: a) culturing an expression
vector or host cell comprising an expression vector, which
comprises at least part of a coding sequence for at least one phage
polypeptide under conditions suitable for the expression of the
polypeptide; and b) recovering the polypeptide from the culture. In
particular aspects, the polypeptide comprises at least one amino
acid sequence selected from the group consisting of SEQ ID NO:1-69,
or modified sequences thereof.
[0021] The invention additionally features a method for producing a
phage polypeptide, e.g., for at least one of SEQ ID NO:1-69, which
comprises a fusion or conjugate with at least one cell inhibitor,
for example, anti-methanogenesis compounds (e.g.,
bromoethanesulphonic acid), antibodies and antibody fragments,
lytic enzymes, peptide nucleic acids, antimicrobial peptides, and
other antibiotics as described in detail herein. Such method
comprises: a) culturing an expression vector or host cell
comprising an expression vector, which comprises a coding sequence
for at least one phage polypeptide under conditions suitable for
the expression of the polypeptide; b) forming the phage fusion or
conjugate (e.g., by expression of the fused sequence or chemical
conjugation to the cell inhibitor); and c) recovering the fusion or
conjugate. In particular aspects, the polypeptide comprises at
least one amino acid sequence selected from the group consisting of
SEQ ID NO:1-69, or modified sequences thereof.
[0022] In addition, the invention features a method of inhibiting
(e.g., inhibiting growth or replication) of a microbial cell, in
particular, a methanogen cell, comprising: a) optionally, producing
or isolating at least one phage polypeptide; and b) contacting the
cell with the phage polypeptide. In a particular aspect, the
polypeptide comprises at least one amino acid sequence selected
from the group consisting of SEQ ID NO:1-69, or a modified sequence
thereof.
[0023] As an added feature, the invention encompasses a method of
inhibiting (e.g., inhibiting growth or replication) of a microbial
cell, in particular, a methanogen cell, comprising: a) optionally,
producing or isolating at least one phage polypeptide, which
further comprises at least one cell inhibitor; and b) contacting
the cell with the phage polypeptide. In a particular aspect, the
polypeptide comprises at least one amino acid sequence selected
from the group consisting of SEQ ID NO:1-69, or a modified sequence
thereof.
[0024] The invention also features a method of detecting and/or
measuring the levels of a phage, or a corresponding phage
polypeptide or polynucleotide, comprising: 1) contacting a sample
from a subject with an antibody raised to a phage polypeptide
(e.g., at least one of SEQ ID NO:1-69, or a modified sequence
thereof) or a corresponding polynucleotide; and 2) determining the
presence or levels of the antibody complex formed with the
polypeptide or polynucleotide in the sample. Such methods can also
be used for detecting and/or measuring the levels of a microbial
cell, in particular, a methanogen cell.
[0025] The invention features, as well, a method of detecting
and/or measuring the levels of a phage, or a corresponding phage
polynucleotide (e.g., a phage coding sequence), comprising: 1)
contacting a sample from a subject with a complementary
polynucleotide (e.g., a sequence complementary to any one of SEQ ID
NO:74-142, or modified sequence thereof); and 2) determining the
presence or levels of the hybridization complex formed with the
phage polynucleotide in the sample. Such methods can also be used
for detecting and/or measuring the levels of a microbial cell, in
particular, a methanogen cell.
[0026] In particular aspects, the methods of the invention utilize
in vivo or in vitro expression components. In other aspects, the
methods employ polypeptides produced by recombinant, synthetic, or
semi-synthetic means, or polypeptides produced by endogenous
means.
[0027] Other aspects and embodiments of the invention are described
herein below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] This invention is described with reference to specific
embodiments thereof and with reference to the figures.
[0029] FIGS. 1A-1B. M. ruminantium prophage .phi.mru showing
putative integration site sequences attL and attR (FIG. 1A), and
predicted phage functional modules and gene structure (FIG.
1B).
[0030] FIG. 1C: Phage induction using sterile air (oxygen
stress).
[0031] FIG. 1D: Initial phage induction using MitomycinC.
[0032] FIG. 1E: Agarose gel electrophoresis of PCR amplicons of
induced (oxygen challenge) and uninduced M. ruminantium. Lanes 2
and 4: 1 kb DNA marker ladder by Invitrogen. Lanes 1 and 3
represent PCRs using primer-pair R1F-L2R on DNA isolated from
induced and uninduced M. ruminantium cultures, respectively.
[0033] FIG. 2. Prophage .phi.mru open reading frame annotation and
comments.
[0034] FIG. 3. Prophage .phi.mru open reading frame annotation,
predicted function, and comments.
[0035] FIGS. 4A-4B. M. ruminantium prophage .phi.mru sequence
information, including coding sequences of phage .phi.mru (FIG.
4A), and amino acid sequences of phage .phi.mru (FIG. 4B).
[0036] FIG. 5. Sequence alignment of phage .phi.mru ORF 2058 with
PeiP from M. marburgensis and PeiW from M. wolfeii.
[0037] FIG. 6: Protein sequence logo of signal peptide sequences
from M. ruminantium created using LogoBar, showing the core
consensus signal.
[0038] FIG. 7: Inhibitory effect of ORF 2058 on resting M.
ruminantium cells.
[0039] FIG. 8: Inhibitory effect of ORF 2058 on M. ruminantium cell
growth and methane production.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0040] "Altered" nucleic acid sequences encoding phage
polypeptides, as used herein, include those with deletions,
insertions, or substitutions of different nucleotides resulting in
a polynucleotide that preferably encodes the same or functionally
equivalent polypeptides. The encoded polypeptide or antibody may
also be "altered" and contain deletions, insertions, or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent polypeptide. Deliberate
amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues as long as the
biological activity (e.g., cell association, cell permeabilisation,
or cell lysis) or immunological activity (e.g., one or more
antibody binding sites) of the polypeptide is retained. For
example, negatively charged amino acids may include aspartic acid
and glutamic acid; positively charged amino acids may include
lysine and arginine; and amino acids with uncharged polar head
groups having similar hydrophilicity values may include leucine,
isoleucine, and valine, glycine and alanine, asparagine and
glutamine, serine and threonine, and phenylalanine and
tyrosine.
[0041] "Amino acid sequence", as used herein, refers to an
oligopeptide, peptide, polypeptide, or protein sequence, and any
fragment thereof, and to naturally occurring, recombinant,
synthetic, or semi-synthetic molecules. The sequences of the
invention comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
100, 150, 200, 250 amino acids, preferably at least 5 to 10, 10 to
20, 20 to 30, 30 to 40, 40 to 50, 50 to 100, 100 to 150, 150 to
200, or 200 to 250, or 250 to 4000 amino acids, and, preferably,
retain the biological activity (e.g., cell association, cell
permeabilisation, or cell lysis) or the immunological activity
(e.g., one or more antibody binding sites) of the original
sequence. Where "amino acid sequence" is recited herein to refer to
an amino acid sequence of a naturally occurring polypeptide
molecule, amino acid sequence, and like terms, are not meant to
limit the amino acid sequence to the complete, original amino acid
sequence associated with the full-length molecule.
[0042] "Amplification", as used herein, refers to the production of
additional copies of a nucleic acid sequence and is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR
Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y.).
[0043] The term "antibody" should be understood in the broadest
possible sense and is intended to include intact monoclonal
antibodies and polyclonal antibodies. It is also intended to cover
fragments and derivatives of antibodies so long as they exhibit the
desired biological activity. Antibodies encompass immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen binding
site that specifically binds (immunoreacts with) an antigen. These
include, but are not limited to, polyclonal, monoclonal, chimeric,
single chain, Fc, Fab, Fab', and Fab.sub.2 fragments, and a Fab
expression library.
[0044] Antibody molecules relate to any of the classes IgG, IgM,
IgA, IgE, and IgD, which differ from one another by the nature of
heavy chain present in the molecule. These include subclasses as
well, such as IgG1, IgG2, and others. The light chain may be a
kappa chain or a lambda chain. Reference herein to antibodies
includes a reference to all classes, subclasses, and types. Also
included are chimeric antibodies, for example, monoclonal
antibodies or fragments thereof that are specific to more than one
source, e.g., one or more mouse, human, or ruminant sequences.
Further included are camelid antibodies or nanobodies. It will be
understood that each reference to "antibodies" or any like term,
herein includes intact antibodies, as well as any fragments,
alterations, derivatives, or variants thereof.
[0045] The terms "biologically active" or "functional," as used
herein, refer to a polypeptide retaining one or more structural,
immunological, or biochemical functions (e.g., cell association,
cell permeabilisation, or cell lysis) sequence.
[0046] The terms "cell inhibitor" or "inhibitor," as used herein,
refer to agents that decrease or block the growth or replication of
microbial cells, especially methanogen cells. A cell inhibitor can
act to decrease or block, for example, cellular division. An
inhibitor can decrease or block, for example, DNA synthesis, RNA
synthesis, protein synthesis, or post-translational modifications.
An inhibitor can also decrease or block the activity of enzymes
involved in the methanogenesis pathway. An inhibitor can also
target a cell for recognition by immune system components.
Inhibition of a cell also includes cell killing and cell death, for
example, from lysis, apoptosis, necrosis, etc. Useful inhibitors
include, but are not limited to, anti-methanogenesis compounds
(e.g., bromoethanesulphonic acid), antibodies and antibody
fragments, lytic enzymes, peptide nucleic acids, antimicrobial
peptides, and other antibiotics as described in detail herein.
[0047] The terms "complementary" or "complementarity," as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base-pairing. For the
sequence A-G-T, the complementary sequence is T-C-A, the reverse
complement is A-C-T and the reverse sequence is T-G-A.
Complementarity between two single-stranded molecules may be
partial, in which only some of the nucleic acids bind, or it may be
complete when total complementarity exists between the single
stranded molecules. The degree of complementarity between nucleic
acid strands has significant effects on the efficiency and strength
of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, which depend upon
binding between nucleic acids strands and in the design and use of
PNA molecules.
[0048] The term "derivative", as used herein, refers to the
chemical modification of a nucleic acid encoding a phage
polypeptide, or a nucleic acid complementary thereto. Such
modifications include, for example, replacement of hydrogen by an
alkyl, acyl, or amino group. In preferred aspects, a nucleic acid
derivative encodes a polypeptide which retains the biological or
immunological function of the natural molecule. A derivative
polypeptide is one which is modified by glycosylation, pegylation,
or any similar process which retains one or more biological
function (e.g., cell association, cell permeabilisation, or cell
lysis) or immunological function of the sequence from which it was
derived.
[0049] The term "homology", as used herein, refers to a degree of
complementarity. There may be partial homology (i.e., I identity)
or complete homology (i.e., 100% identity). A partially
complementary sequence that at least partially inhibits an
identical sequence from hybridizing to a target nucleic acid is
referred to using the functional term "substantially homologous."
The inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or northern blot, solution
hybridization and the like) under conditions of low stringency. A
substantially homologous sequence or hybridization probe will
compete for and inhibit the binding of a completely homologous
sequence to the target sequence under conditions of low stringency.
This is not to say that conditions of low stringency are such that
non-specific binding is permitted; low stringency conditions
require that the binding of two sequences to one another be a
specific (i.e., selective) interaction.
[0050] The term "hybridization", as used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0051] An "insertion" or "addition", as used herein, refers to a
change in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, as compared to the naturally occurring molecule.
[0052] A "methanogen," as used herein, refers to microbes that
produce methane gas, which include Methanobrevibacter,
Methanothermobacter, Methanomicrobium, Methanobacterium, and
Methanosarcina. Specific methanogens include, but are not limited
to, Methanobrevibacter ruminantium, Methanobrevibacter smithii,
Methanobrevibacter acididurans, Methanobrevibacter thaueri,
Methanobacterium bryantii, Methanobacterium formicicum,
Methanothermobacter marburgensis, Methanothermobacter wolfeii,
Methanosphaera stadtmanae, Methanomicrobium mobile, Methanosarcina
barkeri, Methanosarcina mazei, Methanococcoides burtonii, and
Methanolobus taylorii. The Methanobrevibacter ruminantium strain
M1.sup.T is publicly available in depositories at the Leibniz
Institute DSMZ-German Collection of Microorganisms and Cell
Cultures; Braunschweig, Germany (DSM No. DSM1093) and at the
American Type Culture Collection (ATCC; Manassas, Va., USA) (ATCC
No. 35063). All methanogen genera and species are encompassed by
this term.
[0053] "Microbial" cells as used herein, refers to
naturally-occurring or genetically modified microbial cells
including archaebacteria such as methanogens, halophiles, and
thermoacidophiles, and eubacteria, such as cyanobacteria,
spirochetes, proteobacteria, as well as gram positive and gram
negative bacteria.
[0054] The term "modified" refers to altered sequences and to
sequence fragments, variants, and derivatives, as described
herein.
[0055] "Nucleic acid sequence" or "nucleotide sequence" as used
herein, refers to a sequence of a polynucleotide, oligonucleotide,
or fragments thereof, and to DNA or RNA of natural, recombinant,
synthetic, or semi-synthetic origin which may be single or double
stranded, and can represent the sense or antisense strand, and
coding or non-coding regions. The sequences of the invention most
preferably include polypeptide coding sequences that comprise at
least 12, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 300, 450,
600, 750 nucleotides, preferably at least 15 to 30, 30 to 60, 60 to
90, 90 to 120, 120 to 150, 150 to 300, 300 to 450, 450 to 600, or
600 to 750 nucleotides, or at least 1000 nucleotides, or at least
1500 nucleotides. It will be understood that each reference to a
"nucleic acid sequence" or "nucleotide sequence" herein, will
include the original, full-length sequence, as well as any
complements, fragments, alterations, derivatives, or variants,
thereof.
[0056] The term "oligonucleotide" refers to a nucleic acid sequence
comprising at least 6, 8, 10, 12, 15, 18, 21, 25, 27, 30, or 36
nucleotides, or at least 12 to 36 nucleotides, or at least 15 to 30
nucleotides, which can be used, for example, in PCR amplification,
sequencing, or hybridization assays. As used herein,
oligonucleotide is substantially equivalent to the terms
"amplimers," "primers," "oligomers," "oligos," and "probes," as
commonly defined in the art.
[0057] "Polypeptide," as used herein, refers to the isolated
polypeptides of the invention obtained from any species, preferably
microbial, from any source whether natural, synthetic,
semi-synthetic, or recombinant. Specifically, a phage polypeptide
can be obtained from methanogen cells, such as Methanobrevibacter
cells, in particular, M. ruminantium, or M. smithii cells. For
recombinant production, a polypeptide of the invention can be
obtained from microbial or eukaryotic cells, for example,
Escherichia, Streptomyces, Bacillus, Salmonella, yeast, insect
cells such as Drosophila, animal cells such as COS and CHO cells,
or plant cells. It will be understood that each reference to a
"polypeptide," herein, will include the original, full-length
sequence, as well as any fragments, alternations, derivatives, or
variants, thereof.
[0058] The term "polynucleotide," when used in the singular or
plural, generally refers to any nucleic acid sequence, e.g., any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. This includes,
without limitation, single and double stranded DNA, DNA including
single and double-stranded regions, single and double stranded RNA,
and RNA including single and double stranded regions, hybrid
molecules comprising DNA and RNA that may be single stranded or,
more typically, double stranded or include single and double
stranded regions. Also included are triple-stranded regions
comprising RNA or DNA or both RNA and DNA. Specifically included
are mRNAs, cDNAs, and genomic DNAs, and any fragments thereof. The
term includes DNAs and RNAs that contain one or more modified
bases, such as tritiated bases, or unusual bases, such as inosine.
The polynucleotides of the invention can encompass coding or
non-coding sequences, or sense or antisense sequences, or iRNAs
such as siRNAs. It will be understood that each reference to a
"polynucleotide" or like term, herein, will include the full length
sequences as well as any complements, fragments, alterations,
derivatives, or variants thereof.
[0059] "Peptide nucleic acid" or "PNA" as used herein, refers to an
antisense molecule or anti-gene agent which comprises bases linked
via a peptide backbone. The term "ruminant," as used herein, refers
to animals that have a rumen as a special type of digestive organ.
Ruminants include, but are not limited to, cattle, sheep, goats,
buffalo, moose, antelope, caribou, and deer.
[0060] The terms "stringent conditions" or "stringency," as used
herein, refer to the conditions for hybridization as defined by the
nucleic acid, salt, and temperature. These conditions are well
known in the art and may be altered in order to identify or detect
identical or related polynucleotide sequences. See, e.g., Sambrook,
J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al.
(1989) Current Protocols in Molecular Biology, John Wiley &
Sons, New York, N.Y. Numerous equivalent conditions comprising
either low or high stringency depend on factors such as the length
and nature of the sequence (DNA, RNA, base composition), nature of
the target (DNA, RNA, base composition), milieu (in solution or
immobilized on a solid substrate), concentration of salts and other
components (e.g., formamide, dextran sulfate and/or polyethylene
glycol), and temperature of the reactions (within a range from
about 5.degree. C. below the melting temperature of the probe to
about 20.degree. C. to 25.degree. C. below the melting
temperature). One or more factors be may be varied to generate
conditions of either low or high stringency different from, but
equivalent to, the above listed conditions.
[0061] The term "subject" includes human and non-human animals.
Non-human animals include, but are not limited to, birds and
mammals, such as ruminants, and in particular, mice, rabbits, cats,
dogs, pigs, sheep, goats, cows, and horses.
[0062] The terms "substantially purified" or "isolated" as used
herein, refer to nucleic or amino acid sequences that are removed
from their cellular, recombinant, or synthetic environment, and are
at least 60% free, preferably 75% free, and most preferably at
least 90% free or at least 99% free from other components with
which they are associated in a cellular, recombinant, or synthetic
environment.
[0063] "Transformation," as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell. It may
occur under natural or artificial conditions using various methods
well known in the art. Transformation may rely on any known method
for the insertion of foreign nucleic acid sequences into a
prokaryotic or eukaryotic host cell. The method is selected based
on the type of host cell being transformed and may include, but is
not limited to, viral infection, electroporation, heat shock,
lipofection, and particle bombardment. Such "transformed" cells
include stably transformed cells in which the inserted DNA is
capable of replication either as an autonomously replicating
plasmid or as part of the host chromosome. They also include cells
which transiently express the inserted DNA or RNA for limited
periods of time.
[0064] A "variant" of a polypeptide, as used herein, refers to an
amino acid sequence that is altered by one or more amino acids. A
variant polynucleotide is altered by one or more nucleotides. A
variant may result in "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties, e.g.,
replacement of leucine with isoleucine. More rarely, a variant may
result in "nonconservative" changes, e.g., replacement of a glycine
with a tryptophan. Analogous minor variations may also include
amino acid deletions or insertions, or both. Guidance in
determining which amino acid residues may be substituted, inserted,
or deleted without abolishing biological or immunological activity
may be found using computer programs well known in the art, for
example, LASERGENE software (DNASTAR).
[0065] The invention also encompasses variants which retain at
least one biological activity (e.g., cell association, cell
permeabilisation, or cell lysis) or immunological activity of the
polypeptide. A preferred variant is one having substantially the
same or a functionally equivalent sequence, for example, at least
80%, and more preferably at least 90%, sequence identity to a
disclosed sequence. A most preferred variant is one having at least
95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at
least 99.8%, or at least 99.9% sequence identity to a sequence
disclosed herein. The percentage identity is determined by aligning
the two sequences to be compared as described below, determining
the number of identical residues in the aligned portion, dividing
that number by the total number of residues in the inventive
(queried) sequence, and multiplying the result by 100. A useful
alignment program is AlignX (Vector NTI).
DESCRIPTION OF THE INVENTION
[0066] Methane is produced in the foregut of ruminants by
methanogens which act as terminal reducers of carbon in the rumen
system. The multi-step methanogenesis pathway is well elucidated,
mainly from the study of non-rumen methanogens, but the adaptations
that allow methanogens to grow and persist in the rumen are not
well understood. Methanobrevibacter ruminantium is a prominent
methanogen in New Zealand ruminants. As described herein, the
genome of M. ruminantium has been sequenced and shown as
approximately 3.0 Mb in size with a GC content of 33.68%. As an
unexpected finding, the M. ruminantium genome was found to include
a prophage sequence (designated .phi.mru) with distinct functional
modules encoding phage integration, DNA replication and packaging,
capsid proteins, lysis, and lysogenic conversion functions.
[0067] The M. ruminantium phage was identified during high
through-put sequencing, when a 30 to 40 kb region of the genome was
found to be over-represented in the sequenced clones. This
suggested that a part of the genome was present in higher copy
number than normal, and could be attributed to the replication of a
resident phage. The over-represented region was investigated and
detailed bioinformatic analyses of the predicted open reading
frames present indicated that it contained phage-like genes. A low
GC region found at the distal end of the phage sequence (lysogenic
conversion) has been shown to harbour a predicted DNA modification
system by sulphur (dnd) which might provide additional modification
of host or foreign DNA. The M. ruminantium prophage sequence is
described in detail herein. In various aspects of the invention,
the prophage polynucleotides and polypeptides can be used as a
means for inhibiting methanogens and/or methanogenesis in the
rumen, and to further elucidate the role of M. ruminantium in
methane formation.
[0068] The invention therefore encompasses phage polypeptides,
including those comprising at least one of SEQ ID NO:1-69, and
fragments, variants, and derivatives thereof. The invention also
encompasses the use of these polypeptides for targeting and
inhibiting microbial cells, especially methanogen cells. The
invention further encompasses the use of the polypeptides for the
inhibition of growth or replication of such cells. The polypeptides
of the present invention may be expressed and used in various
assays to determine their biological activity. The polypeptides may
be used for large-scale synthesis and isolation protocols, for
example, for commercial production. Such polypeptides may be used
to raise antibodies, to isolate corresponding amino acid sequences,
and to quantitatively determine levels of the amino acid sequences.
The polypeptides of the present invention may also be used as
compositions, for example, pharmaceutical compositions, and as food
supplements, e.g., ruminant feed components. The polypeptides of
the present invention also have health benefits. In heath-related
aspects, inhibitors of methanogens can be used to restore energy to
the subject that is normally lost as methane. In particular
aspects, slow-release ruminal devices can be used in conjunction
with the polypeptides, and compositions (e.g., pharmaceutical
compositions and food supplements) of the invention.
[0069] The polypeptides of the present invention comprise at least
one sequence selected from the group consisting of: (a)
polypeptides comprising at least one amino acid sequence selected
from the group consisting of SEQ ID NO:1-69, or fragments,
variants, or derivatives thereof; (b) polypeptides comprising a
functional domain of at least one amino acid sequence selected from
the group consisting of SEQ ID NO:1-69, and fragments and variants
thereof; and (c) polypeptides comprising at least a specified
number of contiguous residues of at least one amino acid sequence
selected from the group consisting of SEQ ID NO:1-69, or variants
or derivatives thereof. In one embodiment, the invention
encompasses an isolated polypeptide comprising the amino acid
sequence of at least one of SEQ ID NO:1-69. All of these sequences
are collectively referred to herein as polypeptides of the
invention.
[0070] The invention also encompasses polynucleotides that encode
at least one phage polypeptide, including those of SEQ ID NO:1-69,
and fragments, variants, and derivatives thereof. The invention
also encompasses the use of these polynucleotides for preparing
expression vectors and host cells for targeting and inhibiting
microbial cells, especially methanogen cells. The invention further
encompasses the use of the polynucleotides for the inhibition of
growth or replication of such cells. The isolated polynucleotides
of the present invention also have utility in genome mapping, in
physical mapping, and in cloning of genes of more or less related
phage. Probes designed using the polynucleotides of the present
invention may be used to detect the presence and examine the
expression patterns of genes in any organism having sufficiently
homologous DNA and RNA sequences in their cells, using techniques
that are well known in the art, such as slot blot techniques or
microarray analysis. Primers designed using the polynucleotides of
the present invention may be used for sequencing and PCR
amplifications. The polynucleotides of the present invention may
also be used as compositions, for example, pharmaceutical
compositions, and as food supplements, e.g., ruminant feed
components. The polynucleotides of the present invention also have
health benefits. For such benefits, the polynucleotides can be
presented as expression vectors or host cells comprising expression
vectors. In particular aspects, slow-release ruminal devices can be
used in conjunction with the polynucleotides, vectors, host cells,
and compositions (e.g., pharmaceutical compositions and food
supplements) of the invention.
[0071] The polynucleotides of the present invention comprise at
least one sequence selected from the group consisting of: (a)
sequences comprising a coding sequence for at least one amino acid
sequence selected from the group consisting of SEQ ID NO:1-69, or
fragments or variants thereof; (b) complements, reverse sequences,
and reverse complements of a coding sequence for at least one amino
acid sequence selected from the group consisting of SEQ ID NO:1-69,
or fragments or variants thereof; (c) open reading frames contained
in the coding sequence for at least one amino acid sequence
selected from the group consisting of SEQ ID NO:1-69, and their
fragments and variants; (d) functional domains of a coding sequence
for at least one amino acid sequence selected from the group
consisting of SEQ ID NO:1-69, and fragments and variants thereof;
and (e) sequences comprising at least a specified number of
contiguous residues of a coding sequence for at least one amino
acid sequence selected from the group consisting of SEQ ID NO:1-69,
or variants thereof. In one embodiment, the invention encompasses
an isolated polynucleotide comprising a coding sequence for at
least one amino acid sequence selected from the group consisting of
SEQ ID NO:1-69.
[0072] The polynucleotides of the present invention comprise at
least one sequence selected from the group consisting of: (a)
sequences comprising at least one nucleic acid sequence selected
from the group consisting of SEQ ID NO:74-142, or fragments or
variants thereof; (b) complements, reverse sequences, and reverse
complements of a coding sequence for at least one nucleic acid
sequence selected from the group consisting of SEQ ID NO:74-142, or
fragments or variants thereof; (c) open reading frames contained in
the nucleic acid sequence selected from the group consisting of SEQ
ID NO:74-142, and their fragments and variants; (d) functional
domains of a coding sequence of at least one nucleic acid sequence
selected from the group consisting of SEQ ID NO:74-142, and
fragments and variants thereof; and (e) sequences comprising at
least a specified number of contiguous residues of at least one
nucleic acid sequence selected from the group consisting of SEQ ID
NO:74-142, or variants thereof. Oligonucleotide probes and primers
and their variants obtained from any of the disclosed sequences are
also provided. All of these polynucleotides and oligonucleotides
are collectively referred to herein, as polynucleotides of the
invention.
[0073] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
nucleotide sequences encoding the polypeptides of the invention,
some bearing minimal homology to the nucleotide sequences of any
known and naturally occurring gene, may be produced. Thus, the
invention contemplates each and every possible variation of
nucleotide sequence that could be made by selecting combinations
based on possible codon choices. These combinations are made in
accordance with the standard bacterial triplet genetic code as
applied to naturally occurring amino acid sequences, and all such
variations are to be considered as being specifically
disclosed.
[0074] Nucleotide sequences which encode the phage polypeptides, or
their fragments or variants, are preferably capable of hybridizing
to the nucleotide sequence of the naturally occurring sequence
under appropriately selected conditions of stringency. However, it
may be advantageous to produce nucleotide sequences encoding a
polypeptide, or its fragment or derivative, possessing a
substantially different codon usage. Codons may be selected to
increase the rate at which expression of the polypeptide occurs in
a particular prokaryotic or eukaryotic host in accordance with the
frequency with which particular codons are utilized by the host.
For example, codons can be optimized for expression in E. coli in
accordance with known methods. Other reasons for substantially
altering the nucleotide sequence encoding polypeptides and its
derivatives without altering the encoded amino acid sequences
include the production of RNA transcripts having more desirable
properties, such as a greater half-life, than transcripts produced
from the naturally occurring sequence.
[0075] The invention also encompasses production of DNA sequences,
or fragments thereof, which encode the polypeptides, or their
fragments or variants, entirely by synthetic chemistry. After
production, the synthetic sequence may be inserted into any of the
many available expression vectors and cell systems using reagents
that are well known in the art. Moreover, synthetic chemistry may
be used to introduce mutations into a sequence encoding a
polypeptide, or any variants or fragment thereof. Also encompassed
by the invention are polynucleotide sequences that are capable of
hybridizing to the claimed nucleotide sequences, and in particular,
those shown in SEQ ID NO:74-149, or their complements, under
various conditions of stringency as taught in Wahl, G. M. and S. L.
Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R.
(1987; Methods Enzymol. 152:507-511).
[0076] Methods for DNA sequencing which are well known and
generally available in the art and may be used to practice any of
the embodiments of the invention. The methods may employ such
enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (U.S.
Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase Amersham Pharmacia Biotech (Piscataway,
N.J.), or combinations of polymerases and proofreading exonucleases
such as those found in the ELONGASE Amplification System marketed
by Life Technologies (Gaithersburg, Md.). Preferably, the process
is automated with machines such as the Hamilton Micro Lab 2200
(Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ
Research, Watertown, Mass.) the ABI Catalyst and 373 and 377 DNA
Sequencers (Perkin Elmer), or the Genome Sequencer 20.TM. (Roche
Diagnostics).
[0077] The nucleic acid sequences encoding the polypeptides may be
extended utilizing a partial nucleotide sequence and employing
various methods known in the art to detect upstream sequences such
as promoters and regulatory elements, and downstream elements such
as terminators and non-coding RNA structures. For example, one
method which may be employed, "restriction-site" PCR, uses
universal primers to retrieve unknown sequence adjacent to a known
locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). In
particular, genomic DNA is first amplified in the presence of
primer to a linker sequence and a primer specific to the known
region. The amplified sequences are then subjected to a second
round of PCR with the same linker primer and another specific
primer internal to the first one. Products of each round of PCR are
transcribed with an appropriate RNA polymerase and sequenced using
reverse transcriptase.
[0078] Another useful method is inverse PCR, also called IPCR (see,
e.g., Ochman H, Gerber A S, Hartl D L. Genetics. 1988 November;
120(3):621-3). Inverse PCR can be employed when only one internal
sequence of the target DNA is known. The inverse PCR method
includes a series of digestions and self-ligations with the DNA
being cut by a restriction endonuclease. This cut results in a
known sequence at either end of unknown sequences. In accordance
with this method, target DNA is lightly cut into smaller fragments
of several kilobases by restriction endonuclease digestion.
Self-ligation is then induced under low concentrations causing the
phosphate backbone to reform and produce a circular DNA ligation
product. Target DNA is then restriction digested with a known
endonuclease. This generates a cut within the known internal
sequence generating a linear product with known terminal sequences.
This product can then be used for standard PCR conducted with
primers complementary to the known internal sequences.
[0079] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and detection of the emitted
wavelengths by a charge coupled device camera. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer) and
the entire process from loading of samples to computer analysis and
electronic data display may be computer controlled. Capillary
electrophoresis is especially preferable for the sequencing of
small pieces of DNA which might be present in limited amounts in a
particular sample.
[0080] Recently, pyrosequencing has emerged as a useful sequencing
methodology. See, e.g., Ronaghi, M. et al. 1996. Real-time DNA
sequencing using detection of pyrophosphate release. Anal. Biochem.
242: 84-89; Ronaghi, M. et al. 1998. A sequencing method based on
real-time pyrophosphate. Science 281: 363-365; Ronaghi, M. et al.
1999. Analyses of secondary structures in DNA by pyrosequencing.
Anal. Biochem. 267: 65-71; Ronaghi 2001. Genome Res. Vol. 11, Issue
1, 3-11; Nyren The history of pyrosequencing. Methods Mol. Biol.
2007; 373:1-14. Pyrosequencing has the advantages of accuracy,
flexibility, parallel processing, and can be easily automated.
Furthermore, the technique dispenses with the need for labeled
primers, labeled nucleotides, and gel-electrophoresis. In
accordance with this method, polymerase catalyzes incorporation of
nucleotides into a nucleic acid chain. As a result of the
incorporation, pyrophosphate molecules are released and
subsequently converted by sulfurylase to ATP. Light is produced in
the luciferase reaction during which a luciferin molecule is
oxidized. After each nucleotide addition, a washing step is
performed to allow iterative addition. The nucleotides are
continuously degraded by nucleotide-degrading enzyme allowing
addition of subsequent nucleotide. Pyrosequencing has been
successfully applied as a platform for large-scale sequencing,
including genomic and metagenomic analysis (see, e.g., The Genome
Sequencer FLX.TM. from 454 Life Sciences/Roche).
[0081] The SOLiD.TM. System has also been developed for sequencing
(see, e.g., Applied Biosystems. Application Fact Sheet for the
SOLiD.TM. System. Foster City, Calif.). This methodology is based
on sequential ligation of dye-labeled oligonucleotides to clonally
amplified DNA fragments linked to magnetic beads. In this method,
the DNA sequence is generated by measuring serial ligation. The
ligation reaction is based on probe recognition, not sequential
addition, and is therefore less prone to accumulation of errors.
The nature of the chemistry virtually eliminates the possibility of
spurious insertions or deletions. The ligation step and phosphatase
treatment of unligated probes prevents dephasing. In addition,
after seven cycles of ligation, the original primer is stripped
from the template and a new primer is hybridized to begin
interrogating at the n-1 position. Use of this "reset" phase allows
for reduction in systemic noise and allows for longer read lengths.
In addition, two base encoding is used to discriminate between
measurement errors as opposed to true polymorphisms. Changes at a
single position are identified as random errors and can be removed
by the software in data analysis. As an analytical platform, the
SOLiD.TM. System has applications in large-scale sequencing,
digital gene expression, ChIP and methylation studies, and is
particularly useful for detecting genomic variation.
[0082] In another embodiment of the invention, polynucleotides or
fragments thereof which encode polypeptides may be used in
recombinant DNA molecules to direct expression of the polypeptides,
or fragments or variants thereof, in appropriate host cells. Due to
the inherent degeneracy of the genetic code, other DNA sequences
which encode substantially the same or a functionally equivalent
amino acid sequence may be produced, and these sequences may be
used to clone and express phage polypeptides. The nucleotide
sequences of the present invention can be engineered using methods
generally known in the art in order to alter amino acid-encoding
sequences for a variety of reasons, including, but not limited to,
alterations which modify the cloning, processing, and/or expression
of the gene product. DNA shuffling by random fragmentation and PCR
reassembly of gene fragments and synthetic oligonucleotides may be
used to engineer the nucleotide sequences. For example,
site-directed mutagenesis may be used to insert new restriction
sites, alter glycosylation patterns, change codon preference,
introduce mutations, and so forth.
[0083] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding polypeptides may be
ligated to a heterologous sequence to encode a fusion protein. For
example, it may be useful to encode a chimeric sequence that can be
recognized by a commercially available antibody. A fusion protein
may also be engineered to contain a cleavage site located between
the polypeptide of the invention and the heterologous protein
sequence, so that the polypeptide may be cleaved and purified away
from the heterologous moiety.
[0084] In another embodiment, sequences encoding polypeptides may
be synthesized, in whole or in part, using chemical methods well
known in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids
Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res.
Symp. Ser. 225-232). Alternatively, the polypeptide itself may be
produced using chemical methods to synthesize the amino acid
sequence, or a fragment thereof. For example, polypeptide synthesis
can be performed using various solid-phase techniques (Roberge, J.
Y. et al. (1995) Science 269:202-204; Merrifield J. (1963) J. Am.
Chem. Soc. 85:2149-2154) and automated synthesis may be achieved,
for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer).
Various fragments of polypeptides may be chemically synthesized
separately and combined using chemical methods to produce the full
length molecule.
[0085] The newly synthesized polypeptide may be isolated by
preparative high performance liquid chromatography (e.g.,
Creighton, T. (1983) Proteins Structures and Molecular Principles,
WH Freeman and Co., New York, N.Y.). The composition of the
synthetic polypeptides may be confirmed by amino acid analysis or
sequencing (e.g., the Edman degradation procedure; Creighton,
supra). Additionally, the amino acid sequence of the polypeptide,
or any part thereof, may be altered during direct synthesis and/or
combined using chemical methods with sequences from other proteins,
or any part thereof, to produce a variant molecule.
[0086] In order to express biologically active polypeptides, the
nucleotide sequences encoding the polypeptide or functional
equivalents, may be inserted into appropriate expression vector,
i.e., a vector which contains the necessary elements for the
transcription and translation of the inserted coding sequence.
Methods which are well known to those skilled in the art may be
used to construct expression vectors containing sequences encoding
the polypeptide and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook, J. et al.
(2001) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, Plainview, N.Y., and Ausubel, F. M. et al. (2007) Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y.
[0087] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding the polypeptides of the
invention. These include, but are not limited to, microorganisms
such as bacteria transformed with recombinant phage, plasmid, or
cosmid DNA expression vectors; yeast transformed with yeast
expression vectors; insect cell systems infected with virus
expression vectors (e.g., baculovirus); plant cell systems
transformed with virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or with bacterial
expression vectors (e.g., Ti or pBR322 plasmids); or animal cell
systems. For bacteria, useful plasmids include pET, pRSET,
pTrcHis2, and pBAD plasmids from Invitrogen, pET and pCDF plasmids
from Novagen, and Director.TM. plasmids from Sigma-Aldrich. For
methanogens, useful plasmids include, but are not limited to
pME2001, pMV15, and pMP1. In particular, Escherichia coli can be
used with the expression vector pET. The invention is not limited
by the expression vector or host cell employed.
[0088] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector--enhancers, promoters, 5' and
3' untranslated regions--which interact with host cellular proteins
to carry out transcription and translation. Such elements may vary
in their strength and specificity. Depending on the vector system
and host utilized, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters, may be used. For example, when cloning in bacterial
systems, inducible promoters such as the hybrid lacZ promoter of
the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORT1
plasmid (Life Technologies) and the like may be used. The
baculovirus polyhedrin promoter may be used in insect cells.
Promoters or enhancers derived from the genomes of plant cells
(e.g., heat shock, RUBISCO, and storage protein genes) or from
plant viruses (e.g., viral promoters or leader sequences) may be
cloned into the vector.
[0089] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for the polypeptide. For
example, when large quantities of polypeptide are needed, vectors
which direct high level expression of fusion proteins that are
readily purified may be used. Such vectors include, but are not
limited to, the multifunctional E. coli cloning and expression
vectors such as BLUESCRIPT (Stratagene), in which the sequence
encoding a polypeptide may be ligated into the vector in frame with
sequences for the amino-terminal Met and the subsequent 7 residues
of .beta.-galactosidase so that a hybrid protein is produced; pIN
vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.
264:5503-5509); and the like.
[0090] pGEX vectors (Promega, Madison, Wis.) may also be used to
express the polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. Proteins made in such systems may be designed to
include heparin, thrombin, or factor Xa protease cleavage sites so
that the cloned polypeptide of interest can be released from the
GST moiety at will. In the yeast, Saccharomyces cerevisiae, a
number of vectors containing constitutive or inducible promoters
such as alpha factor, alcohol oxidase, and PGH may be used. For
reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods
Enzymol. 153:516-544.
[0091] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding the polypeptides of the
invention. Such signals include the ATG initiation codon and
adjacent sequences. In cases where sequences encoding a
polypeptide, its initiation codon, and upstream sequences are
inserted into the appropriate expression vector, no additional
transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers which are appropriate for the
particular cell system which is used, such as those described in
the literature (Scharf, D. et al. (1994) Results Probl. Cell
Differ. 20:125-162).
[0092] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed polypeptide in the desired fashion. Such
modifications of the sequence include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the polypeptide may also be used to
facilitate correct insertion, folding, and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities are
available from the American Type Culture Collection (ATCC;
Bethesda, Md.) and may be chosen to ensure the correct modification
and processing of the sequence. Specific host cells include, but
are not limited to, methanogen cells, such as Methanobrevibacter
cells, in particular, M. ruminantium, or M. smithii cells. Host
cells of interest include, for example, Rhodotorula, Aureobasidium,
Saccharomyces, Sporobolomyces, Pseudomonas, Erwinia and
Flavobacterium; or such other organisms as Escherichia,
Lactobacillus, Bacillus, Streptomyces, and the like. Specific host
cells include Escherichia coli, which is particularly suited for
use with the present invention, Saccharomyces cerevisiae, Bacillus
thuringiensis, Bacillus subtilis, Streptomyces lividans, and the
like.
[0093] There are several techniques for introducing nucleic acids
into eukaryotic cells cultured in vitro. These include chemical
methods (Feigner et al., Proc. Natl. Acad. Sci., USA, 84:7413 7417
(1987); Bothwell et al., Methods for Cloning and Analysis of
Eukaryotic Genes, Eds., Jones and Bartlett Publishers Inc., Boston,
Mass. (1990), Ausubel et al., Short Protocols in Molecular Biology,
John Wiley and Sons, New York, N.Y. (1992); and Farhood, Annal. NY
Acad. Sci., 716:23 34 (1994)), use of protoplasts (Bothwell, supra)
or electrical pulses (Vatteroni et al., Mutn. Res., 291:163 169
(1993); Sabelnikov, Prog. Biophys. Mol. Biol., 62: 119 152 (1994);
Bothwell et al., supra; and Ausubel et al., supra), use of
attenuated viruses (Davis et al., J. Virol. 1996, 70(6), 3781 3787;
Brinster et al. J. Gen. Virol. 2002, 83(Pt 2), 369 381; Moss, Dev.
Biol. Stan., 82:55 63 (1994); and Bothwell et al., supra), as well
as physical methods (Fynan et al., supra; Johnston et al., Meth.
Cell Biol., 43(Pt A):353 365 (1994); Bothwell et al., supra; and
Ausubel et al., supra).
[0094] Successful delivery of nucleic acids to animal tissue can be
achieved by cationic liposomes (Watanabe et al., Mol. Reprod. Dev.,
38:268 274 (1994)), direct injection of naked DNA or RNA into
animal muscle tissue (Robinson et al., Vacc., 11:957 960 (1993);
Hoffman et al., Vacc. 12:1529 1533; (1994); Xiang et al., Virol.,
199:132 140 (1994); Webster et al., Vacc., 12:1495 1498 (1994);
Davis et al., Vacc., 12:1503 1509 (1994); Davis et al., Hum. Molec.
Gen., 2:1847 1851 (1993); Dalemans et al. Ann NY Acad. Sci. 1995,
772, 255 256. Conry, et al. Cancer Res. 1995, 55(7), 1397-1400),
and embryos (Naito et al., Mol. Reprod. Dev., 39:153 161 (1994);
and Burdon et al., Mol. Reprod. Dev., 33:436 442 (1992)),
intramuscular injection of self replicating RNA vaccines (Davis et
al., J Virol 1996, 70(6), 3781 3787; Balasuriya et al. Vaccine
2002, 20(11 12), 1609 1617) or intradermal injection of DNA using
"gene gun" technology (Johnston et al., supra).
[0095] A variety of protocols for detecting and measuring the
expression of the polypeptides of the invention, using either
polyclonal or monoclonal antibodies specific for the protein are
known in the art. Examples include enzyme-linked immunosorbent
assay (ELISA), radioimmunoassay (RIA), and fluorescence activated
cell sorting (FACS). A two-site, monoclonal-based immunoassay can
be used with monoclonal antibodies reactive to two non-interfering
epitopes on the polypeptide, but a competitive binding assay can
also be used. These and other assays are described, among other
places, in Hampton, R. et al. (1990; Serological Methods, a
laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et
al. (1983; J. Exp. Med. 158:1211-1216).
[0096] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides include oligolabeling, nick translation,
end-labeling or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding the polypeptides, or any
fragments or variants thereof, may be cloned into a vector for the
production of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits Amersham Pharmacia
Biotech, Promega, and US Biochemical. Suitable reporter molecules
or labels, which may be used for ease of detection, include
radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents as well as substrates, cofactors, inhibitors,
magnetic particles, and the like.
[0097] Expression vectors or host cells transformed with expression
vectors may be replicated under conditions suitable for the
expression and recovery of the polypeptide from culture. The
culture can comprise components for in vitro or in vivo expression.
In vitro expression components include those for rabbit
reticulocyte lysates, E. coli lysates, and wheat germ extracts, for
example, Expressway.TM. or RiPs systems from Invitrogen,
Genelator.TM. systems from iNtRON Biotechnology, EcoPro.TM. or
STP3.TM. systems from Novagen, TNT.RTM. Quick Coupled systems from
Promega, and EasyXpress systems from QIAGEN. The polypeptide
produced from culture may be secreted or contained intracellularly
depending on the sequence and/or the vector used. In particular
aspects, expression vectors which encode a phage polypeptide can be
designed to contain signal sequences which direct secretion of the
polypeptide through a prokaryotic or eukaryotic cell membrane.
[0098] Other constructions may include an amino acid domain which
will facilitate purification of the polypeptide. Such domains
include, but are not limited to, metal chelating peptides such as
histidine-tryptophan (e.g., 6.times.-HIS) modules that allow
purification on immobilized metals, protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized
in the FLAG.RTM. extension/affinity purification system (Immunex
Corp., Seattle, Wash.). Useful epitope tags include
3.times.FLAG.RTM., HA, VSV-G, V5, HSV, GST, GFP, MBP, GAL4, and
.beta.-galactosidase. Useful plasmids include those comprising a
biotin tag (e.g., PinPoint.TM. plasmids from Promega), calmodulin
binding protein (e.g., pCAL plasmids from Stratagene), streptavidin
binding peptide (e.g., InterPlay.TM. plasmids from Stratagene), a
c-myc or FLAG.RTM. tag (e.g., Immunoprecipitation plasmids from
Sigma-Aldrich), or a histidine tag (e.g., QIAExpress plasmids from
QIAGEN).
[0099] To facilitate purification, expression vectors can include a
cleavable linker sequences such as those specific for Factor Xa or
enterokinase (Invitrogen, San Diego, Calif.). For example, the
vector can include one or more linkers between the purification
domain and the polypeptide. One such expression vector provides for
expression of a fusion protein comprising a polypeptide of the
invention and a nucleic acid encoding 6 histidine residues
preceding a thioredoxin or an enterokinase cleavage site. The
histidine residues facilitate purification on IMAC (immobilized
metal ion affinity chromatography as described in Porath, J. et al.
(1992) Prot. Exp. Purif. 3: 263-281) while the enterokinase
cleavage site provides a means for purifying the polypeptide from
the fusion protein. A discussion of vectors which contain fusion
proteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.
12:441-453).
[0100] Antibodies of the invention may be produced using methods
which are generally known in the art, for example, for use in
purification or diagnostic techniques. In particular, polypeptides
or polynucleotides may be used to produce antibodies in accordance
with generally known protocols. Such antibodies may include, but
are not limited to, polyclonal, monoclonal, chimeric, and single
chain antibodies, Fab fragments, and fragments produced by a Fab
expression library. Neutralizing antibodies, (i.e., those which
inhibit function) are especially preferred for use with the
invention.
[0101] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others, may be immunized by
injection with a polypeptide, polynucleotide, or any fragment
thereof which has immunogenic properties. Depending on the host
species, various adjuvants may be used to increase immunological
response. Such adjuvants include, but are not limited to, Freund's,
mineral gels such as aluminium hydroxide, and surface active
substances such as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, keyhole limpet hemocyanin, and
dinitrophenol. Among adjuvants used in humans, BCG (bacilli
Calmette-Guerin) and Corynebacterium parvum are especially
preferable.
[0102] It is preferred that the polypeptides or fragments used to
induce antibodies have an amino acid sequence comprising at least
five amino acids and more preferably at least 10 amino acids. It is
also preferable that they are identical to a portion of the amino
acid sequence of the natural protein, and they may contain the
entire amino acid sequence of a small, naturally occurring
molecule. Short stretches of amino acids may be fused with those of
another protein such as keyhole limpet hemocyanin and antibody
produced against the chimeric molecule.
[0103] Monoclonal antibodies may be prepared using any technique
which provides for the production of antibody molecules by
continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique (Kohler, G. et al.
(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol.
Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci.
80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.
62:109-120). Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature (Orlandi, R. et al. (1989)
Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991)
Nature 349:293-299).
[0104] In addition, techniques can be used for the production of
"chimeric antibodies", e.g., the combining of antibody genes to
obtain a molecule with appropriate antigen specificity and
biological activity (Morrison, S. L. et al. (1984) Proc. Natl.
Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature
312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).
Alternatively, techniques described for the production of single
chain antibodies may be adapted, using methods known in the art, to
produce specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobin
libraries (Burton D. R. (1991) Proc. Natl. Acad. Sci.
88:11120-3).
[0105] Those of skill in the art to which the invention relates
will appreciate the terms "diabodies" and "triabodies". These are
molecules which comprise a heavy chain variable domain (VH)
connected to a light chain variable domain (VL) by a short peptide
linker that is too short to allow pairing between the two domains
on the same chain. This promotes pairing with the complementary
domains of one or more other chains and encourages the formation of
dimeric or trimeric molecules with two or more functional antigen
binding sites. The resulting antibody molecules may be monospecific
or multispecific (e.g., bispecific in the case of diabodies). Such
antibody molecules may be created from two or more antibodies using
methodology standard in the art to which the invention relates; for
example, as described by Todorovska et al. (Design and application
of diabodies, triabodies and tetrabodies for cancer targeting. J.
Immunol. Methods. 2001 Feb. 1; 248(1-2):47-66).
[0106] Antibody fragments which contain specific binding sites may
also be generated. For example, such fragments include, but are not
limited to, the F(ab').sub.2 fragments which can be produced by
pepsin digestion of the antibody molecule and the Fab fragments
which can be generated by reducing the disulfide bridges of the
F(ab').sub.2 fragments. Alternatively, Fab expression libraries may
be constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity (Huse, W. D. et al.
(1989) Science 254:1275-1281).
[0107] Various immunoassays may be used for screening to identify
antibodies having binding specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between a polypeptide or
polynucleotide and its specific antibody. A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes is preferred, but a
competitive binding assay may also be employed (Maddox, supra).
[0108] The phage polypeptides described herein have the ability to
target, permeabilise, and/or inhibit cells and are also useful as
carrier molecules for the delivery of additional inhibitory
molecules into microbial cells. The chemistry for coupling
compounds to amino acids is well developed and a number of
different molecule types could be linked to the polypeptides. The
most common coupling methods rely on the presence of free amino
(alpha-amino or Lys), sulfhydryl (Cys), or carboxylic acid groups
(Asp, Glu, or alpha-carboxyl). Coupling methods can be used to link
the polypeptide to the cell inhibitor via the carboxy- or
amino-terminal residue. In some cases, a sequence includes multiple
residues that may react with the chosen chemistry. This can be used
to produce multimers, comprising more than one cell inhibitor.
Alternatively, the polypeptide can be shortened or chosen so that
reactive residues are localized at either the amino or the carboxyl
terminus of the sequence.
[0109] For example, a reporter molecule such as fluorescein can be
specifically incorporated at a lysine residue (Ono et al., 1997)
using N-.alpha.-Fmoc-N.epsilon.-1-(4,4-dimethyl-2,6
dioxocyclohex-1-ylidene-3-methylbutyl)-L-lysine during polypeptide
synthesis. Following synthesis, 5- and 6-carboxyfluorescein
succinimidyl esters can be coupled after 4,4-dimethyl-2,6
dioxocyclohex-1-ylidene is removed by treatment with hydrazine.
Therefore coupling of an inhibitory molecule to the phage
polypeptide can be accomplished by inclusion of a lysine residue to
the polypeptide sequence, then reaction with a suitably derivatised
cell inhibitor.
[0110] EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride) or the carbodiimide coupling method can also be
used. Carbodiimides can activate the side chain carboxylic groups
of aspartic and glutamic acid as well as the carboxyl-terminal
group to make them reactive sites for coupling with primary amines.
The activated polypeptides are mixed with the cell inhibitor to
produce the final conjugate. If the cell inhibitor is activated
first, the EDC method will couple the cell inhibitor through the
N-terminal alpha amine and possibly through the amine in the
side-chain of Lys, if present in the sequence.
[0111] m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) is a
heterobifunctional reagent that can be used to link polypeptides to
cell inhibitors via cysteines. The coupling takes place with the
thiol group of cysteine residues. If the chosen sequence does not
contain Cys it is common to place a Cys residue at the N- or
C-terminus to obtain highly controlled linking of the polypeptide
to the cell inhibitor. For synthesis purposes, it may be helpful
for the cysteine to be placed at the N-terminus of the polypeptide.
MBS is particularly suited for use with the present invention.
[0112] Glutaraldehyde can be used as a bifunctional coupling
reagent that links two compounds through their amino groups.
Glutaraldehyde provides a highly flexible spacer between the
polypeptide and cell inhibitor for favorable presentation.
Glutaraldehyde is a very reactive compound and will react with Cys,
Tyr, and His to a limited extent. The glutaraldehyde coupling
method is particularly useful when a polypeptide contains only a
single free amino group at its amino terminus. If the polypeptide
contains more than one free amino group, large multimeric complexes
can be formed.
[0113] In one aspect, the polypeptides of the invention can be
fused (e.g., by in-frame cloning) or linked (e.g., by chemical
coupling) to cell inhibitors such as antimicrobial agents. Included
among these are antimicrobial peptides, for example,
bactericidal/permeability-increasing protein, cationic
antimicrobial proteins, lysozymes, lactoferrins, and cathelicidins
(e.g., from neutrophils; see, e.g., Hancock and Chapple, 1999,
Antimicrob. Agents Chemother. 43:1317-1323; Ganz and Lehrer, 1997,
Curr. Opin. Hematol. 4:53-58; Hancock et al., 1995, Adv. Microb.
Physiol. 37:135-175). Antimicrobial peptides further include
defensins (e.g., from epithelial cells or neutrophils) and platelet
microbiocidal proteins (see, e.g., Hancock and Chapple, 1999,
Antimicrob. Agents Chemother. 43:1317-1323). Additional
antimicrobial peptides include, but are not limited to, gramicidin
S, bacitracin, polymyxin B, tachyplesin, bactenecin (e.g., cattle
bactenecin), ranalexin, cecropin A, indolicidin (e.g., cattle
indolicidin), and nisin (e.g., bacterial nisin).
[0114] Also included as antimicrobial agents are ionophores, which
facilitate transmission of an ion, (such as sodium), across a lipid
barrier such as a cell membrane. Two ionophore compounds
particularly suited to this invention are the RUMENSIN.TM. (Eli
Lilly) and Lasalocid (Hoffman LaRoche). Other ionophores include,
but are not limited to, salinomycin, avoparcin, aridcin, and
actaplanin. Other antimicrobial agents include penicillin,
Monensin.TM. and azithromycin, metronidazole, streptomycin,
kanamycin, and penicillin, as well as, generally, R-lactams,
aminoglycosides, macrolides, chloramphenicol, novobiocin, rifampin,
and fluoroquinolones (see, e.g., Horn et al., 2003, Applied
Environ. Microbiol. 69:74-83; Eckburg et al., 2003, Infection
Immunity 71:591-596; Gijzen et al., 1991, Applied Environ.
Microbiol. 57:1630-1634; Bonelo et al., 1984, FEMS Microbiol. Lett.
21:341-345; Huser et al., 1982, Arch. Microbiol. 132:1-9; Hilpert
et al., 1981, Zentbl. Bakteriol. Mikrobiol. Hyg. 1 Abt Orig. C
2:21-31).
[0115] Particularly useful inhibitors are compounds that block or
interfere with methanogenesis, including bromoethanesulphonic acid,
e.g., 2-bromoethanesulphonic acid (BES) or a salt thereof, for
example, a sodium salt. Sodium molybdate (Mo) is an inhibitor of
sulfate reduction, and can be used with bromoethanesulphonic acid.
Other anti-methanogenesis compounds include, but are not limited
to, nitrate, formate, methyl fluoride, chloroform, chloral hydrate,
sodium sulphite, ethylene and unsaturated hydrocarbons, acetylene,
fatty acids such as linoleic and cis-oleic acid, saturated fatty
acids such as behenic and stearic acid, and, also lumazine (e.g.,
2,4-pteridinedione). Additional compounds include
3-bromopropanesulphonate (BPS), propynoic acid, and ethyl
2-butynoate.
[0116] Further included as antimicrobial agents are lytic enzymes,
including phage lysozyme, endolysin, lysozyme, lysin, phage lysin,
muralysin, muramidase, and virolysin. Useful enzymes exhibit the
ability to hydrolyse specific bonds in the bacterial cell wall.
Particular lytic enzymes include, but are not limited to,
glucosaminidases, which hydrolyse the glycosidic bonds between the
amino sugars (e.g., N-acetylmuramic acid and N-acetylglucosamine)
of the peptidoglycan, amidases, which cleave the
N-acetylmuramoyl-L-alanine amide linkage between the glycan strand
and the cross-linking peptide, and endopeptidases, which hydrolyse
the interpeptide linkage (e.g., cysteine endopeptidases) and
endoisopeptidases that attack pseudomurein of methanogens from the
family Methanobacteriacaea.
[0117] The polypeptides encoded by ORF 2058 or ORF 2055, described
in detail herein and below, are useful as rumen methanogen-specific
lytic enzymes. The native enzymes can be prepared from freshly
.phi.mru-lysed M. ruminantium cells. Alternatively, ORF 2058 or ORF
2055 can be cloned in an expression vector and expressed in a
heterologous host such as Escherichia coli. This was accomplished
previously with PeiP and PeiW and the recombinant proteins were
shown to be active against Methanothermobacter cell walls under
reducing conditions (Luo et al., 2002). ORF 2058 or ORF 2055 lytic
enzymes or any other lytic enzyme can be used in compositions, for
example, as a feed additive for ruminants or it can be incorporated
into a slow release capsule or bolus device for delivery over a
longer time period within the rumen. The lytic enzymes can be used
either in combination or sequentially with other methanogen
inhibitor(s) to avoid adaptation of the host methanogens and
resistance to the enzymes. Random and/or targeted mutations in the
enzymes can also be used to avoid adaptation. The lytic/lysogenic
switch components (e.g., ORF 1981 and ORF 1983-ORF 1986) can be
used in a similar manner as the lytic enzymes.
[0118] Additionally, PNAs are included as antimicrobial agents.
PNAs are peptide-nucleic acid hybrids in which the phosphate
backbone has been replaced by an achiral and neutral backbone made
from N-(2-aminoethyl)-glycine units (see, e.g., Eurekah Bioscience
Collection. PNA and Oligonucleotide Inhibitors of Human Telomerase.
G. Gavory and S. Balasubramanian, Landes Bioscience, 2003). The
bases A, G, T, C are attached to the amino nitrogen on the backbone
via methylenecarbonyl linkages (P. E. Nielsen et al., Science 1991.
254: 1497-1500; M. Egholm et al., Nature 1993. 365: 566-568). PNAs
bind complementary sequences with high specificity, and higher
affinity relative to analogous DNA or RNA (M. Egholm et al.,
supra). PNA/DNA or PNA/RNA hybrids also exhibit higher thermal
stability compared to the corresponding DNA/DNA or DNA/RNA duplexes
(M. Egholm et al., supra). PNAs also possess high chemical and
biological stability, due to the unnatural amide backbone that is
not recognized by nucleases or proteases (V. Demidov et al.,
Biochem Pharmacol 1994. 48: 1310-1313). Typically, PNAs are at
least 5 bases in length, and include a terminal lysine. PNAs may be
pegylated to further extend their lifespan (Nielsen, P. E. et al.
(1993) Anticancer Drug Des. 8:53-63).
[0119] In one particular aspect, the polypeptides of the invention
can be fused (e.g., by in-frame cloning) or linked (e.g., by
chemical coupling) to cell inhibitors such as antibodies or
fragments thereof. The antibodies or antibody fragments can be
directed to microbial cells, or particularly methanogen cells, or
one or more cell components. For example, cell surface proteins,
e.g., extracellular receptors, can be targeted. Included are
immunoglobulin molecules and immunologically active portions of
immunoglobulin (Ig) molecules, i.e., molecules that contain an
antigen binding site that specifically binds (immunoreacts with) an
antigen.
[0120] The polypeptides of the invention find particular use in
targeting a microbial cell, in particular, a methanogen cell. In
certain aspects, the polypeptides can be used to associate with or
bind to the cell wall or membrane, permeabilise the cell, and/or
inhibit growth or replication of the cell. As such, the
polypeptides can be used for transient or extended attachment to
the cell, or to penetrate the cell wall or membrane and/or
accumulate in the intracellular environment. It is understood that
the phage polypeptides, as well as the corresponding
polynucleotides, expression vectors, host cells, and antibodies of
the invention, can be used to target various microbes, for example,
Methanobrevibacter ruminantium, which is the primary methanogen in
ruminants, and Methanobrevibacter smithii, which is the primary
methanogen in humans. To effect targeting, the microbial cell can
be contacted with the phage polypeptide as isolated from one or
more natural sources, or produced by expression vectors and/or host
cells, or synthetic or semi-synthetic chemistry as described in
detail herein. For enhanced permeabilisation, the polypeptide can
be fused or linked to one or more signal sequences (predicted
consensus sequence:
[ML]KKKK[K]{0,1}X{0,9}[IL][IFL][IL][IL][IS][LIA]X{0,4}[LIVF][LIAV][LI][IL-
V][LAIV][ILFV][LIVF][SAL][ILV][GSA][AS][VAI][SA]A, see FIG. 6). See
also Perez-Bercoff, .ANG.., Koch, J. and Burglin, T. R. (2006)
LogoBar: bar graph visualization of protein logos with gaps.
Bioinformatics 22, 112-114. In particular aspects, the polypeptide
is delivered to subjects as composition described in detail herein,
for example, through use of a slow-release device for
ruminants.
[0121] In certain embodiments, the polypeptide is fused or linked
to a cell inhibitor, for example, an anti-methanogenesis compound
(e.g., bromoethanesulphonic acid), an antibody or antibody
fragment, lytic enzyme, peptide nucleic acid, antimicrobial
peptide, or other antibiotic. The polypeptide-inhibitor is
delivered to subjects as a composition to inhibit growth and/or
replication of microbial cells, in particular, methanogen cells.
The composition comprises, for example: a) an isolated phage, phage
particle, phage genome, or alteration, fragment, variant, or
derivative thereof; b) an isolated phage polypeptide, or an
alteration, fragment, variant, or derivative thereof; c) an
isolated polynucleotide, or an alteration, fragment, variant, or
derivative thereof; d) an expression vector comprising this
polynucleotide; or e) a host cell comprising this expression
vector. The compositions of the invention can be specifically
packaged as part of kits for targeting, permeabilising, and/or
inhibiting microbial cells, especially methanogen cells, in
accordance with the disclosed methods. The kits comprise at least
one composition as set out herein and instructions for use in
targeting or permeabilising cells, or inhibiting cell growth or
replication, for methanogens or other microbes.
[0122] As an additional embodiment, the invention relates to a
pharmaceutical composition in conjunction with a pharmaceutically
acceptable carrier, for use with any of the methods discussed
above. Such pharmaceutical compositions may comprise a phage
polypeptide, in combination with a cell inhibitor. Alternatively,
the pharmaceutical compositions may comprise an expression vector
or host cell as described in detail herein. The compositions may be
administered alone or in combination with at least one other agent,
such as stabilizing compound, which may be administered in any
sterile, biocompatible pharmaceutical carrier, including, but not
limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a subject alone, or in
combination with other agents, drugs (e.g., antimicrobial drugs),
or hormones.
[0123] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.). The pharmaceutical compositions utilized in this
invention may be administered by any number of routes including,
but not limited to, oral, intravenous, intramuscular,
intra-arterial, intramedullary, intrathecal, intraventricular,
transdermal, subcutaneous, intraperitoneal, intranasal, enteral,
topical, sublingual, or rectal means.
[0124] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the subject.
Pharmaceutical preparations for oral use can be obtained through
combination of active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are carbohydrate or
protein fillers, such as sugars, including lactose, sucrose,
mannitol, or sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums including arabic and tragacanth; and proteins such as gelatin
and collagen. If desired, disintegrating or solubilising agents may
be added, such as the crosslinked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate.
[0125] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders, such as lactose or starches, lubricants, such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers. Dragee cores may
be used in conjunction with suitable coatings, such as concentrated
sugar solutions, which may also contain gum arabic, talc,
polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to the
tablets or dragee coatings for product identification or to
characterize the quantity of active compound, i.e., dosage.
[0126] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions. For topical or nasal
administration, penetrants appropriate to the particular barrier to
be permeated are used in the formulation. Such penetrants are
generally known in the art.
[0127] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes. The pharmaceutical composition may be
provided as a salt and can be formed with many acids, including but
not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric,
malic, succinic, etc. Salts tend to be more soluble in aqueous or
other protonic solvents than are the corresponding free base forms.
In other cases, the preferred preparation may be a lyophilized
powder which may contain any or all of the following: 1-50 mM
histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5
to 5.5, that is combined with buffer prior to use. After
pharmaceutical compositions have been prepared, they can be placed
in an appropriate container and labeled for treatment of an
indicated condition. For administration of a composition of the
invention, such labeling would include amount, frequency, and
method of administration.
[0128] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
For any compound, the therapeutically effective dose can be
estimated initially either in cell assays, e.g., in microbial
cells, or in particular, in methanogen cells, or in animal models,
usually mice, rabbits, dogs, or pigs, or in ruminant species such
as sheep, cattle, deer, and goats. The animal model may also be
used to determine the appropriate concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration. Normal dosage amounts
may vary from 0.1 to 100,000 micrograms, up to a total dose of
about 1 g, or more, depending upon the route of administration.
Guidance as to particular dosages and methods of delivery is
provided in the literature and generally available to practitioners
in the art. Those skilled in the art will employ different
formulations for polynucleotides than for polypeptides. Similarly,
delivery of polynucleotides or polypeptides will be specific to
particular cells, conditions, locations, etc.
[0129] Phage-based therapeutics are known, and methods of
manufacture of such compositions are published in the art. Phage
therapeutics have been described, for example, for targeting
Staphylococcus (e.g., S. aureus), Pseudomonas (e.g., P.
aeruginosa), Escherichia (e.g., E. coli), Klebsiella (e.g., K.
ozaenae, K. rhinoscleromatis scleromatis and K. pneumonia),
Proteus, Salmonella, Shigella (see, e.g., Carlton, R. M. (1999).
Archivum Immunologiae et Therapiae Experimentalis, 47: 267-274;
Liu, J. et al. (2004). Nat. Biotechnol. 22, 185-191; Projan, S.
(2004). Nat. Biotechnol. 22, 167-168; Sulakvelidze, A., Alavidze,
Z. and Morris, J. G. (2001). Antimicrobial Agents and Chemotherapy,
45 (3): 649-659; Weber-Dabrowska, Mulczyk, M. and Gorski, A.
(2000). Archivum Immunologiae et Therapiae Experimentalis, 48:
547-551. Phage therapies have inherent advantages over traditional
anti-microbials, in that phage are highly specific and don't affect
the normal microflora of the human body; phage do not infect
eukaryotic cells, and have no known serious side effects; phage can
localize to the site of infection; and phage can replicate
exponentially, so treatments require only a small dose and are
generally low in cost (see, e.g., Sulakvelidze et al., supra). For
current review, see Fischetti V A, Nelson D, Schuch R. Reinventing
phage therapy: are the parts greater than the sum? Nat. Biotechnol.
2006 December; 24(12):1508-11.
[0130] Peptide- and polypeptide-based therapeutics have also been
described, for example, for denileukin, difitox, octreotide,
vapreotide, lanreotide, RC-3940 series peptides, decapeptyl,
lupron, zoladex, cetrorelix (see, e.g., Lu et al., 2006, AAPS J
8:E466-472), hemocidins, staphopains (see, e.g., Dubin et al.,
2005, Acta Biochemica Polonica, 52:633-638), as well as
indolicidin, defensins, lantibiotics, microcidin B17, histatins,
and maganin (see, e.g., Yeaman and Yount, 2003, Pharmacol Rev
55:27-55). General guidance for peptide and polypeptide
therapeutics can also be found in Degim et al., 2007, Curr Pharm
Des 13:99-117 and Shai et al., 2006, Curr Prot Pept Sci, 7:479-486.
Recently approved peptide-based drugs include Hematide.TM.
(synthetic peptide-based erythropoiesis-stimulating agent, Affymax,
Inc.), Exenatide (synthetic exendin-4, Amylin/Eli Lilly), Natrecor
(nesiritide, natriuretic peptide, Scios), Plenaxis (abarelix,
Praecis Pharmaceuticals), and SecreFlo (secretin, Repligen).
[0131] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active agent or to maintain the desired effect. Factors
which may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time, and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0132] Particularly useful for the compositions of the invention
(e.g., pharmaceutical compositions) are slow release formulas or
mechanisms. For example, intra-ruminal devices include, but are not
limited to, Time Capsule.TM. Bolus range by Agri-Feeds Ltd., New
Zealand, originally developed within AgResearch Ltd., New Zealand,
as disclosed in WO 95/19763 and NZ 278977, and CAPTEC by Nufarm
Health & Sciences, a division of Nufarm Ltd., Auckland, New
Zealand, as disclosed in AU 35908178, PCT/AU81/100082, and Laby et
al., 1984, Can. J. Anim. Sci. 64 (Suppl.), 337-8, all of which are
incorporated by reference herein. As a particular example, the
device can include a spring and plunger which force the composition
against a hole in the end of a barrel.
[0133] As a further embodiment, the invention relates to a
composition for a water supplement, e.g., drenching composition, or
food supplement, e.g., ruminant feed component, for use with any of
the methods discussed above. In particular aspects, the food
supplement comprises at least one vegetable material that is
edible, and a peptide or polypeptide of the invention.
Alternatively, the food supplement comprises at least one vegetable
material that is edible, and a polypeptide or peptide, or a
polynucleotide encoding a peptide or polypeptide disclosed herein,
for example, as an expression vector or host cell comprising the
expression vector. In particular, the composition further includes
a cell inhibitor, as fused or linked to the resultant sequence. The
preferred vegetable material include any one of hay, grass, grain,
or meal, for example, legume hay, grass hay, corn silage, grass
silage, legume silage, corn grain, oats, barley, distillers grain,
brewers grain, soy bean meal, and cotton seed meal. In particular,
grass silage is useful as a food composition for ruminants. The
plant material can be genetically modified to contain one or more
components of the invention, e.g., one or more polypeptides or
peptides, polynucleotides, or vectors.
[0134] In another embodiment, antibodies which are raised to the
polypeptides or polynucleotides of the invention may be used to
determine the presence of microbes, especially methanogens, or in
assays to monitor levels of such microbes. The antibodies useful
for diagnostic purposes may be prepared in the same manner as those
described above. Diagnostic assays include methods which utilize
the antibody and a label to detect a polypeptide in human body
fluids or extracts of cells or tissues. The antibodies may be used
with or without modification, and may be labeled by joining them,
either covalently or non-covalently, with a reporter molecule. A
wide variety of reporter molecules which are known in the art may
be used, several of which are described above.
[0135] A variety of protocols for measuring levels of a polypeptide
or polynucleotide are known in the art (e.g., ELISA, RIA, FACS, and
blots, such as Southern, Northern, Western blots), and provide a
basis for determining the presence or levels of a microbe,
especially a methanogen. Normal or standard levels established by
combining body fluids or cell extracts taken from normal subjects,
e.g., normal humans or ruminants, with the antibody under
conditions suitable for complex formation. The amount of standard
complex formation may be quantified by various methods, but
preferably by photometric means. Quantities of polypeptide or
polynucleotide expressed in subject, control, and treated samples
(e.g., samples from treated subjects) are compared with the
standard values. Deviation between standard and subject values
establishes the parameters for determining the presence or levels
of the microbe.
[0136] In a particular embodiment of the invention, the
polynucleotides may be used for diagnostic purposes using
particular hybridization and/or amplification techniques. The
polynucleotides which may be used include oligonucleotides,
complementary RNA and DNA molecules, and PNAs. The polynucleotides
may be used to detect and quantitate gene expression in samples in
which expression may be correlated with the presence or levels of a
microbe. The diagnostic assay may be used to distinguish between
the absence, presence, and alteration of microbe levels, and to
monitor levels during therapeutic intervention.
[0137] In one aspect, hybridization with PCR probes may be used to
identify nucleic acid sequences, especially genomic sequences,
which encode the polypeptides of the invention. The specificity of
the probe, whether it is made from a highly specific region, e.g.,
10 unique nucleotides in the 5' regulatory region, or a less
specific region, e.g., in the 3' coding region, and the stringency
of the hybridization or amplification (maximal, high, intermediate,
or low) will determine whether the probe identifies only naturally
occurring sequences, alleles, or related sequences. Probes may also
be used for the detection of related sequences, and should
preferably contain at least 50% of the nucleotides from any of the
coding sequences. The hybridization probes of the subject invention
may be DNA or RNA and derived from the nucleotide sequence of SEQ
ID NO:74-142, or complements, or modified sequences thereof, or
from genomic sequences including promoter, enhancer elements, and
introns of the naturally occurring sequence.
[0138] Means for producing specific hybridization probes for DNAs
include the cloning of nucleic acid sequences into vectors for the
production of mRNA probes. Such vectors are known in the art,
commercially available, and may be used to synthesize RNA probes in
vitro by means of the addition of the appropriate RNA polymerases
and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a variety of reporter groups, for example,
radionuclides such as .sup.32P or .sup.355, or enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like. The polynucleotides may be used in
Southern or northern analysis, dot blot, or other membrane-based
technologies; in PCR technologies; or in dipstick, pin, ELISA
assays, or microarrays utilizing fluids or tissues from subject
biopsies to detect the presence or levels of a microbe. Such
qualitative or quantitative methods are well known in the art.
[0139] In a particular aspect, the nucleic acid sequences may be
useful in various assays labelled by standard methods, and added to
a fluid or tissue sample from a subject under conditions suitable
for hybridization and/or amplification. After a suitable incubation
period, the sample is washed and the signal is quantitated and
compared with a standard value. If the amount of signal in the test
sample is significantly altered from that of a comparable control
sample, the presence of altered levels of nucleotide sequences in
the sample indicates the presence or levels of the microbe. Such
assays may also be used to evaluate the efficacy of a particular
treatment regimen in animal studies, in clinical trials, or in
monitoring the treatment of a subject.
[0140] In order to provide a basis for the diagnosis of the
presence or levels of a microbe, a normal or standard profile for
expression is established. This may be accomplished by combining
body fluids or cell extracts taken from normal subjects, with a
polynucleotide or a fragment thereof, under conditions suitable for
hybridization and/or amplification. Standard levels may be
quantified by comparing the values obtained from normal subjects
with those from an experiment where a known amount of a
substantially purified polynucleotide is used. Standard values
obtained from normal samples may be compared with values obtained
from samples from subjects treated for microbial growth. Deviation
between standard and subject values is used to establish the
presence or levels of the microbe.
[0141] Once the microbe is identified and a treatment protocol is
initiated, hybridization and/or amplification assays may be
repeated on a regular basis to evaluate whether the level of
expression in the subject begins to decrease relative to that which
is observed in the normal subject. The results obtained from
successive assays may be used to show the efficacy of treatment
over a period ranging from several days to months.
[0142] Particular diagnostic uses for oligonucleotides designed
from the nucleic acid sequences may involve the use of PCR. Such
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably consist of two
nucleotide sequences, one with sense orientation (5'.->.3') and
another with antisense orientation (3'.->.5'), employed under
optimized conditions for identification of a specific nucleotide
sequence or condition. The same two oligomers, nested sets of
oligomers, or even a degenerate pool of oligomers may be employed
under less stringent conditions for detection and/or quantitation
of closely related DNA or RNA sequences.
[0143] Methods which may also be used to quantitate expression
include radiolabeling or biotinylating nucleotides, coamplification
of a control nucleic acid, and standard curves onto which the
experimental results are interpolated (Melby, P. C. et al. (1993)
J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal.
Biochem. 229-236). The speed of quantitation of multiple samples
may be accelerated by running the assay in an ELISA format where
the oligomer of interest is presented in various dilutions and a
spectrophotometric or colorimetric response gives rapid
quantitation.
[0144] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotides described herein may be
used as targets in a microarray. The microarray can be used to
monitor the expression level of large numbers of genes
simultaneously (to produce a transcript image), and to identify
genetic variants, mutations and polymorphisms. This information may
be used to determine gene function, to understand the genetic basis
of disease, to diagnose disease, and to develop and monitor the
activities of therapeutic agents. In one embodiment, the microarray
is prepared and used according to methods known in the art such as
those described in PCT application WO 95/11995 (Chee et al.),
Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and
Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93:
10614-10619).
[0145] In one aspect, the oligonucleotides may be synthesized on
the surface of the microarray using a chemical coupling procedure
and an ink jet application apparatus, such as that described in PCT
application WO 95/251116 (Baldeschweiler et al.). In another
aspect, a "gridded" array analogous to a dot or slot blot (HYBRIDOT
apparatus, Life Technologies) may be used to arrange and link cDNA
fragments or oligonucleotides to the surface of a substrate using a
vacuum system, thermal, UV, mechanical or chemical bonding
procedures. In yet another aspect, an array may be produced by hand
or by using available devices, materials, and machines (including
multichannel pipettors or robotic instruments; Brinkmann, Westbury,
N.Y.) and may include, for example, 24, 48, 96, 384, 1024, 1536, or
6144 spots or wells (e.g., as a multiwell plate), or more, or any
other multiple from 2 to 1,000,000 which lends itself to the
efficient use of commercially available instrumentation.
[0146] In order to conduct sample analysis using the microarrays,
polynucleotides are extracted from a biological sample. The
biological samples may be obtained from any bodily fluid (blood,
urine, saliva, phlegm, gastric juices, etc.), cultured cells,
biopsies, or other tissue preparations. To produce probes, the
polynucleotides extracted from the sample are used to produce
nucleic acid sequences which are complementary to the nucleic acids
on the microarray. If the microarray consists of cDNAs, antisense
RNAs are appropriate probes. Therefore, in one aspect, mRNA is used
to produce cDNA which, in turn and in the presence of fluorescent
nucleotides, is used to produce fragments or antisense RNA probes.
These fluorescently labeled probes are incubated with the
microarray so that the probe sequences hybridize to the cDNA
oligonucleotides of the microarray. In another aspect, nucleic acid
sequences used as probes can include polynucleotides, fragments,
and complementary or antisense sequences produced using restriction
enzymes, PCR technologies, and oligolabeling kits (Amersham
Pharmacia Biotech) well known in the area of hybridization
technology.
[0147] In another embodiment of the invention, the polypeptides of
the invention or functional or immunogenic fragments or
oligopeptides thereof, can be used for screening libraries of
compounds in any of a variety of drug screening techniques. The
fragment employed in such screening may be free in solution,
affixed to a solid support, borne on a cell surface, or located
intracellularly. The formation of binding complexes, between the
polypeptide and the agent being tested, may be measured.
[0148] One technique for drug screening which may be used provides
for high throughput screening of compounds having suitable binding
affinity to the polypeptide of interest as described in published
PCT application WO 84/03564. In this method, large numbers of
different small test compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. The test
compounds are reacted with the polypeptide, or fragments thereof,
and washed. Bound polypeptide is then detected by methods well
known in the art. Purified polypeptide can also be coated directly
onto plates for use in the aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies can be used
to capture the polypeptide and immobilize it on a solid
support.
[0149] In another technique, one may use competitive drug screening
assays in which neutralizing antibodies capable of binding the
polypeptide specifically compete with a test compound for binding
to the polypeptide. In this manner, the antibodies can be used to
detect the presence of a test compound which shares one or more
antigen binding sites with the antibody.
EXAMPLES
[0150] The examples described herein are for purposes of
illustrating embodiments of the invention. Other embodiments,
methods, and types of analyses are within the scope of persons of
ordinary skill in the molecular diagnostic arts and need not be
described in detail hereon. Other embodiments within the scope of
the art are considered to be part of this invention.
Example 1
Genome Size Estimation
[0151] Methanobrevibacter ruminantium strain M1.sup.T (DSM1093) was
grown on BY+ medium (basal medium, Joblin et al., 1990) which
consists of [g/l] NaCl (1), KH.sub.2PO.sub.4 (0.5),
(NH.sub.4).sub.2SO.sub.4 (0.25), CaCL.sub.2.2H.sub.2O (0.13),
MgSO.sub.4.7H.sub.2O (0.2), K.sub.2HPO.sub.4 (1), clarified rumen
fluid (300 ml), dH.sub.2O (360 ml), NaHCO.sub.3 (5), resazurin (0.2
ml), L-cysteine-HCl (0.5), yeast extract (2), and Balch's trace
elements solution (10 ml) (added trace elements; Balch et al.,
1979) which consists of (g/1) nitrilotriacetic acid (1.5),
MgSO.sub.4.7H.sub.2O (3), MnSO.sub.4.H.sub.2O (0.5), NaCl (1),
FeSO.sub.4.7H.sub.2O (0.1), CoCl.sub.2.6H.sub.2O (0.1), CaCl.sub.2
(0.1), ZnSO.sub.4.7H.sub.2O (0.1), CuSO.sub.4.5H.sub.2O (0.01),
AlK(SO.sub.4).sub.2.12H.sub.2O (0.01), H.sub.3BO.sub.3 (0.01),
Na.sub.2MoO.sub.4.2H.sub.2O (0.01), NiSO.sub.4.6H.sub.2O (0.03),
Na.sub.2SeO.sub.3 (0.02), and Na.sub.2Wo.sub.4.2H.sub.2O (0.02).
Genomic DNA was extracted by freezing cell pellets in liquid
N.sub.2 and grinding using a pre-chilled, sterilised mortar and
pestle. Cell homogenates were imbedded in agarose plugs and
subsequent manipulations were carried out in the plugs to reduce
the physical shearing of genomic DNA. Digests were performed with
restriction endonucleases and DNA fragments were separated using
pulsed-field gel electrophoresis (PFGE).
Example 2
DNA Cloning and Sequencing
[0152] The DNA of the M. ruminantium genome was sequenced by
Agencourt Biosciences Corporation (Massachusetts, USA) using a
random shotgun cloning approach (Fleischmann et al., 1995) and by
Macrogen Corporation (Rockville, Md., USA) using pyrosequencing.
Briefly, libraries of M. ruminantium DNA were constructed in
Escherichia coli by random physical disruption of genomic DNA and
separation of fragments by gel electrophoresis. Large fragments in
the 40 Kb range were retrieved from the gel and used to generate a
large insert fosmid library. DNA fragments in the 2 to 4 Kb range
were recovered and used to generate a small insert plasmid library.
Clones resulting from both large and small insert libraries were
grown, and their fosmid or plasmid DNA was recovered and sequenced
using high throughput sequencing technology. A sufficient number of
clones were sequenced to give a theoretical 8 fold coverage of the
M. ruminantium genome. Pyrosequencing was performed on randomly
sheared genomic DNA fragments to give a final theoretical 10 fold
coverage.
Example 3
Sequence Assembly and Prophage Annotation
[0153] DNA sequences were aligned to find sequence overlaps and
assembled into contiguous (contig) sequences using Paracel Genome
Assembler (Paracel Inc, CA, USA) and the Staden package (Staden et
al., 1998) in combination with sequence from both standard and
inverse PCRs. Contigs were analysed using the open reading frame
(ORF) finder GLIMMER (Gene Locator Interpolated Markov Model ER
Delcher et al., 1999) and each ORF was analysed by gapped BLASTP
(Basic Local Alignment Search Tool (Altschul et al., 1997) against
the National Center for Biotechnology Information (NCBI)
non-redundant nucleotide and protein databases. The contigs from
the 8 fold draft phase sequence were joined at random by artificial
linking of sequences to generate a "pseudomolecule" and submitted
to The Institute for Genomic Research (TIGR, DC, USA) for
autoannotation. The contigs assembled from the 10 fold
pyrosequencing were reanalysed using GLIMMER and ORFs were
autoannotated using GAMOLA (Global Annotation of Multiplexed
On-site Blasted DNA sequences; Alternann and Klaenhammer, 2003).
ORFs were categorised by function using the clusters of orthologous
proteins (COG) database (threshold 1e-02) (hypertext transfer
protocol://world wide web.pnas.org/cgi/content/full/102/11/3906;
Tatusov et al., 2001).
[0154] Protein motifs were determined by HMMER (hypertext transfer
protocol://hmmer.wustl.edu) using PFAM HMM and TIGRFAM libraries,
with global and local alignment (hypertext transfer
protocol://pfam.wustl.edu) and standard and fragment-mode TIGRFAM
HMMs models (hypertext transfer protocol://world wide
web.tigr.org/TIGRFAMs) respectively (threshold 1e-02). tRNAs were
identified by using TRNASCAN-SE (Lowe and Eddy, 1997) and
nucleotide repeats were identified using the KODON software package
(Applied Maths, Austin, Tex., USA) and REPUTER (Kurtz and
Schleiermacher, 1999). Automated annotations were subsequently
verified manually. Genome atlas visualizations were constructed
using GENEWIZ (Jensen et al., 1999) and underlying data structures
were generated by customised in-house developed algorithms. Pathway
reconstructions from the predicted M. ruminantium ORFeome were
carried out in conjunction with the KEGG (Kyoto Encyclopedia of
Genes and Genomes, Kanehisa et al., 2004) on-line database using
in-house developed software (PathwayVoyager; Alternann and
Klaenhammer, 2005).
Example 4
Sequencing Results and Analysis
[0155] Size estimation of the M. ruminantium genome by restriction
enzyme digestion of genomic DNA and sizing of fragments via PFGE,
indicated a single chromosome of approximately 2.5-2.9 Mb. Initial
sequencing of large and small insert clones (6 fold draft coverage)
and assembly of the sequence into contigs indicated that a 40 Kb
region of the genome was highly over-represented (>20 fold),
particularly within the small insert library. This was possibly due
to a high copy number plasmid (although no extrachromosomal DNAs
had been identified) or a lysogenic bacteriophage that had
replicated during the growth of the culture used for DNA
extraction. Because of this large sequence bias, additional
sequencing was carried out (2 fold theoretical genome coverage) for
only small insert clones yielding a final 8 fold coverage from
Sanger sequencing. The 8 fold draft phase sequence was assembled
into 756 contigs which were linked via 105 scaffolds. Further
pyrosequencing was carried out to an additional .about.10 fold
coverage and incorporation of these sequences into the assembly
resulted in the contig number dropping to 27. Subsequent gap
closure using inverse and long range PCR techniques reduced the
contig number to 14, with one misassembly remaining.
[0156] During the high-throughput sequencing phase, a bias was
observed in the sequence coverage towards a region (.about.50 Kb)
of significantly higher G+C content immediately adjacent to a low
G+C region (.about.12 Kb). Analysis of the genome sequence via
GAMOLA and GeneWiz led to the discovery of a prominent high-GC
region located immediately adjacent to a large low-GC spike.
Detailed analyses of the high G+C region revealed the presence of
gene-products with similarities to a phage-related integrase, the
large subunit of the phage terminase, a phage portal protein, a
phage capsid protein, and a predicted peptidase acting as phage
lysin (FIG. 3). These gene products were used as anchor points for
the overall structure of the predicted M. ruminantium prophage,
designated .phi.mru. Based on analyses of DNA secondary structures,
the likely phage integration sites attL and attR were identified
(FIG. 1A). Phage integration at the att site appears to have
disrupted a putative membrane protein encoded by ORFs 1980 and
2069, and this gene may harbour the original integration site for
the .phi.mru phage genome, attB.
[0157] The general structure (FIG. 1B) and DNA sequence (FIG. 4A)
of .phi.mru were determined based on commonly recognized modular
structure of phage genomes combined with similarities to sequence
and functional databases. See, e.g., Alternann E, Klein J R,
Henrich B. Primary structure and features of the genome of the
Lactobacillus gasseri temperate bacteriophage (phi)adh. Gene. 1999
Aug. 20; 236(2):333-46; Desiere F, Lucchini S, Canchaya C, Ventura
M, Brussow H. Antonie Van Leeuwenhoek. Comparative genomics of
phages and prophages in lactic acid bacteria. 2002 August;
82(1-4):73-91. The predicted .phi.mru phage ORFeome was
successfully classified into modules encoding phage integration,
DNA replication and packaging, phage structural proteins, and a
lysis cassette, and approximately 40% of the phage ORFs were
functionally characterised. A terminator-like structure in a large
non-coding region (244 bp), flanked by a large number of direct and
indirect repeats and determined within the DNA replication module
was characterised as a putative origin of DNA replication. Several
genes within the phage genome sequence were predicted on the
antisense strand and these coincided with low-GC regions. It is to
be determined if these genes inactivate phage function or indicate
misassembly within the phage genome.
[0158] The low-GC region between the predicted phage lysin and
attR, was found to harbour a DNA sulphur modification system, dnd
(degradation during electrophoresis), including a type II
restriction m6 adenine DNA methyltransferase and a transcriptional
regulator likely to be specific for the dnd system. Furthermore,
non-coding RNA structures were identified both within and flanking
the phage genome. Within the predicted DNA replication module, an
rbcL was identified. rbcL represents a 5' UTR RNA stabilising
element from Chlamydomonas reinhardtii. The family is thought to be
involved in the stabilisation of the rbcL gene which codes for
large subunit of ribulose-1,5-bisphosphate carboxylase. Mutations
in this family can lead to a 50-fold acceleration in transcript
degradation.
[0159] Flanking the phage genome, three group I intron structures
were identified. Group I catalytic introns are large self-splicing
ribozymes. They catalyse their own excision from mRNA, tRNA and
rRNA precursors in a wide range of organisms. The core secondary
structure consists of 9 paired regions (P1-P9). These fold to
essentially two domains--the P4-P6 domain (formed from the stacking
of P5, P4, P6 and P6a helices) and the P3-P9 domain (formed from
the P8, P3, P7 and P9 helices). The secondary structure mark-up for
this family represents only this conserved core. Group I catalytic
introns often have long ORFs inserted in loop regions. These
non-coding RNA structures are located in the non-coding regions
between upstream of ORF 1980 (SEQ ID NO:74), downstream of ORF 2065
(SEQ ID NO:141) and attR and upstream of ORF 2069 (SEQ ID
NO:142).
Example 5A
Phage Genes
[0160] The discovery of a prophage sequence within the M.
ruminantium genome sequence was unexpected. There have been no
previous reports of Methanobrevibacter ruminantium strain M1 (DSM
1093) being susceptible to either lytic or lysogenic phage,
although there have been reports of phage being identified for
other Methanobrevibacter species (Baresi and Bertani, 1984; Knox
and Harris, 1986). The sequence of the .phi.mru prophage is
significantly higher in G+C content than the surrounding M.
ruminantium genome suggesting that it originated from another
organism. The observed levels of homology do not suggest an obvious
host from which it originated, and indicates that .phi.mru is
unlike any other phage encountered to date.
[0161] The .phi.mru DNA sequence is inserted within a predicted M.
ruminantium membrane protein and is flanked by DNA sequences with
secondary structures consistent with attL and attR sites. Despite
the lack of strong homology to other known proteins, all of the
functional module characteristics of a phage could be identified
within the .phi.mru sequence. An interesting feature of the
sequence is a low G+C region at the 3' end which shows homology to
proteins involved in a DNA sulphur modification system (dnd)
system. These genes are located upstream of the attR attachment
site of .phi.mru and were therefore likely brought into the M.
ruminantium genome during phage integration. The region encodes
several dnd associated ORFs (dnd 1, 2, and 3), and a Type II
methylase subunit along with a putative transcriptional
regulator.
[0162] The dnd phenotype sensitises its DNA to degradation during
electrophoresis. Analyses of respective dnd ORF functions suggested
an incorporation of sulphur or a sulphur-containing substance into
the hosts' genome. The Dnd phenotype was also discovered to exist
in DNA of widespread bacterial species of variable origin and
diverse habitat. Similarly organized gene clusters were found in
several bacterial genomes representing different genera and in cDNA
of marine organisms, suggesting such modification as a widespread
phenomenon. A coincidence between the Dnd phenotype and DNA
modification by sulphur was demonstrated to occur in several
representative bacterial genomes by the in vivo (35)S-labelling
experiments (Zhou X, He X, Liang J, Li A, Xu T, Kieser T, Heimann J
D, Deng Z. A novel DNA modification by sulphur. Mol. Microbiol.
2005 September; 57(5):1428-38).
[0163] Type II R\M systems are the simplest and the most prevalent.
Instead of working as a complex, the methyltransferase and
endonuclease are encoded as two separate proteins and act
independently. There is no specificity protein. Both proteins
recognize the same recognition site, and therefore compete for
activity. The methyltransferase acts as a monomer, methylating the
duplex one strand at a time. The endonuclease acts as a homodimer,
which facilitates the cleavage of both strands. Cleavage occurs at
a defined position close to or within the recognition sequence. At
this point it is unclear how the predicted functionality acts
together with the dnd system. Yet, it is clear that the phage has
utility as a gene delivery vehicle. In particular, the lysogenic
conversion region can be used as the locus of gene replacement.
[0164] It is likely that the dnd system was transported to M.
ruminantium by the phage. As such, the role of the .phi.mru dnd
system in protecting or modifying M. ruminantium or foreign DNA is
unknown. Another interesting feature of the .phi.mru sequence is
the number of ORFs encoded on the antisense strand. These ORFs
correspond with low GC regions and have weak BLAST matches to
proteins from a variety of organisms. This could suggest that these
genes have been accumulated within the .phi.mru genome since its
integration into M. ruminantium. It is not clear if these ORFs
represent an ongoing accumulation of insertions that may eventually
lead to phage inactivation and domestication or if .phi.mru is
fully active.
[0165] One .phi.mru gene of particular interest to methane
mitigation is ORF2058 located in the lysis cassette. ORF 2058 is
annotated as a peptidase and has a Protein Family (Pfam) match
(Score:-13.7, E value:0.00054) to Peptidase C39 family proteins.
These proteins are cysteine peptidases and are part of the larger
clan of CA peptidases as defined by the MEROPS peptidase database
(Rawlings et al., 2006). The C39 peptidase family are usually
associated with ABC transporters and function as maturation
proteases during the export and processing of bacteriocins. The CA
peptidase clan also includes the viral cysteine endopeptidases such
as the C71 archaeal phage endoisopeptidases that cleave the
crosslinking peptides of the archaeal cell wall. The cell walls of
methanogenic archaea belonging to the Methanobacteriales family
contain parallel chains of pseudomurein, a polymer of
N-acetyl-L-talosaminurinic acid crosslinked by a peptide. The C71
pseudomurein endoisopeptidases are able to cleave the cell wall
peptide crosslinks of archaea and lyse the cells.
[0166] Based on the location and synteny with the pseudomurein
endoisopeptidase from Methanothermobacter marburgensis phage
.psi.M2 (FIG. 3), ORF 2058 may have a role as a methanogen lysin
gene which encodes the lytic enzyme involved in cell lysis prior to
release of phage progeny. Alignment of ORF 2058 with PeiP from M.
marburgensis and PeiW from M. wolfeii (FIG. 5) shows low overall
homology between the proteins. However there is conservation of the
histidine and aspartic acid residues involved in the
endoisopeptidase catalytic triad and a cysteine residue in ORF 2058
is positioned near the conserved cysteine of PeiP and PeiW which
makes up the third conserved site of the catalytic triad (Makarova
et al., 1999, Luo et al., 2002). Furthermore, the Gly-His-Tyr motif
surrounding the catalytic His residue in PeiP and PeiW is also
found in ORF 2058. These observations indicate that ORF 2058 is a
.phi.mru lysin gene which functions to lyse M. ruminantium cells
during the phage lytic cycle. The differences observed between ORF
2058 and PeiP and PeiW may reflect different archaeal cell wall
peptide crosslinks and therefore peptidase substrate
specificity.
Example 5B
Phage Induction
[0167] Methanobrevibacter ruminantium strain M1.sup.T (DSM1093) was
grown on BY+ medium (basal medium, Joblin et al., 1990) which
consists of [g/l] NaCl (1), KH.sub.2PO.sub.4 (0.5),
(NH.sub.4).sub.2SO.sub.4 (0.25), CaCL.sub.2.2H.sub.2O (0.13),
MgSO.sub.4.7H.sub.2O (0.2), K.sub.2HPO.sub.4 (1), clarified rumen
fluid (300 ml), dH.sub.2O (360 ml), NaHCO.sub.3 (5), resazurin (0.2
ml), L-cysteine-HCl (0.5), yeast extract (2), and Balch's trace
elements solution (10 ml) (added trace elements; Balch et al.,
1979) which consists of (g/l) nitrilotriacetic acid (1.5),
MgSO.sub.4.7H.sub.2O (3), MnSO.sub.4.H.sub.2O (0.5), NaCl (1),
FeSO.sub.4.7H.sub.2O (0.1), CoCl.sub.2.6H.sub.2O (0.1), CaCl.sub.2
(0.1), ZnSO.sub.4.7H.sub.2O (0.1), CuSO.sub.4.5H.sub.2O (0.01),
AlK(SO.sub.4).sub.2.12H.sub.2O (0.01), H.sub.3BO.sub.3 (0.01),
Na.sub.2MoO.sub.4.2H.sub.2O (0.01), NiSO.sub.4.6H.sub.2O (0.03),
Na.sub.2SeO.sub.3 (0.02), and Na.sub.2Wo.sub.4.2H.sub.2O
(0.02).
[0168] At optical densities (OD), measured at a wavelength of 600
(OD.sub.600), between 0.10 and 0.14, M. ruminantium was challenged
with 1 ml and 2 ml of sterile air (.about.160 to 320 .mu.l oxygen),
respectively (FIGS. 10) and 2 pg/ml MitomycinC (FIG. 1D). Typical
lysis curves could be observed for both challenges, with latent
times of .about.90 min for air challenge. Initial results for
MitomycinC challenge indicate a very short latent period. To verify
the excision of the phage from the host genome, 2 oligonucleotides
were designed, facing both phage attachment sites, respectively
(R1F: caaagagagattaaagaagcagacg; SEQ ID NO:146 and L2R
agtagtgttggaatcagtgaaaagg; SEQ ID NO:147). This primer pair only
produces an amplicon if the phage genome recircularises upon
excision.
[0169] FIG. 1E depicts the initial excision experiments when M.
ruminantium was challenged with air. Upon induction, a clear and
unambiguous amplicon of the expected size was found, indicating
successful excision and recircularisation. A similar, albeit weaker
band was also found in uninduced M. ruminantium cells, indicating
that .phi.mru has the ability to spontaneously excise during
normal, unchallenged growth.
Example 5C
Lytic Enzyme Bioassays
[0170] The polypeptide encoded by ORF 2058 is useful as a rumen
methanogen-specific lytic enzyme and has been sub-cloned in an E.
coli expression vector for production of recombinant protein. ORF
2058 was amplified by PCR using the primers Mbbrum11for 22
(1122For, cac cat ggt tag att cag cag aga c; SEQ ID NO:148) and
Mbbrum11rev22 (1122Rev, tca tgc agg aca gac aac ata gta g; SEQ ID
NO:149) in 150 .mu.L reaction volume containing: 121.5 ng M.
ruminantium strain M1 genomic DNA; 0.2 .mu.M 1122For and 1122Rev
primers; 15 .mu.L Accuprime Pfx buffer (with dNTPs, InVitrogen);
2.4 .mu.L Accuprime Pfx (InVitrogen).PCR conditions were 95.degree.
C. for 2 min initial denaturation followed by 35 cycles of
95.degree. C. for 15 seconds, 55.degree. C. for 30 seconds and
68.degree. C. for 40 seconds. No final extension was used. The PCR
product was purified and quantified using a Nanodrop (Thermo
Scientific, GA, USA).
[0171] ORF 2058 cloning: The PCR-amplified ORF 2058 was cloned into
either pET 100 or pET 151-D Topo vectors (InVitrogen) according to
the manufacturer's recommendations, and transformed into chemically
competent TOP 10 cells (InVitrogen). Transformants were analysed by
colony PCR, and plasmid DNA purified and sequenced. Clones with DNA
sequences matching that of ORF 2058 were selected.
[0172] ORF 2058 expression: Plasmid DNA from clones containing
verified ORF 2058 inserts were transformed via electroporation into
electro-competent BL21* or Rosetta 2 cells. The best growth
conditions for expression of soluble ORF 2058 protein was found to
be in LB media, with induction being carried out between 0.48-0.6
Absorbance 600 nm using 0.5 mM IPTG and continuing growth for
approximately six hours at 30.degree. C. Cells were then harvested
by centrifugation and frozen at -20.degree. C.
[0173] Cell lysis: The cell pellet was thawed and resuspended in
the following buffer (pH 7.5): 300 mM NaCl, 2 mM DTT, 10 mM
imidazole, 20 mM Tris, 20% glycerol, 1% Triton-X, 5 mM CaCl.sub.2,
and 10 mM MgCl.sub.2. Lysozyme was added to 1 mg/ml final
concentration, followed by incubation on ice with gentle agitation
for 30 min. DNase I and RNase I were each added to 5 pg/ml final
concentration followed by incubation on ice with gentle agitation
for 30 min. The cell lysate was centrifuged at 12,000 rpm for 15
min and the crude lysate was filtered through a 0.8 .mu.m
filter.
[0174] Nickel affinity chromatography: The filtered supernatant
from the cell lysis procedure was applied to an 80 mL nickel
affinity column and eluted using a 20 mM to 250 mM imidazole
gradient in the following buffer (pH 7.5): 300 mM NaCl, 2 mM DTT,
20 mM Tris, and 20% glycerol. Fractions eluted from the column
containing the expressed ORF 2058 protein were concentrated using a
Millipore ultra filtration cell with a 10,000 kDa molecular weight
cut-off membrane. The ORF 2058 construct in pET100 expressed in E.
coli BL21* cells was eluted from the nickel column by the following
elution buffer, pH 8.2 (20 mM Tris, 250 mM imidazole, 300 mM NaCl,
10 mM b-mercaptoethanol, 10% glycerol), and the enzyme was stored
in a buffer in which additional glycerol and dithiothreitol were
added to achieve a final concentration of 40% glycerol, 1 mM
dithiothreitol, pH 8.2)
[0175] Desalting: Desalting of the concentrated protein expressed
from the pET 151 construct in Rosetta 2 cells was performed using a
250 mL BioGel P6 DG (BioRad, CA, USA) column with the following
buffer (pH 7.0):20 mM MOPS, 1 mM DTT, 300 mM NaCl, and 20%
glycerol. Fractions from the column were concentrated as described
above and the final sample was filtered and snap-frozen in liquid
nitrogen before being stored at -20.degree. C.
[0176] Lysis of resting M. ruminantium cells: Five ml cultures of
M. ruminantium M1 (DSM 1093) were grown in BY+ medium in Hungate
tubes to late log phase and cells were collected by centrifugation
of the Hungate tubes at 5,000.times.g at room temperature for 30
minutes. The tubes were moved into an anaerobic chamber (95%
CO.sub.2-- 5% H.sub.2 atmosphere, Coy Laboratory Products, MI, USA)
where the supernatant was discarded and the cells from 10 ml
culture were resuspended in 1 ml MOPS buffer pH 6.8 (50 mM MOPS, 5
mM CaCl.sub.2, 1 mM dithiothreitol). The cell suspension was
adjusted to an OD (600 nm) of .about.0.12 by dilution with
additional MOPS buffer.
[0177] The standardised cell suspension (50 .mu.l) was dispensed
into a microtitre plate and varying concentrations of ORF 2058
lytic enzyme (prepared from the pET 100 construct) were added and
the total volume of the reaction was made up to 250 ml with buffer.
The cell and protein mixtures were incubated at 37.degree. C. and
OD readings were recorded. The effects of the enzyme additions (pg
enzyme added per assay) on resting M. ruminantium cells are shown
in FIG. 7. The enzyme additions decreased the OD 600 nm readings of
the suspended cells in a dose-dependant manner compared to the
control cells without added enzyme. This indicates that the ORF
2058 lytic enzyme is able to attack and lyse resting cells of M.
ruminantium under anaerobic conditions.
[0178] Lysis of growing M. ruminantium cells: M. ruminantium was
grown in RM02 medium. RM02 medium was composed of the following
ingredients (g/L): KH.sub.2PO.sub.4 (1.4), (NH.sub.4).sub.2SO.sub.4
(0.6), KCl (1.5), trace element solution SL10 (1 ml),
selenite/tungstate solution, (1 ml), 0.1% (w/v) resazurin solution
(4 drops). The components were mixed and boiled under O.sub.2-free
100% CO.sub.2 and cooled in an ice bath while bubbling with 100%
CO.sub.2. After cooling NaHCO.sub.3 (4.2 g) and
L-cysteine.HCl.H.sub.2O (0.5 g) were added and 9.5 ml of the medium
was dispensed into Hungate tubes while gassing the tubes with 100%
CO.sub.2. The tubes were autoclaved and stored in the dark for 24 h
before using. Prior to inoculation, NoSubRFV (0.5 ml per tube,
containing substrates, yeast extract, vitamins) was added. After
inoculation tubes were gassed with 80% CO.sub.2120% H.sub.2 to 25
lb/in.sup.2. M. ruminantium was grown to mid-log (OD 600 nm
.about.0.1) at which point ORF 2058 lytic enzyme (prepared from the
pET 151 D Topo clone) was added to cultures at varying
concentrations. Incubation of cultures continued and OD readings
were recorded. The effect of the enzyme additions on M. ruminantium
growth and methane formation (% methane production relative to the
no-addition control after 217 hours growth are indicated in
brackets) are shown in FIG. 8. The results show that the ORF 2058
lytic enzyme dramatically affected the growth of M. ruminantium in
a dose-dependant manner, decreasing the OD 600 nm of growing
cultures within 2 hours of addition. The two highest levels of
enzyme addition also reduced methane formation to an extent similar
to that of chloroform addition (100 .mu.l/10 ml culture
addition).
Example 6
Overview
[0179] An unexpected discovery from the sequencing of the M
ruminantium genome was the presence of a prophage sequence.
Analysis of the genome sequence identified a region of unusually
high GC content which contained a number of phage-related genes.
The overall structure of the predicted prophage was identified by
further bioinformatic analyses and designated as .phi.mru.
Approximately 40% of the phage genes were assigned to discrete
functional groups including phage integration, DNA replication and
packaging, phage structural proteins and lysis. DNA sequences
flanking the phage genome were found to represent potential sites
for phage integration (attL and attR).
[0180] The phage appears to have inserted itself into a M.
ruminantium putative membrane protein which likely harbours the
original methanogen integration site for the .phi.mru phage genome,
attB. Furthermore, a terminator-like structure found within the DNA
replication module is thought to represent an origin of phage DNA
replication. A low-GC region at the 3' end of the phage genome
harbours what appears to be a DNA modification system by sulphur,
including a gene that is likely to control the expression of the
dnd system. These genes were probably carried into the M.
ruminantium genome during phage integration and their role with
respect to modifying phage, host or foreign DNA remains to be
elucidated. The retention of the dnd system by M ruminantium
suggests it has imparted a benefit to the host. However the role of
the .phi.mru dnd system in modifying M ruminantium or foreign DNA
is still under investigation.
[0181] Another interesting feature of the .phi.mru sequence is the
number of genes encoded on the antisense strand which correspond
with low GC regions and have weak matches to proteins from a
variety of organisms. This suggests that these genes have
accumulated within the .phi.mru genome since its integration into
M. ruminantium and it may be that these genes represent an ongoing
build up of insertions that might eventually lead to phage
inactivation and phage domestication. The high GC content of the
.phi.mru phage sequence compared to the M. ruminantium genome
suggests that it originated from another organism. However, the
previous host is not obvious as the .phi.mru proteins appear
somewhat unique by comparison to other phage encountered to
date.
[0182] The .phi.mru genes of notable interest in regard to methane
mitigation are those located within the lysis cassette. One gene in
particular encodes a protein with similarity to family C39
peptidases. This peptidase family includes, among others, viral
cysteine endopeptidases such as the C71 archaeal phage
endoisopeptidases that cleave the crosslinking peptides of
pseudomurein which makes up Methanobrevibacter cell walls. Based on
gene location within the phage genome and synteny with pseudomurein
endoisopeptidases from other non-rumen methanogen phage genomes,
this gene may have a role as a lysin gene encoding the lytic enzyme
involved in cell lysis prior to release of phage progeny. This gene
and its encoded enzyme are of obvious interest as possible control
mechanism for M. ruminantium and other rumen methanogens with
similar cell walls.
[0183] Ruminant phage and their enzymes that are involved in lysing
host cells represent significant opportunities for controlling both
methanogen populations and other community members (bacteria,
protozoa and fungi) in the rumen. In addition, it is possible to
identify key host enzyme targets that are susceptible to inhibition
by phage proteins through understanding the life cycles of phage.
The inventors have surveyed the composition of rumen phage in cows,
sheep and deer and shown them to display temporal variation in
numbers and type. New Zealand methanogen isolates that are affected
by phage have also been identified. Pure cultures of methanogens
have been used to evaluate phage lytic enzymes, and culture-based
and PCR-based techniques have been developed to screen for novel
phage. Purified phage from rumen samples have been shown to be
amenable to random DNA sequence analysis which enables phage
enzymes to be discovered.
[0184] There are several advantages to the use of phage or their
enzymes in mitigation techniques for lowering methane emissions.
Phage are natural members of the rumen microbial community and,
thus, would not be viewed as antibiotic treatment (and could more
easily overcome any regulatory constraints). Phage are usually
specific for a narrow range of hosts potentially enabling the
selected targeting of methanogens. Phage therapy is now recognised
as a treatment for antibiotic resistant organisms and generally
regarded as safe. Once produced, phage are usually relatively
stable. Introduction of methanogens strains into the rumen that are
susceptible to phage could have long-term beneficial effects,
particularly if inoculation occurs at an early age (e.g., in young
lambs and calves). Certain methanogens are known to either contain
phage genomes, be susceptible to lytic phage, or undergo autolysis
(suggestive of lytic enzymes) including Methanobrevibacter smithii
(strain PS), Methanobacterium bryantii and Methanobrevibacter
strain MF-1. One notable example of phage being used to inhibit
agriculturally problematic organisms is the use of phage to target
Escherichia coli. 0157:H7.
[0185] Methanobrevibacter ruminantium was chosen for genome
sequencing because of its prevalence in the rumen under a variety
of dietary conditions (based on cultivation and molecular detection
data), the availability of cultures, its amenity to routine growth
in the laboratory, and the relatively large amount of previous
studies and background literature available for this organism. The
present invention provides important data regarding the M.
ruminantium genome, and constructs a detailed picture of the phage
within the rumen. The .phi.mru prophage sequence provides specific
reagents for inhibition of M. ruminantium and for future genetic
manipulations to assist in determining gene function. The phage can
be used to block conserved functions/components among methanogens
to prevent or reduce methane formation in the rumen.
REFERENCES
[0186] Alternann E, Klaenhammer T R (2005) PathwayVoyager: pathway
mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG)
database. BMC Genomics 6:60-66. [0187] Alternann, E Klaenhammer T R
(2003) GAMOLA: a new local solution for sequence annotation and
analyzing draft and finished prokaryotic genomes. OMICS: A journal
of integrative biology 7, 161-169. [0188] Altschul S F, Madden T L,
Schaffer A A, Zhang J, Zhang Z, Miller W, Lipman D J (1997) Gapped
BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Research 25, 3389-3402. [0189] Balch W E,
Fox G E, Magrum L J, Woese C R, Wolfe R S (1979) Methanogens:
reevaluation of a unique biological group. Microbiological Reviews
43, 260-296. [0190] Baresi, L. and Bertani, G. 1984. Isolation of a
bacteriophage for a methanogenic bacterium. In Abstracts of the
Annual Meeting of the American Society for Microbiology. Washington
D.C.: American Society for Microbiology, p. 133. [0191] Bickle, T.
A. and D. H. Kruger. 1993. Biology of DNA restriction. Microbiol.
Rev. 57:434-450. [0192] Bult C J, et al. (1996) Complete genome
sequence of the methanogenic archaeon, Methanococcus jannaschii.
Science 273, 1058-1073. [0193] Coutinho P M, Henrissat B (1999)
Carbohydrate-active enzymes: an integrated database approach. In
`Recent Advances in Carbohydrate Bioengineering` (Eds H J Gilbert,
G Davies, B Henrissat and B Svensson) pp. 3-12 (The Royal Society
of Chemistry, Cambridge) (Carbohydrate Active Enzymes database,
hyper text transfer protocol://world wide web.cazy.org/). [0194]
Delcher A L, Harmon D, Kasif S, White O, Salzberg S L (1999)
Improved microbial gene identification with GLIMMER. Nucleic Acids
Research 27, 4636-4641. [0195] Fleischmann, R D, et al. (1995)
Whole-genome random sequencing and assembly of Haemophilus
influenzae Rd. Science 269, 496-512. [0196] Fricke W F, Seedorf H,
Henne A, Kruer M, Liesegang H, Hedderich R, Gottschalk G, Thauer R
K (2006) The genome sequence of Methanosphaera stadtmanae reveals
why this human intestinal archaeon is restricted to methanol and
H.sub.2 for methane formation and ATP synthesis. Journal of
Bacteriology 188, 642-658. [0197] Godde J S, Bickerton A (2006) The
repetitive DNAe called CRISPRs and their associated genes: evidence
of horizontal transfer among prokaryotes. Journal of Molecular
Evolution 62, 718-729. [0198] Haft D H, Selengut J, Mongodin E F,
Nelson K E (2005) A guild of 45 CRISPR-associated (Cas) protein
families and multiple CRISPR/Cas subtypes exist in prokaryotic
genomes. PLoS Computational Biology 1:474-483 [0199] Jansen R,
Embden J D, Gaastra W, Schouls L M (2002) Identification of genes
that are associated with DNA repeats in prokaryotes. Molecular
Microbiology 43, 1565-1575. [0200] Jansen R, van Embden J D,
Gaastra W, Schouls L M (2002) Identification of a novel family of
sequence repeats among prokaryotes. OMICS: A journal of integrative
biology 6, 23-33. [0201] Jensen L J, Friis C, Ussery D W (1999)
Three views of microbial genomes. Research in Microbiology 150,
773-777. [0202] Joblin K N, Naylor G E, Williams A G (1990) Effect
of Methanobrevibacter smithii on xylanolytic activity of anaerobic
ruminal fungi. Applied and Environmental Microbiology 56,
2287-2295. [0203] Kanehisa M, Goto S, Kawashima S, Okuno Y, Hattori
M (2004) The KEGG resource for deciphering the genome. Nucleic
Acids Research 32, D277-D280. [0204] Kiener, A., Konig, H., Winter,
J. and Leisinger, T. 1987. Purification and use of Methanobacterium
wolfeii pseudomurein endopeptidase for lysis of Methanobacterium
thermoautotrophicum. J. Bacteriol. 169, 1010-1016. [0205] Knox, M.
R. and Harris, J. E. 1986. Isolation and characterisation of a
bacteriophage of Methanobrevibacter smithii. In Abstracts of the
XIV International Congress on Microbiology. Manchester:
International Union of Microbiological Societies. [0206] Kurtz S,
Schleiermacher C (1999) REPuter: fast computation of maximal
repeats in complete genomes. Bioinformatics 15, 426-427. [0207]
Lowe T M, Eddy S R (1997) tRNAscan-SE: a program for improved
detection of transfer RNA genes in genomic sequence. Nucleic Acids
Research 25, 955-964. [0208] Loenen, W. and N. Murray. 1986.
Modification enhancement by restriction alleviation protein (Ra1)
of bacteriophage lambda. J. Mol. Biol. 190:11-22. [0209] Lucchini,
S., F. Desiere, and H. Brussow. 1999. Comparative genomics of
Streptococcus thermophilus phage species supports a modular
evolution theory. J. Virol. 73:8647-8656. [0210] Luo, Y. N.,
Pfister, P., Leisinger, T. and Wasserfallen, A. 2002. Pseudomurein
endoisopeptidases PeiW and PeiP, two moderately related members of
a novel family of proteases produced in Methanothermobacter
strains. FEMS Microbiol. Lett. 208, 47-51. [0211] Makarova, K. S.,
Aravind, L. and Koonin, E. V. 1999. A superfamily of archaeal,
bacterial, and eukaryotic proteins homologous to animal
transglutaminases. Protein Sci. 8, 1714-1719. [0212] Makarova K S,
Grishin N V, Shabalina, S A, Wolf Y I, Koonin E V (2006) A putative
RNA-interference-based immune system in prokaryotes: computational
analysis of the predicted enzymatic machinery, functional analogies
with eukaryotic RNAi, and hypothetical mechanisms of action.
Biology Direct 1:7-32. [0213] New Zealand Statistics 2005 (world
wide web.stats.govt.nz) [0214] New Zealand's Greenhouse Gas
Inventory 1990-2004. The National Inventory Report and Common
Reporting Format. (2006) Ministry for the Environment. hyper text
transfer protocol://world wide
web.mfe.govt.nz/publications/climate/nir-apr06/nir-apr06.pdf.
[0215] Reeve J N, Nolling J Morgan R M, Smith D R (1997)
Methanogenesis: genes, genomes and who's on first? Journal of
Bacteriology 179, 5975-5986. [0216] Rawlings, N. D., Morton, F. R.
and Barrett, A. J. 2006. MEROPS: the peptidase database. Nucleic
Acids Res. 34, D270-D272. [0217] Samuel B S, Hansen E E, Manchester
J K, Coutinho P M, Henrissat B, Fulton R, Latreille P, Kim K,
Wilson R K, Gordon J I (2007) Genomic adaptations of
Methanobrevibacter smithii to the human gut. Proceedings of the
National Academy of Sciences USA 104, 10643-10648. [0218] Smith D
R, et al. (1997) Complete genome sequence of Methanobacterium
thermoautotrophicum .quadrature.H: Functional analysis and
comparative genomics. Journal of Bacteriology 179, 7135-7155.
[0219] Smith P H, Hungate R E (1958) Isolation and characterization
of Methanobacterium ruminantium n. sp. Journal of Bacteriology 75,
713-718. [0220] Staden R, Beal K F, Bonfield J K (1998) The Staden
Package. Methods in Molecular Biology Bioinformatics Methods and
Protocols 132, 115-130. [0221] Tatusov R L, Natale D A, Garkavtsev
I V, Tatusova T A, Shankavaram U T, Rao B S, Kiryutin B, Galperin M
Y, Fedorova N D, Koonin E V (2001) The COG database: new
developments in phylogenetic classification of proteins from
complete genomes Nucleic Acids Research 29, 22-28.
[0222] All publications and patents mentioned in the above
specification are herein incorporated by reference.
[0223] Where the foregoing description reference has been made to
integers having known equivalents thereof, those equivalents are
herein incorporated as if individually set forth. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. It is
appreciated that further modifications may be made to the invention
as described herein without departing from the spirit and scope of
the invention.
Sequence CWU 1
1
1511258PRTMethanobrevibacter ruminantium 1Val Arg Asn Met Lys Asn
Lys Ser Leu Ile Leu Ile Ser Leu Leu Leu 1 5 10 15 Leu Ile Thr Ile
Ile Ser Ile Gly Ser Val Val Ala Thr Asp Asn Glu 20 25 30 Glu Ile
Asn Met Asp Asn Ile Asn Asn Ile Asp Asn Asn Glu Asp Ile 35 40 45
Ala Asn Ile Asp Asn Val Asp Asn Val Asp Asn Ser Asn Ile Asn Asn 50
55 60 Pro Thr Asp Ile Arg Ile Asp Asn Ser Asn Leu Asn Arg Glu Thr
Glu 65 70 75 80 Leu Asp Ser Asn Leu Asn Lys Ser Asn Gln Ile Arg Glu
Asp Glu Leu 85 90 95 Glu Gln Ser Asn Ala Lys Ser Asn Leu Lys Ser
Ser Lys Leu Ser Ser 100 105 110 Thr Ile Thr Val Asp Gly Ser Asp Glu
Asn Gln Met Ser Asn Pro Thr 115 120 125 Ile Gln Ser Ala Ile Asp Ser
Ala Asn Ala Gly Asp Thr Ile Ile Ile 130 135 140 Thr Gly Lys Ser Tyr
Val His Cys His Phe Ile Val Asn Lys Pro Leu 145 150 155 160 Thr Ile
Ile Ser Glu Ile Gly Thr Ser Met Ser Pro Cys Pro Ser Asn 165 170 175
Thr Lys Gly Ser Gly Ala His Gly Ile Phe Tyr Ile Ser Pro Glu Ala 180
185 190 Ser Gly Thr Val Leu Lys Gly Phe Asn Leu Thr Asn Thr Tyr Gly
Asp 195 200 205 Tyr Asp Asp Tyr Gly Ile Leu Ile Arg Gly Ala Glu Asn
Val Glu Ile 210 215 220 Ile Asn Cys Thr Ile Asn Thr Val Ser Asp Gly
Asp Gly Ile Arg Ile 225 230 235 240 Glu Asn Ala Thr Asn Thr Lys Ile
Ala Asp Cys Leu Ile Lys Asp Ser 245 250 255 Asn Ile
2255PRTMethanobrevibacter ruminantium 2Met Lys Thr Phe Arg Gln Gln
Leu Leu Glu Asp Pro Glu Phe Gln Asn 1 5 10 15 Tyr Leu Leu Gln Arg
Pro Asn Leu Thr Glu Ser Ser Leu Gln Ser Tyr 20 25 30 Leu Asn Ala
Ala Thr Asn Phe Val Arg Phe Thr Gly Glu Pro Phe Tyr 35 40 45 Lys
Thr Val His Glu Leu Arg Ser Gln Gln Asn Asp Arg Ile Glu Asn 50 55
60 Asn Ile Ile Ile Arg Phe Asn Pro Asn Gln Ser Arg Ile Asn Ile Met
65 70 75 80 Gln Phe Glu Phe Ile Glu Tyr Leu Lys Gly Arg Gly Cys Thr
Glu Val 85 90 95 Ser Ile Asp Ser Tyr Val Arg Tyr Met Arg Thr Ile
Leu Ser Thr Leu 100 105 110 Gly Ile Ile Leu Pro Lys Ser Pro Lys Leu
Asp Asp Thr Pro Gln Asp 115 120 125 Trp Tyr Leu Leu Thr Lys Asp Asp
Ile Lys Tyr Val Leu Asp Thr Ala 130 135 140 Asn Leu Gln Tyr Lys Ala
Val Ile Asn Phe Ala Ala Val Thr Gly Leu 145 150 155 160 Arg Val Arg
Asp Met Arg Ser Leu Thr Ile Lys Asp Phe Met Thr Ala 165 170 175 Thr
Glu Glu Tyr His Gly Cys Thr Glu Val Glu Asp Phe Leu Asp Ser 180 185
190 Ala Pro Asp Gly Met Ile Gly Phe Trp Glu Leu Phe Pro Gln Lys Thr
195 200 205 Arg Lys Phe Arg Leu Pro Cys Lys Val Cys Asn Thr Pro Glu
Ser Ser 210 215 220 Asp Leu Leu Leu Phe Ser Leu Asn Glu Arg Val Lys
Tyr Phe Glu Trp 225 230 235 240 Lys Asn Glu Lys Asp Gly Thr Asp Leu
Lys Ile Thr Lys Asn Asp 245 250 255 398PRTMethanobrevibacter
ruminantium 3Met Asn Thr Leu Lys Ile Glu Cys Ser Lys Asp Asp Tyr
Ile Ile Lys 1 5 10 15 Thr Ala Lys Ala Asn Ser Glu Asn Glu Leu Lys
Val Glu Val Pro Glu 20 25 30 Asn Trp Asn Cys Asp Tyr Val Asn Ala
Val Leu Trp Glu Glu Asp Ile 35 40 45 Cys Glu Val Leu Glu Lys Gly
Asp Glu Arg Met Leu Leu Ile Pro Met 50 55 60 Cys Gly Glu Leu Leu
Leu Glu Gly Val Gln Glu Asp Glu Tyr Ile Lys 65 70 75 80 Tyr Ile Cys
Leu Pro Val Lys Tyr Gln Asp Gln Met Val Leu Ile Ala 85 90 95 Lys
Ile 4255PRTMethanobrevibacter ruminantium 4Met Gly Met Lys Lys Val
Lys Ile Glu Ser Lys Ala Lys Asn Asn Met 1 5 10 15 Thr Arg Asn Glu
Lys Leu Phe Tyr Lys Leu Tyr Asp Asp Leu Tyr Asn 20 25 30 Glu Asp
Arg Leu Ile Leu Phe Ser Thr Tyr Phe Asn Ile Tyr Asp Lys 35 40 45
Phe Ile Phe Lys Lys Asp Ile Ile His Tyr Val Leu Met Asn Tyr Ser 50
55 60 Glu Asn Glu Ile Ile Glu Ala Met Lys Lys Ile Asp Glu Ile Gln
Ser 65 70 75 80 Glu Gly Ile Asp Ile Lys Thr Phe Ile Pro Lys Lys Tyr
Cys Pro Lys 85 90 95 Cys Lys Lys Val Met Asp Ser Tyr Gly Lys Ile
Cys Pro Asp Cys Gly 100 105 110 Thr Ile Leu Ile Glu Asp Glu Lys Lys
Ile Gln Glu Leu Gln Ala Lys 115 120 125 Asp Lys Ala Tyr Glu Glu Tyr
Leu Glu Lys Glu Tyr Asn Ile Tyr Leu 130 135 140 Gln Asn Ser Tyr His
Asn Met Ile Gln Gly Ser Tyr Thr Ile Lys Ile 145 150 155 160 Arg Thr
Arg Lys Pro Lys Thr Glu Thr Glu Ile Val Arg Ile Pro Ala 165 170 175
Gln Thr Val Thr Ser Arg Gly Lys Phe Asn Ser Ile Ser Gln His Tyr 180
185 190 Pro Ser Glu Thr Tyr Thr Arg Gln Lys Val Thr Arg Ala Lys Tyr
Lys 195 200 205 Glu Cys Arg Val Leu Phe Asp Lys Glu Lys Met Ile Leu
Asn Ile Asp 210 215 220 Gly Thr Ala Thr Lys Leu Tyr Tyr Asp Glu Val
Cys Glu Leu Glu Tyr 225 230 235 240 Pro Glu Gln Glu Val Asn Glu Leu
Val Thr Leu Thr Leu His Asn 245 250 255 5228PRTMethanobrevibacter
ruminantium 5Met Lys Thr Gln Asp Leu Ile Asn Ile Ile Asn Asp Glu
Glu Ser Pro 1 5 10 15 Val Phe Leu Asn Arg Glu Val Phe Glu Met Asp
Tyr Val Pro Asp Ile 20 25 30 Tyr Lys Tyr Arg Asp Glu Gln Leu Ala
Lys Met Ala Met Tyr Cys Asn 35 40 45 Ser Ile Pro Asp Asn Ile Ala
Pro Lys Asn Leu Gln Leu Cys Gly Gly 50 55 60 Asn Ala Thr Gly Lys
Thr Thr Thr Leu Lys Gln Phe Phe Lys Met Leu 65 70 75 80 Asn Glu Ala
Phe Pro Asn Ile Val Thr Val Tyr Ile Asn Cys Gln Leu 85 90 95 Phe
Asn Thr Glu Asn Thr Val Tyr Gly Lys Ile Tyr Asn Lys Leu Tyr 100 105
110 Gly Val Lys Gly Ser Ile Asn Gly Lys Ser Asn Thr Met Leu Phe Asp
115 120 125 Lys Ile Val Ala Arg Leu Lys Lys Glu Asn Lys Ile Leu Ile
Ile Gly 130 135 140 Leu Asp Asp Phe Asp Ser Phe Lys Ser Arg Asp Gly
Leu Asn Lys Met 145 150 155 160 Leu Tyr Asn Phe Leu Arg Ile His Glu
Ala Glu Glu Gly Ile Gln Ile 165 170 175 Cys Ile Phe Thr Val Ser Asn
Lys Gly Glu Ser Glu Ser Leu Leu Leu 180 185 190 Pro Ser Arg Gln Ser
Ser Thr Gly Phe Arg Tyr Ser Leu Thr Ser Thr 195 200 205 Pro Trp Ser
Arg Cys Thr Thr Tyr Trp Thr Thr Gly Ala Pro Ser Val 210 215 220 Ser
Ile Leu Ala 225 6255PRTMethanobrevibacter ruminantium 6Met Phe Leu
Glu Lys Cys Asp Gly Lys Asp Thr Ile Ser Met Ser Leu 1 5 10 15 Gln
Glu Lys Met Asn Leu Ile Leu Glu Thr Met Glu Ser Lys Gly Ser 20 25
30 Pro Phe Ile Ser Cys Ile His Cys Asn Ile Pro Ile Asn Glu Ala Glu
35 40 45 Glu Trp Tyr Lys Asn Gly Glu Ile Gly Asp Gln Asp Phe Ile
Asn Phe 50 55 60 Tyr Asp Asp Val Asn Leu Ile Glu Glu Ser Phe Gly
Phe Glu Ile Tyr 65 70 75 80 Lys Lys Ser Glu Tyr Pro Thr Leu His Thr
Gln Ser Ile Asn Gln Ile 85 90 95 Ala Ser Thr Tyr Pro Met Asn Arg
Thr Gln Asn Glu Lys Thr Pro Leu 100 105 110 Phe Leu Arg Arg Glu Arg
Lys Leu Tyr Glu Ile Thr Asn Ile Phe Lys 115 120 125 Ser His Ser Thr
Asn Glu Ile Phe Ile Ser Met Asp Ser Lys Ser Lys 130 135 140 Leu Lys
His Glu Ile Lys Tyr Asp Phe Thr Leu Lys Glu Leu Asn Glu 145 150 155
160 Ile Phe Lys Asn Tyr Leu Glu Glu Asp Cys Ser Ile Tyr Ile Leu Asn
165 170 175 Asp Asn Arg Ala Phe Val Met Thr Leu Gly His Phe Gln Phe
Glu Phe 180 185 190 Asp Val Phe Gly Ser Ala Lys Glu Ser Tyr Val Phe
Asp Val Glu Ile 195 200 205 Asp Asp Glu Lys Tyr Asp Lys Ile Phe Ile
Arg Ser Ser Tyr Tyr Phe 210 215 220 Asn Ile Pro Val Asp Glu Leu Asp
Gly Leu Ala Lys Ile Leu Lys Gln 225 230 235 240 Lys Arg Ile Ile Glu
Glu Gly Phe Phe Glu Gly Gln Phe Leu Tyr 245 250 255
768PRTMethanobrevibacter ruminantium 7Met Ile Ile Phe Leu Lys Leu
Lys Phe Gly Arg Val Ile Met Glu Lys 1 5 10 15 Leu Ile Glu Ile Asp
Gly Val Gln Tyr Thr Glu Ala Gln Ile Arg Arg 20 25 30 Ala Leu Ala
Ile Glu Arg Asp Val Arg Ser Pro Asn Phe Val Asp Met 35 40 45 Leu
Leu Gly Lys Ile Lys Pro Ser Glu Leu Ala Ser Arg Val Ser Glu 50 55
60 Lys Gly Asp Ala 65 851PRTMethanobrevibacter ruminantium 8Met Cys
Tyr Val Gly Asn Thr Arg Thr Leu Val Tyr His Thr Glu Asp 1 5 10 15
Cys Phe Cys Asn His Trp Leu Leu Asn Glu Asn Lys Thr Ile Leu Glu 20
25 30 Glu Lys Pro Val Asp Met Lys Pro Cys Ser Phe Cys Lys Pro Gln
Phe 35 40 45 Asp Thr Glu 50 977PRTMethanobrevibacter ruminantium
9Met Leu Asp Met Val Ala Glu Met Val Glu Asn Ile Arg Lys Gly Glu 1
5 10 15 Gly Asp Gly Tyr Ser Ile Tyr Pro Pro Phe Ser Cys Ile Val Phe
Leu 20 25 30 Gln Gly Lys Lys Tyr Ser Glu Cys Cys Cys Lys Ala Glu
Ala Arg Asp 35 40 45 Gln Lys Phe Ala Leu Val Asn Leu Val Gly Phe
Arg Arg Glu Asp Val 50 55 60 Lys Val Ile Asp Pro Arg Thr Asn Glu
Glu Leu Phe Val 65 70 75 1053PRTMethanobrevibacter ruminantium
10Met Ser Met Leu Ala Asp Phe Glu Pro Ala Arg Leu His Lys Arg Thr 1
5 10 15 Trp Ala Glu Arg His Asp Val Glu Ile Leu Ala Val Ile Cys Leu
Ala 20 25 30 Ile Ser Ile Ala Met Leu Leu Leu Phe Phe Ala Leu Ala
Glu Pro Thr 35 40 45 Val Ala Gly Val Ile 50
11150PRTMethanobrevibacter ruminantium 11Met Thr Lys Glu Phe Glu
Asp Phe Met Arg Arg Asn Thr Gly Leu Leu 1 5 10 15 Val Phe Leu Arg
Trp Asp Thr Val Ala Tyr Leu Lys Tyr Leu Glu Ser 20 25 30 His Tyr
Asp Glu Arg Lys Tyr Glu Cys Ala Tyr Arg Leu Leu Glu Ala 35 40 45
Ile Asp Asn Leu Phe Asp Phe Tyr Gln Ile Thr Phe Ser Lys Lys Thr 50
55 60 Glu Arg Glu Ile Pro Asp Gln Leu Phe Glu Lys Asp Lys Ile Asn
Lys 65 70 75 80 Gly Phe Leu Ser His Ile Gly Lys Ala Cys Lys Lys Gln
Ser Asp Phe 85 90 95 Tyr Gly Gln Arg Trp Lys Ser Thr Arg Gly Ile
Arg Asp Ala Tyr Gly 100 105 110 His Tyr Ser Ala Gly Asn Phe Leu Phe
Ala Asp Ile Tyr Gly Cys Leu 115 120 125 His Arg Ile Ser Asp Asp Cys
Tyr Arg Ile Leu Asn Phe Ser Glu Tyr 130 135 140 Glu Ile Glu Glu Lys
Ala 145 150 12251PRTMethanobrevibacter ruminantium 12Met Asn Glu
Ile Val Thr Thr Thr Asn Glu Asn Asn Val Pro Val Asp 1 5 10 15 Val
Asp Tyr Ala Ile Glu Glu Trp Lys Ala Tyr Gln Arg Leu Thr Arg 20 25
30 Glu Leu Leu Asp Glu Thr Asp Tyr Gln Thr His Arg Gly Arg Lys Tyr
35 40 45 Lys Thr Lys Ser Ala Trp Gln Lys Tyr Ala Arg Ala Phe Asn
Ile Asn 50 55 60 Thr Gln Ile Ile Asp Lys Glu Ile Val Lys Asn Asp
Lys Gly Ile Val 65 70 75 80 Ile Glu Ala Glu Tyr Thr Val Arg Ala Thr
Leu Pro Asn Gly Arg Phe 85 90 95 Val Glu Ser Asp Gly Ser Cys Asp
Arg Arg Glu Ser Gly Lys Arg Glu 100 105 110 Met Ser Asn His Ser Ile
Lys Ala Thr Ala Lys Thr Arg Ala Thr Asn 115 120 125 Arg Ala Ile Ser
Glu Leu Ile Gly Ala Gly Asp Val Ser Ala Asp Glu 130 135 140 Leu Asp
Pro Ala Phe Asp Lys Val Gln His Ser Lys Thr Asn His Val 145 150 155
160 Ile Glu Ala Glu Val Ala Glu Ile Ile Glu Ser Pro Tyr Asp Lys Asn
165 170 175 Ala Gly Phe Glu Thr Ala Asp Lys Ile Glu Pro Val Asp Glu
Asp Pro 180 185 190 Val Cys Lys Asn Trp Val Lys Thr Ile Cys Lys Thr
Ile Lys Ala Glu 195 200 205 Gly Lys Pro Cys Leu Lys Gly Val Leu Ile
Gln Lys Ala Arg Thr Ile 210 215 220 Gly Thr Met Thr Asp Glu Glu Arg
Asn Arg Leu Ile Glu Tyr Ile Lys 225 230 235 240 Thr Leu Pro Lys Gly
Glu Val Asn Leu Asp Asp 245 250 1376PRTMethanobrevibacter
ruminantium 13Met Ile Glu Cys Ile Gln Glu Glu Gly Asp Phe Thr Asp
Trp Glu Val 1 5 10 15 Pro Ser Ser Ser Ser Asp Ile Lys Tyr Ile Val
Ser Val Asp Asp Glu 20 25 30 Gly Asn Leu Phe Cys Ser Cys Pro Asp
Phe Tyr Tyr Arg Lys Ser Arg 35 40 45 Met Asn Pro His Ile Ser Asn
Pro Glu Ser Tyr Cys Lys His Ile Arg 50 55 60 Gln Val Leu Glu Glu
Asp Asn Arg Leu Gln Met Leu 65 70 75 14255PRTMethanobrevibacter
ruminantium 14Met Asp Asn Ile Asn Lys Thr Lys Thr Ser Leu Ala Lys
Phe Glu Glu 1 5 10 15 Phe Phe Ser Thr Val Tyr Lys Asp Glu Val Met
Glu Val Leu Glu Lys 20 25 30 Tyr Pro Glu Glu Arg Thr Leu Val Val
Asp Tyr Glu Asn Leu Glu Met 35 40 45 Phe Asp Pro Asp Leu Ala Asp
Leu Leu Ile Glu Lys Pro Asp Glu Val 50 55 60 Ile Ala Ala Ser Gln
Lys Ala Ile Lys Asn Ile Asp Pro Leu Met Lys 65 70 75 80 Asp Pro Lys
Leu Asp Ile Lys Phe Lys Asn Val Ser Asn Cys Ile Asp 85 90 95 Phe
Val Asn Ala Asp Ser Lys Tyr Ile Gly Lys Leu Ile Ser Phe Glu 100 105
110 Ala Lys Val Met Glu Ala Lys Glu Pro Lys Pro Ile Leu Asp Ile Ala
115 120 125 Val Tyr Glu Cys Arg Gly Cys Met Ser Leu Arg Glu Ile Pro
Gln Thr 130 135 140 Ile Asn Ser Ser Leu Glu Pro Ser Leu Cys Pro Glu
Cys
Gly Gly Arg 145 150 155 160 Ser Phe Arg Leu Leu Gln Asp Glu Ser Glu
Phe Leu Glu Ser Gln Leu 165 170 175 Leu Ile Val Ser Ser Asp Asp Thr
Ser Lys Ser Leu Lys Val Leu Leu 180 185 190 Leu Arg Asp Glu Cys Ser
Phe Asp Leu Tyr Ser Met Gly Gln Glu Val 195 200 205 Arg Ile Thr Gly
Ile Leu Lys Ser Phe Ser Ser Asn Tyr Gly Tyr Glu 210 215 220 Tyr Phe
Leu Glu Cys Asn Leu Ile Glu Ile Leu Asn Asp Ser Glu Asp 225 230 235
240 Ser Glu Tyr Asp Glu Tyr Gly Asn Arg Asn Ser Pro Glu Tyr Arg 245
250 255 15143PRTMethanobrevibacter ruminantium 15Met Ala Asn Lys
Ile Arg Val Asn Leu Thr Val Asp Pro Asn Leu Trp 1 5 10 15 Gln Leu
Ala Lys Asp Lys Leu Pro Cys Ser Arg Ser Glu Phe Phe Glu 20 25 30
Asn Gln Leu Lys Met Phe Leu Gly Ile Glu Asp Asp Glu Ser Glu Ile 35
40 45 Ile Lys Asp Ile Gln Thr Lys Glu Asn Glu Ile Asn Ala Leu Arg
Asp 50 55 60 Lys Leu Cys His Val Arg Lys Ser Lys Gln Leu Lys Leu
Glu Ser Asn 65 70 75 80 Lys Ser Met Glu Lys Ala Met Ala Ser Leu Asn
Arg Met His Lys Lys 85 90 95 Tyr Gly Lys Ile Gly Glu Asn Gln Ile
Arg Asn Leu Ala His Val His 100 105 110 Lys Val Asp Phe Asp Asp Leu
Lys Lys Glu Cys Gln Asp Asn Cys Met 115 120 125 Asn Ile Phe Glu Phe
Ala Glu Val Pro Lys His Asp Ser Val Met 130 135 140
16134PRTMethanobrevibacter ruminantium 16Met Thr Ile Gly Ser Leu
Asp Asn Phe Gly Lys Ser Asp Gly Glu Asn 1 5 10 15 Met Asn Pro Glu
Asp Phe Asp Cys Ser Val Phe Phe Glu Met Tyr Lys 20 25 30 Ala Leu
Phe Glu Ile Leu Asp Val Glu Val Gly Ser Phe Ala Glu Leu 35 40 45
Leu Asp Val Tyr Lys Asn Val Glu Met Asp Tyr Thr Leu Lys Arg His 50
55 60 Ala Leu Lys Gln Lys Glu Ile Leu Tyr Trp Phe Asn Thr Asp Trp
Lys 65 70 75 80 Glu Glu Leu Gly Lys Glu Lys Pro Thr Glu Lys Asp Lys
Glu Lys Trp 85 90 95 Ile Arg Gln Lys Ile Gly Tyr Asp Ser Phe Val
Val Glu Gln Leu Glu 100 105 110 Val Lys Leu Lys His Ile Arg Arg Met
Tyr Glu Thr Ala Leu Lys His 115 120 125 Ser Phe Glu Ala Ile Lys 130
1791PRTMethanobrevibacter ruminantium 17Met Asn Val Lys Thr Val Met
Asn Asp Leu Ile Gly Leu Ser Lys Glu 1 5 10 15 Phe Glu Gly Val Glu
Tyr Glu Ile Glu Ser Lys Asn Ser Ile Tyr Phe 20 25 30 Tyr Ser Phe
Pro Lys Tyr Met Lys Glu Gly Ile Val Ile Leu Lys Tyr 35 40 45 Ser
Ala Ile Tyr Asp Leu His Thr Ile Leu Lys Gly Met Asp Gly Ile 50 55
60 Ile Val Asp Ile Leu Glu Val Glu Asp Asn Pro Gly Asp Glu Lys Lys
65 70 75 80 Asp Leu Leu Tyr Val Gln Ile Glu Val Lys Glu 85 90
1877PRTMethanobrevibacter ruminantium 18Met Ile Ser Asp Glu Trp Glu
Glu Glu Tyr Tyr Val Lys Val Asn Glu 1 5 10 15 Glu Leu Glu Gln Val
Glu Val Arg Phe Phe Ser Lys Val Asp Arg Leu 20 25 30 Val Phe Ala
Gln Ser Tyr Ser Ser Ser Ser Ser Phe Ser Phe Glu Glu 35 40 45 Ala
Glu Ile Leu Cys Asp Arg Ile Cys Asp Ile Leu Thr Asn Asp Leu 50 55
60 Gly Asn Ala Lys Tyr Tyr Leu Gly Gly Lys Asp Glu Leu 65 70 75
1949PRTMethanobrevibacter ruminantium 19Met Asn Tyr Arg Glu Trp Val
Ala Ser Gln Phe Glu Leu Arg Met Thr 1 5 10 15 Glu Asp Gly Arg Val
Cys Phe Lys Thr Pro Cys Ser Tyr Tyr Thr Phe 20 25 30 Ser Lys Glu
Asp Phe Glu Ile Ile Arg Glu Met Phe Leu Asn Phe Glu 35 40 45 Ser
2057PRTMethanobrevibacter ruminantium 20Met Asp Arg Ile Lys Glu Leu
Asn Val Cys Gly Thr Cys Lys His Ser 1 5 10 15 His Leu Ile Pro Asp
Ile Asn Gly Glu Ile Ala Val Asn Ile Cys Arg 20 25 30 Ile Gly Ser
Glu Ala Val Asn Lys Asp Gly Gly Leu Thr Tyr Cys Val 35 40 45 Asp
Trp Thr Pro Arg Arg Lys Leu Ser 50 55 21111PRTMethanobrevibacter
ruminantium 21Met Leu Ser Lys Lys Glu Ala Ile Gln Met Thr Leu Asp
Asn Glu Lys 1 5 10 15 His Tyr Pro Val Lys Cys Lys Tyr Cys Gly Lys
Pro Phe Thr Lys Ser 20 25 30 His Asn Arg Gln Met Tyr Cys Ser Asp
Ser Cys Arg Arg Asn Ala Leu 35 40 45 Arg Glu Gln Lys Ala Arg Tyr
Gln Ala Lys Arg Arg Leu Lys Ile Lys 50 55 60 Gln Lys Val Leu Ile
Val Asp Glu Tyr Lys Lys Tyr Gly Leu Gly Ser 65 70 75 80 Tyr Gly Thr
Ser Ala Asn Gly His Arg Lys Asn Asn Phe Ser His Glu 85 90 95 Tyr
Met Ala Ile Gln Lys Glu Met Lys Arg Ile Gly Leu Lys Arg 100 105 110
22183PRTMethanobrevibacter ruminantium 22Met Tyr Leu Ala Lys Phe
Cys Pro Asn Cys Gly Asn Lys Val Glu Glu 1 5 10 15 Asn Asp Lys Phe
Cys Ile Tyr Cys Gly Asn Lys Leu Arg Val Ile Ile 20 25 30 Pro Glu
Lys Lys Val Lys Arg Ser Ser Asn Ser Ile Asn Asp Glu Lys 35 40 45
Thr Ser Lys Tyr Val Glu Val Ile Asp Gly Leu Met Arg Tyr Lys Val 50
55 60 Phe Pro Ser Ser Leu Pro Val Lys Tyr Ile Ile Tyr Lys Val Asn
Tyr 65 70 75 80 Gly Thr Thr Ser Asp Glu Ile Lys Asn Ile Leu Glu Asn
Gly Asn Tyr 85 90 95 Asn Tyr Lys Ile Asn Ile His Tyr Phe Leu Gln
Asn Lys Lys Leu Tyr 100 105 110 Phe Arg Ser Pro Lys Asn Pro Asn Met
Phe Phe Lys Phe His Asn Asp 115 120 125 Arg Phe Asn Glu Glu Met Arg
Lys Asp Asn Lys Glu Ile Ile His Ile 130 135 140 Ser Ser Tyr Ala Arg
Val His Arg Pro Ser Leu Thr Lys Ile Phe Asn 145 150 155 160 Phe Asn
Gln Glu Lys Thr Phe Ile Leu Ala Asn Lys His Phe Glu Glu 165 170 175
Asn Ile Arg Lys Thr Arg Leu 180 2377PRTMethanobrevibacter
ruminantium 23Met Ile Tyr Asp Lys Ala Thr Val Thr Ser Leu Ile Val
Ala Ile Leu 1 5 10 15 Leu Pro Leu Met Ser Met Leu Gly Ile Gly Glu
Leu Thr Gln Asn Tyr 20 25 30 Ile Leu Ala Ile Val Ser Gly Met Ile
Ala Leu Val Val Trp Tyr Tyr 35 40 45 Asn Glu Lys His Asn Ser Asp
Leu Val Ser Gly Thr Thr Lys Cys Asp 50 55 60 Cys Glu Leu Cys Tyr
Gly Gly Asp Asp Glu Ala Leu Ile 65 70 75 24112PRTMethanobrevibacter
ruminantium 24Met Lys His Leu Tyr Glu Ile Ile Pro Tyr Arg Arg Thr
Val Trp Ile 1 5 10 15 Thr Gly Phe Leu Lys Thr Thr Val Ser Ser Ala
Met Ile Thr Thr Gly 20 25 30 Val Val Ile Leu Phe Asn Ser Ile Thr
Glu His Pro Tyr Phe Met Glu 35 40 45 Trp Asp Glu Ile Gly Ile Val
Leu Gly Ile Val Ser Ile Thr Ile Ala 50 55 60 Cys Ile Tyr Ile Ala
Met Ile Asp Arg Trp Lys Glu Arg Arg Lys Lys 65 70 75 80 Glu Glu Leu
Asp Thr Ile Glu Asp Tyr Ile Asn Arg Lys Ala Glu Glu 85 90 95 Ile
Ala Asn Met Lys Val Leu Arg Lys Leu Glu Glu Leu Glu Glu Glu 100 105
110 25204PRTMethanobrevibacter ruminantium 25Met Ile Glu Ile Ser
Thr Ile Lys Ile Thr Asp Ile Lys Pro Ala Glu 1 5 10 15 Tyr Asn Pro
Arg Ile Met Ser Gln Leu Glu His Thr Lys Leu Arg Asn 20 25 30 Ser
Met Glu Thr Phe Gly Val Val Asp Pro Ile Ile Ile Asn Leu Lys 35 40
45 Asn Asn His Ile Ile Gly Gly His Gln Arg Tyr Glu Val Leu Leu Asp
50 55 60 Lys Ser Met Glu Asp Asn Glu Phe Ile Lys Glu Leu His Leu
Ile Arg 65 70 75 80 Leu Gly Asp Val Gly Trp Ala Phe Pro Glu Ser Asp
Leu Glu Val Glu 85 90 95 Asp Asp Asp His Glu Lys Ala Leu Asn Leu
Ala Leu Asn Asn Ile Glu 100 105 110 Gly Glu Trp Asp Leu Pro Lys Leu
Glu Pro Ile Leu Thr Asp Leu Lys 115 120 125 Asp Val Gly Phe Asp Ile
Glu Leu Thr Gly Phe Ser Asp Ile Glu Leu 130 135 140 Thr Glu Leu Asn
Leu Glu Asn Asn Leu Val Phe Ala Glu Glu Phe Glu 145 150 155 160 Pro
Asp Glu Ser Glu Glu Asp Val Asp Leu Glu Asp Ile Tyr Asp Glu 165 170
175 Pro Val Lys Glu Met Leu Gln Cys Pro Ala Cys Asp His Val Asp Val
180 185 190 Val Lys Arg Phe Lys Arg Val Asp Ser Gln Gly Asp 195 200
26152PRTMethanobrevibacter ruminantium 26Met Asp Val Asp Asn Leu
Leu Gly Tyr Asp Lys Val Leu Glu Leu Arg 1 5 10 15 Ser Leu Leu Glu
Glu Val Thr Asp Lys Val Ile Pro Val Trp His Lys 20 25 30 Asn Arg
Gly Ile Lys Asp Phe Lys Gln Met Cys Gln Asp Tyr Asn Phe 35 40 45
Val Ser Ile Ser Gly Trp Arg Asn Glu Asp Val Lys Asp Asp Gln Phe 50
55 60 Ile His Phe Val Arg His Ala His Arg Asn Gly Cys Arg Ile His
Gly 65 70 75 80 Leu Gly Leu Thr Arg Arg Lys Val Leu Asp Arg Val Pro
Phe Asp Ser 85 90 95 Val Asp Ser Ser Ser Trp Leu Gln Thr Ile Leu
Tyr Ala Arg Leu Gly 100 105 110 Gln Lys Gln Leu Asp Ser Lys Phe Ala
Thr Glu Arg Arg Gly Asp Leu 115 120 125 Ala Val Leu Ser Tyr Ile Lys
Trp Met Lys Thr Gln Glu Glu Tyr Tyr 130 135 140 Lys Lys Trp Arg His
Tyr His Asp 145 150 27218PRTMethanobrevibacter ruminantium 27Met
Ile Asn Glu Arg Ile Arg Pro Tyr Leu Asn Phe Ser Phe Asn Asp 1 5 10
15 Lys Lys Val Ile Phe Val Thr Leu Phe Val Val Ser Asn Leu Ile Ser
20 25 30 Asn Leu Leu Ala Ile Lys Val Phe Asn Leu Gly Phe Trp Gly
Leu Thr 35 40 45 Thr Asp Cys Gly Asn Leu Leu Phe Pro Leu Gly Tyr
Leu Met Ala Asp 50 55 60 Val Ile Thr Glu Val Tyr Gly Glu Arg Thr
Ala Arg Arg Val Ile Leu 65 70 75 80 Leu Gly Leu Phe Ala Asn Ile Leu
Leu Ile Val Ala Thr Thr Leu Thr 85 90 95 Val Tyr Met Pro Tyr Pro
Ser Tyr Trp Thr Gly Gln Gly Ala Tyr Ala 100 105 110 Tyr Met Phe Gly
Phe Thr Pro Arg Ile Val Leu Ala Gly Phe Ile Ala 115 120 125 Tyr Leu
Val Gly Gln Phe Val Asn Ala Arg Leu Met Val Leu Ile Lys 130 135 140
Lys Trp Thr Asn Ser Lys Tyr Leu Phe Met Arg Thr Ile Gly Ser Thr 145
150 155 160 Leu Gly Gly Glu Leu Cys Asp Ser Cys Ile Cys Ser Ser Ile
Ala Tyr 165 170 175 Tyr Gly Ile Val Pro Asn Ser Gly Ile Leu Leu Phe
Ile Leu Met Gln 180 185 190 Tyr Val Val Lys Val Thr Trp Glu Val Val
Met Gln Pro Leu Thr Tyr 195 200 205 Lys Ser Ile Ala Trp Ala Arg Lys
Asp Gly 210 215 28194PRTMethanobrevibacter ruminantium 28Met Pro
Glu Pro Trp Glu Arg Gln Arg Asp Glu Asn Gly Lys Leu Glu 1 5 10 15
Pro Ile Lys Ala Phe Glu Tyr Phe Thr Glu Tyr Leu Thr Met Asp Lys 20
25 30 Pro Arg Ser Met Arg Val Leu Cys Glu Arg Leu Gly Lys Lys Asp
Gly 35 40 45 Tyr Ile Arg Gln Leu His Ala Tyr Ser Ser Thr Trp Asn
Trp Val Glu 50 55 60 Arg Ala Glu Ala Tyr Asp Glu His Ile Ile Leu
Lys Lys Arg Leu Arg 65 70 75 80 Lys Glu Lys Phe Tyr Asp Glu Leu Val
Glu Ser Glu Leu Pro Asn Leu 85 90 95 Arg Lys Arg Leu Glu Tyr Tyr
Asn Lys Asn Met Asn Asp Ile Glu Thr 100 105 110 Asp Met Thr Thr Lys
Pro Thr Ser Lys Ala His Ala Tyr Asp Lys Asn 115 120 125 Ser Lys Ala
His Ser Thr Thr Leu Asn Glu Ile Leu Leu Met Ile Gly 130 135 140 Lys
Pro Thr Glu Ile Lys Glu Thr Ser Leu Glu Ala Asp Ile Glu Ser 145 150
155 160 Asp Asn Lys Ile Asp Leu Glu Asn Arg Val Glu Val Asp Ile Thr
Ser 165 170 175 Asp Glu Phe Met Glu Ser Glu Leu Glu Tyr Met Arg Lys
Met Ile Glu 180 185 190 Glu Lys 29255PRTMethanobrevibacter
ruminantium 29Met Lys Asp Ile Val Asn His Tyr Gly Tyr Leu Ser Pro
Tyr Lys Pro 1 5 10 15 Thr Ile Arg Ser Asp Ser Lys Ala Lys Asn Lys
Phe Lys Leu Asn Glu 20 25 30 Pro Tyr Arg Gly Gln Met Leu Ser Ala
Gly Ala Gly Gly Ser Ile Met 35 40 45 Gly Tyr Gly Ala Gly Leu Leu
Ile Val Asp Asp Pro Ile Lys Asn Val 50 55 60 Ala Glu Ala Glu Ser
Lys Val Arg Gln Ala Lys Leu Lys Asp Trp Trp 65 70 75 80 Gly Gly Thr
Ile Lys Ser Arg Val Gln Arg Arg Ser Asn Gly Leu Pro 85 90 95 Pro
Ile Lys Ile Val Ile Ala Gln Arg Leu His Leu Lys Asp Leu His 100 105
110 Gly Ile Ile Lys Glu Thr Glu Pro Thr Ile Pro Ala Asn Asp Ala Phe
115 120 125 Arg Ile Leu Arg Asn Gly Gly Ser Ile Asp Pro Asn Thr Trp
Val Asp 130 135 140 Phe Asn Leu Pro Ala Ile Cys Asp Ser Glu Asp Asp
Ile Leu Gly Arg 145 150 155 160 Lys Ile Gly Glu Val Leu Trp Glu Glu
Gln Arg Asp Tyr Glu Trp Leu 165 170 175 Met Ala Glu Lys Arg Ser Met
Gly Ser Tyr Leu Phe Asn Ser Ile Tyr 180 185 190 Gln Gly Gln Pro Val
Glu Arg Asp Gly Glu Ile Phe Lys Arg Glu Trp 195 200 205 Phe Gln Asp
Glu Val Asn His Lys Leu Thr Cys Leu Ile Asp Pro Lys 210 215 220 Asp
Ile Pro Lys Asp Leu Pro Gln Leu Arg Tyr Trp Asp Phe Gly Ala 225 230
235 240 Ser Gly Asp Ala Gly Asp Gly Thr Ser Ala Ile Leu Thr Ser Tyr
245 250 255 30261PRTMethanobrevibacter ruminantium 30Met Ser Lys
Lys Gln Glu Met Met Arg Ile Glu Arg Leu Lys His Tyr 1 5 10 15 Ala
Tyr Gln Thr Gly Leu Ile Ile Pro Ile Phe Lys Asn His Asp Leu 20 25
30 Ile Lys Lys Ile Glu Asn Gly Lys Ile Thr
Asn Thr Asp Glu Ile Lys 35 40 45 Ile Tyr Ile Glu Glu Asn Glu Lys
Gln Ile Lys Arg Arg Arg Glu Phe 50 55 60 Ile Ser Ile Ile Tyr Asp
Asn Cys Lys Tyr Phe Lys Tyr Asp Ser Ile 65 70 75 80 Cys Tyr Lys Leu
Ile Ser Lys Val Asn Asn Phe Glu Ile Lys Ser Leu 85 90 95 Glu Glu
Leu Met Asn Glu Ile Glu Ser Glu Lys Lys Lys Asn Gly Phe 100 105 110
Arg Lys Asn Ile Glu Glu Lys Glu Leu Ile Lys Glu Asn Glu Asn Lys 115
120 125 Gly Arg Leu Lys Glu Tyr Val Arg Ile Val Arg Arg Glu Tyr Gly
Leu 130 135 140 Asp Phe Thr Ser Val Lys Lys Leu Lys Leu Lys Ile Asp
Asn Asn Lys 145 150 155 160 Ile Arg Ser Lys Glu Glu Leu Asn Glu Glu
Ile Ile Glu Glu Lys Lys 165 170 175 Lys Thr Glu Leu Arg Arg Ile Val
Tyr Asp Ser Asp Leu Asn Trp Asp 180 185 190 Leu Lys Trp Glu Leu Glu
Ser Lys Ile Arg His Asn Glu Ile Thr Thr 195 200 205 Lys Glu Glu Leu
Ile Lys Glu Ile Arg Arg Ile Glu Phe Ile Asn Ile 210 215 220 Ile Tyr
Asp Asn Asn Arg Asp Phe Lys Leu Asp Tyr Val Thr Ile Gln 225 230 235
240 Lys Leu Val Ser Lys Ile Gly Ser Asn Glu Ile Thr Thr Lys Glu Glu
245 250 255 Leu Ile Lys Glu Met 260 31255PRTMethanobrevibacter
ruminantium 31Met Ser Lys Asn Ala Lys Ala Asp Ala Phe Val Val Thr
Thr Glu Asp 1 5 10 15 Gly Ser Tyr Asp Ile Val Asp Ala Asp Val Leu
Glu Arg Tyr Ala Ile 20 25 30 Lys Ser Glu Ser Asp Glu Thr Gly Ser
Lys Gln Leu Lys Thr Asp Gly 35 40 45 Trp Glu Tyr Asp Asp Thr Leu
Leu Glu Pro Leu Tyr Asp Pro Leu Gln 50 55 60 Leu Cys Glu Leu Leu
Glu Ile Asn Thr Tyr His Glu Asn Cys Val Asp 65 70 75 80 Val Val Ala
Arg Asp Ser Ala Gly Ile Gly Tyr Asp Ile Val Pro Val 85 90 95 Thr
Gly Glu Lys Glu Lys Glu Leu Asn Lys Pro Lys Leu Thr Asn Phe 100 105
110 Leu Glu Asn Ile Glu Pro Asn Ile Asn Glu Leu Leu Tyr Gln Met Asn
115 120 125 Tyr Asp Arg Arg Ala Thr Gly Tyr Gly Ala Leu Glu Leu Ile
Arg Lys 130 135 140 Asp Lys Ser Lys Ser Glu Pro Val Asn Leu Ser His
Ile Ser Ser Tyr 145 150 155 160 Thr Leu Arg Arg Thr Ser Asp Gly Lys
Arg Val Lys Gln Arg Val Gly 165 170 175 Thr Lys Thr Val Trp Phe Val
Ile Tyr Gly Lys Asn Tyr Asp Lys Glu 180 185 190 Gly Asn Leu Cys Asp
Val His Ser Glu Thr Gly Glu Phe His Pro Tyr 195 200 205 Asn Ser Leu
Ser Lys Glu Glu Arg Ala Asn Glu Leu Leu Trp Thr Met 210 215 220 Glu
Tyr Thr Thr Lys Ser Lys Tyr Tyr Gly Leu Pro Lys Ile Val Gly 225 230
235 240 Ala Ile Pro Ala Ile Tyr Ser Asp Ile Ser Arg Ser Lys Tyr Asn
245 250 255 32254PRTMethanobrevibacter ruminantium 32Met Met Ala
Leu Lys Val Arg His Ser Lys Arg Gln Ile Gln Asn Ile 1 5 10 15 Lys
Arg Glu Tyr Arg Arg Arg Leu Ile Leu Glu Glu Gln Cys Ser Arg 20 25
30 Glu Ile Ala Asp Phe Phe Arg Arg Leu Glu Arg Lys Ile His Lys Val
35 40 45 Met Asp Glu His Trp Glu Ser Glu Leu Gly Leu Phe His Leu
Asn Lys 50 55 60 Val Ser Asp Ile Ile Gln Asp Ser Arg Gln Glu Tyr
Tyr Asp Ile Leu 65 70 75 80 Phe Lys Tyr Cys Lys Asp Ser Tyr Met Lys
Gly Arg Glu Ala Thr Glu 85 90 95 Arg Arg Phe Asn Arg Lys Leu Glu
Asn Ile Ser Met Lys Ala Asp Val 100 105 110 Asn Ile Thr Arg Leu Glu
Asp Leu Phe Lys Pro Asp Pro Thr Ile Arg 115 120 125 Tyr Asn Leu Asn
Asn Lys Val Phe Gln Ala Ser Ala His Thr Met Asp 130 135 140 Arg Val
Asp Asn Arg Ile Met Glu Asn Ile Thr Gln Ser Tyr Asp Asp 145 150 155
160 Gly Leu Gly Ile Asp Glu Ala Lys Asp Arg Leu Thr Val Glu Tyr Asn
165 170 175 Gly Leu Lys Ser Trp Glu Ala Gln Arg Ile Ala Arg Thr Glu
Ile Asn 180 185 190 Ser Ala Gln Asn Asp Gly Ala Phe Asp Val Tyr Gly
Glu Leu Gly Val 195 200 205 Glu Tyr His Gln Trp Trp Thr Ala Gln Asp
Glu Arg Val Arg Glu Thr 210 215 220 Pro Gln Ala Asp His Arg Glu Leu
His Gly Lys Ile Val Lys Val Gly 225 230 235 240 Asn Ser Phe Ser Asn
Gly Leu Gln Tyr Pro Gly Asp Arg Thr 245 250
3387PRTMethanobrevibacter ruminantium 33Met Ile Ile Glu Ile Pro Gly
Asn Asp Arg Thr Glu Glu Ile Asn Leu 1 5 10 15 Pro Asn Gly Gln Phe
Val Leu Ile Thr Tyr Leu Gln Glu Asn Asp Met 20 25 30 Met Ser Leu
Pro Asp Gly Lys Tyr Val Cys Pro Phe Arg Met Ile Gln 35 40 45 Leu
Phe Thr Glu Glu Gly Gly Glu Leu Ile Gly Glu Cys Ile Glu Glu 50 55
60 Asn Pro His Tyr Asn Thr Arg Phe Tyr Asn Thr Val Phe Glu His Leu
65 70 75 80 Asp Glu Gly Ile Glu Tyr Arg 85
34179PRTMethanobrevibacter ruminantium 34Met Leu Arg Val Val Glu
Arg Thr Tyr Tyr Gln Gln Glu Glu Ile Lys 1 5 10 15 Thr Leu Asp Cys
Arg Ile Arg Glu Ala Gly Val Asn Thr Tyr Ser Leu 20 25 30 Ala Arg
Gln Gly Ala Val Asp Tyr Pro Thr Tyr Glu Ser Tyr Asn Glu 35 40 45
Val Arg Glu Glu Arg Ile Lys Glu Ala Lys Glu Lys Tyr Gly Glu Ser 50
55 60 Tyr Tyr Tyr His Trp Arg Asp Val Glu Thr Tyr Phe Tyr Tyr Leu
Gly 65 70 75 80 Arg Phe Phe Ser Asp Leu Glu Glu Ile Glu Lys Tyr Leu
Glu Arg Thr 85 90 95 Val Thr Tyr Lys Pro His Arg Glu Glu Leu Lys
Glu Ala Met Glu Arg 100 105 110 Leu Asp Lys Arg Phe Glu Glu Val Ile
Asn Glu Phe Trp Tyr Ser Leu 115 120 125 Glu Glu Tyr Glu Asp Ile Thr
Glu Asp Val Leu Glu Gln Leu Lys Glu 130 135 140 Gly Asp Cys Thr Val
His Cys Glu Phe Leu Ile Lys Glu Phe Lys Lys 145 150 155 160 Phe Val
Asn Val Ile Cys Arg Ile Ile Lys Gln Asn Glu Ile Asn His 165 170 175
Lys Glu Phe 3596PRTMethanobrevibacter ruminantium 35Met Asp Ala Ile
Asn Val Ile Asn Gln Asn Lys Ile Leu Val Asp Val 1 5 10 15 Leu Tyr
Arg Gly Thr Val Asn Leu Ile Asp Ile Val Ile Gly Asp Ala 20 25 30
Leu Val Tyr Asp Asn Pro Thr Val Val Val Lys Cys Tyr Thr Thr Asp 35
40 45 Leu Ala Phe Ala Thr Thr Glu Ile Asp Glu Ile Val Leu Glu Asn
Glu 50 55 60 Glu Phe Glu Leu Leu Ile Thr Tyr Glu Asp Gly Glu Phe
Ser Ile Leu 65 70 75 80 Leu Lys Ser His Asn Leu Gly Glu Leu Gly Tyr
Ile Glu Trp Val Ile 85 90 95 36101PRTMethanobrevibacter ruminantium
36Met Val Phe Val Asp Leu Cys Glu Asn Glu Ile Ala Asp Met Val Glu 1
5 10 15 Ser Phe Tyr Arg Asn Gly Asp Gly Ser Ser Arg Ile Leu Thr Asn
Arg 20 25 30 Ala Val Gly Glu Ile Val Glu His Tyr Cys Ser Phe Glu
Val Asp Gly 35 40 45 Arg Ile Thr Thr Asn Leu Arg Asp Phe Leu Leu
Tyr Ser Val Val Leu 50 55 60 Tyr Asp Thr Ile Gly Glu Ala Val Asp
Asp Gly Val Asn Leu Glu Glu 65 70 75 80 Val Phe Ile Ile Glu Asp Arg
Asn Cys Tyr Thr Gly Lys Ser Gln Val 85 90 95 Ile Leu Ile Gly Gly
100 37185PRTMethanobrevibacter ruminantium 37Met Val Asn Trp Leu
Lys Ala Ile Gly Asp Asn Phe Ser Val Asp Tyr 1 5 10 15 Leu Leu Leu
Ala Leu Phe Ser Ser Gly Asp Leu Ile Leu Val Ala Ile 20 25 30 Val
Leu Asn Ser Tyr Gly Val Ile Ser Pro Glu Asn Val Arg Glu Leu 35 40
45 Val Ile Asp Tyr Ile Ser Tyr Arg Lys Val Asp Ile Phe Trp Arg His
50 55 60 Leu Arg Arg Pro Arg Met Ser Phe Glu Asp Tyr Val Leu Asp
Asn Phe 65 70 75 80 Glu Glu Met Glu Thr Gly Glu Leu Thr Arg Glu Gln
Val Val Glu Phe 85 90 95 Val Ser Arg Gln Glu Arg Lys Gly Leu Thr
Phe Cys Asn Glu Ile Phe 100 105 110 Ile Ala Val Pro Leu Lys Lys Gly
Ser Lys Asp Asp Ile Val Glu Ile 115 120 125 Leu Trp Asn Glu Tyr Phe
Val Glu Asp Tyr Lys Glu Asn Trp Leu Glu 130 135 140 Gln His Glu Asn
Leu Gly Trp Asn Asp Trp Lys Lys Leu Leu Lys Lys 145 150 155 160 Glu
Ile Val Glu Asn Gly Gly Asp Asp Phe Gln Ile Phe Arg Asn His 165 170
175 Leu Ile Asp Cys Val Leu Met Glu Tyr 180 185
3863PRTMethanobrevibacter ruminantium 38Met Thr Met Lys Val Thr Phe
Glu Asp Glu Asn Gly Glu Arg Thr Val 1 5 10 15 Glu Phe Gly Asp Asp
Val Asp Phe Val Leu Ile Glu Ser Asp Asp Asp 20 25 30 Gly Asn Ile
Glu Ile Arg Glu Gly Asp Trp Glu Leu Asp Gly Asp Ser 35 40 45 Asp
Asp Asp Trp Glu Glu Tyr Asp Asp Trp Asp Glu Glu Glu Phe 50 55 60
3993PRTMethanobrevibacter ruminantium 39Met Asp Glu Phe Val Glu Thr
Leu Phe Asp Thr Tyr Trp Lys Val Asn 1 5 10 15 Glu Asn Gly Glu Tyr
Met Ser Leu Thr Asp Cys Gly Asp Phe Tyr Ile 20 25 30 Ala Lys Val
Ala Pro Cys Val Arg Asn Trp Ser Ile Val Ile Glu Cys 35 40 45 Asn
Cys Phe Cys Phe His Cys Lys Glu Phe Val Tyr His Glu Asn Gly 50 55
60 Ala Ile Leu Glu Ile Gly Met Glu Ile Ser Ser Leu Tyr Leu Ser Gln
65 70 75 80 Met Glu Ile Lys Asp Leu Lys Ile Tyr Met Ile Asp Cys 85
90 40119PRTMethanobrevibacter ruminantium 40Met Phe Leu Glu Gly Ile
Phe Glu Gln Asp Gly Glu Asn Val Arg Glu 1 5 10 15 Gln Val Ile Tyr
Trp Arg Lys Ser Asn Gln Val His Asn Trp Phe Val 20 25 30 Val Asn
Ala Gln Asp Gly Glu Asp Asn Cys Gln Pro His Ser Val Ser 35 40 45
Arg Glu Gln Leu Glu Glu Leu Arg Asp Leu Cys Arg Ala Val Leu Ala 50
55 60 Asp Asn Asp Lys Ala Glu Glu Leu Leu Pro Thr Arg Pro Gly Phe
Phe 65 70 75 80 Phe Gly Ala Ile Asp Tyr Asp Glu Trp Tyr Tyr Tyr Asp
Leu Gln Tyr 85 90 95 Thr Val Glu Lys Ile Asp Glu Val Leu Lys Asp
Asp Arg Tyr Leu Tyr 100 105 110 Phe Glu Tyr Cys Ser Trp Trp 115
4157PRTMethanobrevibacter ruminantiumMOD_RES(42)..(42)Any amino
acid 41Met Gln Leu Val Val Glu Gly Glu Asn Met Glu Cys Pro Cys Asp
Asn 1 5 10 15 Cys Glu Met Leu Arg Glu Asn Glu Pro Ile Lys Val Ile
Asp Trp Lys 20 25 30 Ser Asn Ser Pro Phe Gly Asn Gly Ile Xaa Ile
His Phe Trp Arg Glu 35 40 45 Arg Leu Gln Asp Val Gly Tyr Asn Gly 50
55 42116PRTMethanobrevibacter ruminantium 42Met Glu Leu Met Thr Arg
Glu Met Glu Gly Lys Leu Lys Ser Phe Pro 1 5 10 15 Phe Tyr Ser Gln
Asp Gly Lys Gly Asp Asp Ala Ile Val Val Met Lys 20 25 30 Phe Phe
Asn Pro Tyr Gly Leu Gly Thr Trp Tyr Val Leu Glu Ala Glu 35 40 45
Lys Gln Glu Asn Gly Asp Tyr Leu Phe Phe Gly Tyr Val Glu Ser Pro 50
55 60 Ile Thr Pro Glu Phe Asn Glu Tyr Gly Tyr Phe Ser Leu Ser Glu
Leu 65 70 75 80 Glu Asn Leu Lys Ile Pro Ile Lys Ile Asn Gly Ile Thr
Val Ser Tyr 85 90 95 Gly Arg Ile Glu Arg Asp Leu Tyr Phe Glu Arg
Val Arg Ile Gly Asp 100 105 110 Ile Ile Gly Asn 115
4374PRTMethanobrevibacter ruminantium 43Met Phe Gly Gln Lys Lys Glu
Phe Val Lys Lys Met Tyr His Val Gly 1 5 10 15 Asp Val Val Glu Leu
Val His Met Asp Asp Ala Gln Ala Pro Pro Ser 20 25 30 Gly Thr Arg
Gly Glu Ile Leu Phe Val Asp Asp Ile Gly Gln Ile His 35 40 45 Val
Arg Trp Glu Asn Gly Ser Gly Leu Ala Leu Ile Tyr Gly Glu Asp 50 55
60 Arg Phe Lys Val Val Glu Arg Lys Gly Glu 65 70
4450PRTMethanobrevibacter ruminantium 44Met Asp Leu Leu Tyr Leu Tyr
Asp Asp Leu Thr Ala Arg Arg Glu Val 1 5 10 15 Tyr Asp Ser Val Gly
Leu Ser Phe Val Val Lys Tyr Lys Phe Ser Ser 20 25 30 Arg Lys Glu
Ala Gln Asp Phe Ala Leu Lys Tyr Gly Ala Glu Leu Ile 35 40 45 Glu
Glu 50 4562PRTMethanobrevibacter ruminantium 45Met Ile Glu Arg Arg
Arg Leu Gly Met Lys Tyr Asp Ile Phe Thr Ile 1 5 10 15 Leu Asp Glu
Ile Ser Arg Lys Leu Asp Asp Gly Glu Leu Ser Asp Glu 20 25 30 Gln
Val Asp Phe Leu Leu Gln Met Glu Ile Leu Val Glu Glu Gly Thr 35 40
45 Ile Thr Asp Glu Gln Ala Gln Asp Val Met Asn Gly Asp Tyr 50 55 60
4655PRTMethanobrevibacter ruminantium 46Met Met Lys Leu Ser Leu Lys
Glu Leu Gly Glu Glu Ile Glu Met Ile 1 5 10 15 Leu Ala Glu Gly Gly
Leu Thr Phe Asp Gln Val Asp Tyr Leu Leu Tyr 20 25 30 Leu Glu Thr
Cys Ile Ala Asp Gly Ser Ile Thr Glu Glu Gln Lys Arg 35 40 45 Glu
Ile Ile Cys Arg Asp Phe 50 55 4777PRTMethanobrevibacter ruminantium
47Met Val Arg Glu Gln Glu Arg Leu Ile Met Tyr Leu Glu Ile Asp Glu 1
5 10 15 Val Lys Cys Glu Asn Ile Asp Arg Ile Glu Phe Asp Asp Val Ala
Met 20 25 30 Glu Ile Val Leu Thr Asp Glu Lys Val Tyr Glu Arg Ile
Lys Arg Trp 35 40 45 Leu Lys Ser Asn Glu Ile Asp Tyr Asp Cys Arg
Glu Asp Arg Tyr Phe 50 55 60 Ala Asn Leu Ile Glu Tyr Val Ile Arg
Ile Thr Trp Trp 65 70 75 4871PRTMethanobrevibacter ruminantium
48Met Val Thr Val Arg Asp Val Gly Phe Thr Ile Glu Glu Arg Phe Phe 1
5 10 15 Leu Thr Ala Gln Glu Leu Glu Tyr Ser Glu Val Gly Glu Glu His
Glu 20 25 30 Ser Val Ile Asp Arg Ala Ile Ala Leu Leu Tyr Thr Lys
Leu Arg Thr 35 40 45 Arg Asp Phe Glu
Phe Thr Asp Glu Glu Arg Glu Leu Leu Glu Asp Ala 50 55 60 Phe Val
Ile Val Ser Asp Gln 65 70 49255PRTMethanobrevibacter ruminantium
49Met Ala Lys Glu Asn Val Ile Asp Tyr Lys Ile Glu Arg Gln Asn Asp 1
5 10 15 Asn Thr Trp Ser Tyr Leu Tyr Val Thr Glu Arg Gly Arg Gly Asn
Ile 20 25 30 Ile Ala Ser Ser Phe Gly Glu Leu Arg Gln Lys Val Leu
Lys Arg Gly 35 40 45 Leu Pro Trp Asn Asp Ile Ser Asn Leu Phe Thr
Lys Lys Ser Ser Asp 50 55 60 Ser Asn Val Arg Arg Glu Ser Val Lys
Asn Ile Asp Asp Glu Ser Val 65 70 75 80 Leu Ala Asp Val Ala Lys Lys
Ser Ser Asp Ser Asn Val Arg Leu Glu 85 90 95 Ala Val Arg Lys Ile
Ser Asp Asn Tyr Val Leu Ile Asp Ile Val Lys 100 105 110 Asn Ala Ser
Asp Tyr Asp Val Arg Arg Glu Ala Val Arg Lys Ile Asn 115 120 125 Asp
Ser Ser Val Leu Glu Asp Ile Ala Lys Asn Asn Asn Asp Glu Asn 130 135
140 Val Arg Leu Glu Ala Val Arg Asn Ile Asn Asp Glu Ser Val Leu Glu
145 150 155 160 Asn Ile Ser Lys Asn Ala Ser Asp Ser Lys Val Arg Ile
Glu Ala Ile 165 170 175 Lys Lys Ile Asn Asp Glu Thr Ile Ile Ile Lys
Leu Ala Lys Asn Asn 180 185 190 Asn Asp Glu Asp Val Arg Ile Glu Ala
Val Arg Lys Ile Asn Asp Lys 195 200 205 Thr Val Ile Ile Asp Phe Ala
Lys Asn Ala Ser Asp Ser Lys Val Arg 210 215 220 Arg Glu Ala Val Arg
Lys Ile Asn Asp Ser Ser Val Leu Ala Tyr Val 225 230 235 240 Leu Lys
Asn Asp Pro Ser Trp Ile Val Arg Ile Glu Ala Val Arg 245 250 255
50231PRTMethanobrevibacter ruminantium 50Met Val Val Lys Cys Pro
Asn Cys Phe Ser Pro Arg Val Ser Lys Cys 1 5 10 15 Glu Asp Thr Asn
Ile Lys Trp Gln Cys Asp Lys Cys Lys Cys Lys Phe 20 25 30 Asn His
Gly Ala Phe Asp Ile Asn Ala Glu Met Glu Lys Val Glu Gln 35 40 45
Leu Thr Ile Glu Lys Ile Glu Arg Glu Arg Glu Arg Thr Glu Gln Phe 50
55 60 Glu Arg Ala Ile Lys Glu Ala Lys Glu Gln Phe Glu Arg Glu Arg
Thr 65 70 75 80 Glu Gln Phe Glu Arg Glu Arg Lys Glu Arg Leu Glu Arg
Glu Lys Arg 85 90 95 Glu Lys Glu Arg Glu Lys Ile Glu Lys Glu Arg
Glu Arg Lys Glu Arg 100 105 110 Leu Glu Arg Asn Arg Ile Lys Ile Glu
Glu Arg Glu Arg Glu Arg Ile 115 120 125 Lys Arg Asn Glu Arg Ile Arg
Arg Glu Asn Glu Arg Asn Arg Ile Lys 130 135 140 Ser Asp Lys Arg Glu
Arg Glu Lys Ser Glu Glu Lys Arg Ile Lys Arg 145 150 155 160 Asn Glu
Arg Ile Arg Lys Ala Asn Glu Arg Asn Ser Ile Lys Arg Glu 165 170 175
Lys Arg Glu Arg Glu Arg Glu Arg Asn Val Met Thr Ile Asp Glu Tyr 180
185 190 Tyr Arg Ser Ile Gly Tyr Gly Ser Thr Gly Lys Ser Lys Val Trp
Ser 195 200 205 Ala Ile Ile Ile Pro Ile Leu Leu Val Ile Cys Ile Ile
Leu Ile Leu 210 215 220 Met Phe Tyr Gly Gly Gly Met 225 230
51248PRTMethanobrevibacter ruminantium 51Met Thr Glu Gly Gln Ile
Arg Gln Ile Ala His Glu Tyr Leu Ala Asn 1 5 10 15 Tyr Ser Leu Val
Asp Lys Asn His Glu Phe Phe Glu Thr Arg Glu Val 20 25 30 Ile Gly
Val Pro Val Glu Ser Tyr Ile Thr Asn Glu Pro Ile Ser Leu 35 40 45
Lys Gly Leu Asp Gly Thr Val Asn Glu Tyr Pro Lys Gly Thr Trp Ile 50
55 60 Ala Thr Thr Arg Ile Thr Asp Glu Glu Glu Met Glu Lys Ala Leu
Asn 65 70 75 80 Gly Glu Tyr Thr Gly Tyr Ser Ile Thr Thr Val Ser Lys
Lys Phe Ala 85 90 95 Asp Lys Gln Ile Gln Leu Pro Arg Arg Val Leu
Met Lys Asp Ile Lys 100 105 110 Asp Pro Val Gly Phe Thr Ile Ser Leu
Val Arg Lys Pro Cys Val Arg 115 120 125 Gly Ala Lys Phe Cys Ser Met
Lys Glu Asp Ile Glu Asn Gly Asp Val 130 135 140 Val Ser Glu Asn Ile
Asp Asp Lys Leu Glu Glu Glu Thr Lys Gly Phe 145 150 155 160 Val Gln
Ser Ile Lys Gly Ile Phe Asn Lys Glu Asp Lys Asp Glu Asp 165 170 175
Lys Asn Pro Glu Asp Glu Asp Ile Glu Leu Asp Ile Lys Ala Ile Val 180
185 190 Asp Glu Val Thr Lys Asp Phe Val Asn Thr Asp Asp Phe Glu Thr
Phe 195 200 205 Lys Asn Glu Leu Glu Lys Ala Leu Ser Asp Lys Phe Glu
Thr Leu Gly 210 215 220 Ala Glu Leu Phe Lys Ser Leu Lys Lys Ser Leu
Glu Lys Asp Lys Ala 225 230 235 240 Glu Glu Ala Lys Lys Ser Gly Arg
245 52280PRTMethanobrevibacter ruminantium 52Met Gly Asn Glu Ala
Thr Leu Asn Gln Leu Val Asn Glu Gln Glu Lys 1 5 10 15 Ala Val Phe
Lys Ser Met Arg Thr Asp Met Glu Thr Gly Lys Ala Val 20 25 30 Leu
Asn Val Glu Gln Leu Gly Tyr Phe Leu Arg Glu Ala Thr Leu Asp 35 40
45 Asn Thr Ile Leu Arg Asp Ala Asp Phe Lys Leu Met Lys Ser Phe Lys
50 55 60 Lys His Leu Asn Arg Val Gly Ile Asn Gly Arg Val Leu Thr
Asn Gly 65 70 75 80 Tyr Asp Val Asn Gly Glu Thr Asp Pro Glu Ile Pro
Ala Ala Asp Val 85 90 95 Asp Phe Gly Ala Asn Glu Leu Asp Val Lys
Lys Leu Lys Ala Met Cys 100 105 110 Glu Ile Glu Asp Asp Glu Lys Glu
Asp Asn Met Thr Gln Ala Gln Phe 115 120 125 Glu Gln Thr Leu Leu Gln
Met Met Gly Glu Arg Ile Gly Glu Asp Leu 130 135 140 Glu Tyr Trp Ala
Leu Phe Ala Asp Ser Glu Val Ala Arg Ser Asp Asp 145 150 155 160 Pro
Leu Leu Asn Thr Asn Asp Gly Trp Leu Lys Lys Cys Ala Asn His 165 170
175 Ile Ser Ser Arg Ser Ile Ala Pro Ser Asn Gly Met Phe Asp Ile Glu
180 185 190 Asp Gly Pro Glu Ala Met Phe Asp Ala Met Ile Lys Ala Leu
Pro Pro 195 200 205 Arg Phe Arg Lys Asn Arg Arg Met Leu Lys Phe Tyr
Val Pro Phe Glu 210 215 220 Val Glu Asp Ala Tyr Arg Asn Ile Leu Ile
Asn Arg Gly Thr Gly Leu 225 230 235 240 Gly Asp Ser Ala Gln Ile Gly
Phe Asn Ala Leu Ser Tyr Lys Gly Ile 245 250 255 Pro Ile Glu His Cys
Ser Thr Leu Asp Asp Glu Asp Gly Arg Gly Met 260 265 270 Leu Gly Asn
Arg Val Cys Ser Met 275 280 53147PRTMethanobrevibacter ruminantium
53Met Arg Arg Arg Cys Leu Asn Ser Pro Glu His Asn Gly Met Ile Ser 1
5 10 15 His Ile Ile Val Leu Leu Ile Cys Phe Ile Gly Leu Val Glu Ala
Ile 20 25 30 Leu Met Ala Leu Val Asp Trp Glu Asp Leu Ala Ile Ser
Val Arg Lys 35 40 45 Ser Pro Arg Lys Leu Tyr Asn Val Leu Lys Asp
Glu Leu Gly Leu Pro 50 55 60 Glu Trp Asn Glu Leu Ser Val Ile Glu
Arg Arg Ser Met Lys Lys Arg 65 70 75 80 Tyr Ala Val Ile Arg Asp Ser
Phe Pro Glu Leu Pro Pro Trp Glu Glu 85 90 95 Leu Ser Val Ile Asp
Arg Arg Ser His Lys Arg Leu Tyr Lys Leu Ile 100 105 110 Lys Ser Val
Tyr Asp Gly Asp Tyr Asp Asp Ser Pro Ser Leu Glu Gly 115 120 125 Pro
Pro Ala Ala Val Gly Pro Gln Lys Glu Ile Pro Leu Glu Glu Ala 130 135
140 Glu Tyr Pro 145 54137PRTMethanobrevibacter ruminantium 54Met
Thr Trp Ile Gly Thr Glu Asp Val Ile Glu Phe Thr Gly Val Lys 1 5 10
15 Pro Gln Thr Phe Arg Phe Glu Lys Gly Asp Thr Ser Ser Leu Glu Thr
20 25 30 Leu Leu Glu Lys Trp Ile Leu Gln Ala Glu Gly Leu Ile Ile
Ser Tyr 35 40 45 Cys Asn Tyr Asp Phe Asn Asp Leu Glu Glu Ile Pro
Pro Ala Val Val 50 55 60 Asn Val Cys Leu Arg Leu Thr Ala Asn Met
Val Ala Leu Ala Gln Ala 65 70 75 80 Arg Lys Asp Thr Pro Val Ile Gln
Val Lys Glu Trp Asn Val Gln Thr 85 90 95 Val Ser Ser Asn Ile Phe
Ser Asn Asp Leu Lys Arg Asp Leu Thr Pro 100 105 110 Phe Val His Glu
Arg Lys Ser Tyr Lys Gly Asp Glu Ile Asp Phe Phe 115 120 125 Val Ile
Thr Gly Asp Asp Asp Ser Trp 130 135 55148PRTMethanobrevibacter
ruminantium 55Met Val Lys Leu Gln Ile Asp Val Glu Glu Leu Lys Pro
Leu Glu Pro 1 5 10 15 Arg Phe Lys Lys Val Ala Lys Arg Thr Val Val
Leu Thr Ala Asn Glu 20 25 30 Leu Gln Arg Asn Leu Lys Lys Leu Ser
Pro Val Asp His Gly Arg Leu 35 40 45 Gln Gly Ser Trp Val Ile Phe
Gln Thr Gly Glu Leu Glu Arg Thr Val 50 55 60 Lys Ser Ser Ala Lys
Tyr Ala Ile Phe Val Asn Asp Gly Thr Gly Leu 65 70 75 80 Tyr Gly Pro
Leu Gly His Lys Ile Arg Pro Lys Asn Gly Lys Phe Leu 85 90 95 Ala
Phe Thr Pro Asn Lys Gly Lys Phe Lys Gly Lys Leu Val Val Val 100 105
110 Pro Trp Thr Arg Gly Gln Lys Pro Gln Arg Phe Val Glu Arg Ser Met
115 120 125 Glu Met Thr Glu Arg Arg Val Gln Glu Phe Met Ile Arg Ala
Met Met 130 135 140 Glu Met Asp Ser 145 56169PRTMethanobrevibacter
ruminantium 56Met Arg Phe Val Asn Thr Ala Ser Leu Val Pro Gln Thr
Val Lys Ala 1 5 10 15 Tyr Leu Glu Arg Glu Ile Cys Glu Gly Gly Leu
Leu Glu Asp Val Glu 20 25 30 Thr Leu Ile Pro Ser Val Asn Ser Asp
Val Pro Val Asp Pro Pro Ala 35 40 45 Ile Trp Ile Val Gln His Pro
Thr Thr Arg Trp Ser Gly Ser Gln Pro 50 55 60 Asn Leu Ser Asn Lys
Ile Ala Met Ser Val Pro Phe Glu Phe Val Cys 65 70 75 80 Val Glu Tyr
Ser Asp Asp Leu Glu Glu Ala Glu Ile Leu Gly Ile Ser 85 90 95 Leu
Ala Ser Arg Val Gly Ser Ser Leu Met Lys Asn Phe Asn Lys Val 100 105
110 Lys Val Asp Asp Ser Met Pro Asn Arg Phe Phe His Lys Leu Glu Phe
115 120 125 Glu Thr Leu Tyr Pro Val Gly Glu Val Thr Val Val Gly Lys
Ser Glu 130 135 140 Arg Ile Pro Ala Thr Ser Ile Ile Phe Asn Phe Val
Tyr Val Val Asp 145 150 155 160 Trp Leu Lys Cys Asn Arg Arg Tyr Asp
165 57255PRTMethanobrevibacter ruminantium 57Met Gly Ile Arg Val
Val Gly Met Lys Glu Glu Ala Arg Tyr Gly Val 1 5 10 15 Ala Glu Ser
Ala Pro Asp Phe His Gln Glu Val Ser Lys Ala Lys Ala 20 25 30 Ser
Leu Asn Ser Thr Pro Asn Thr Lys Ser Ser Gly Ser Arg Met Lys 35 40
45 Lys Lys Ala Arg Ala Gly Val Tyr Lys Pro Thr Ala Asn Ile Glu Gly
50 55 60 Glu Val Asp Leu Lys Arg Ile Gly His Tyr Leu Lys Ala Phe
Leu Asp 65 70 75 80 Asn Tyr His Phe Thr Asp Gly Gly Ser Asn Pro Asn
Val His Glu Phe 85 90 95 Trp Gly Gly Glu Asn Asn Lys Leu Ser Ser
Phe Thr Leu Trp Val Thr 100 105 110 Phe Asp Ile Phe Glu Lys Thr Ile
Val Gly Ser Leu Leu Asp Asn Phe 115 120 125 Lys Met Glu Val Ser Asp
Glu Tyr Met Lys Phe Thr Ala Asp Phe Val 130 135 140 Tyr Lys Thr Glu
Glu Ser Asp Glu Ile Glu Asn Ile Glu Leu Tyr Lys 145 150 155 160 Val
Lys Leu Leu Asp Gly Asp Trp Ala Leu Met Phe Tyr Asp Val Ser 165 170
175 Val Glu Ile Asp Glu Asn Ala Pro Pro Gly Ile Val Ser Ser Phe Ser
180 185 190 Phe Asp Gly Lys Asn Asn Ile Asn Val Asp Lys Thr Ile Gly
Leu Gly 195 200 205 Ser Arg Gly Pro Gln Arg Lys Ala Ala Ala Gln Gly
Arg Asp Ile Ser 210 215 220 Ile Ser Phe Val Ser Thr Leu Glu Arg Glu
Thr Leu Glu Leu Ile Gln 225 230 235 240 Lys Ala Glu Tyr Gly Glu Val
Gly Thr Glu Pro Ser Glu Cys Lys 245 250 255
58146PRTMethanobrevibacter ruminantium 58Met Val Val Val Lys Lys
Ser Asp Ile Leu Lys Gly Val Lys Lys Ile 1 5 10 15 Glu Lys Val Lys
Ile Glu Ala Leu Asp Gly Asp Glu Met Tyr Leu Arg 20 25 30 Pro Leu
Ser Gln Ala Glu Ile Asn Glu Val Asp Glu Ile Glu Ala Lys 35 40 45
Ala Met Gly Ile Phe Glu Thr Asn Glu Thr Ala His Arg Gly Arg Arg 50
55 60 Gln Lys Pro Lys Ser Val Val Glu Ser Lys Gly Lys Ile Asn Leu
Glu 65 70 75 80 Leu Gln Gln Lys Ala Gln His Gln Ala Lys Thr Lys Ala
Ile Phe Leu 85 90 95 Ser Leu Asp Asn Glu Lys Asn Val Gly Glu Glu
Ala Trp Ser Glu Thr 100 105 110 Glu Ile Glu Gln Met Pro His Lys Leu
Phe Glu Glu Leu Phe Asn His 115 120 125 Val Lys Arg Leu Ser Gly Ile
Glu Leu Asp Glu Asp Asp Val Asp Thr 130 135 140 Phe His 145
59255PRTMethanobrevibacter ruminantium 59Met Pro Ser Ser Asn Val
Met Asn Ile Ile Val Lys Ala Glu Asp Met 1 5 10 15 Ala Ser Ser Val
Ala Gln Lys Val Glu Asn Ser Phe Arg Lys Leu Gly 20 25 30 Asn Thr
Ile Asp Ser Thr Phe Thr Thr Ser Leu Ser Asn Thr Lys Phe 35 40 45
Asn Gln Glu Leu Thr Ser Phe Gly Thr Asp Leu Asp Lys Val Thr Gln 50
55 60 Arg Leu Lys Gln Val Gly Val Asn Gly Gln Ser Ser Phe Asn Gln
Leu 65 70 75 80 Thr Asn Ala Glu Arg Arg Thr Leu Glu Lys Leu Ser Glu
Phe Asp Pro 85 90 95 Val Ser Ala Gln Val Leu Gln His Leu Ser Arg
Ile Gly Ile Thr Gly 100 105 110 Gln Gln Thr Phe Asn Gln Leu Ser Val
Ser Glu Gln Lys Ser Leu Met 115 120 125 Asn Met Lys Ser Thr Ala Gln
Gln Leu Glu Glu Val Asn Ser Lys Leu 130 135 140 Arg Val Val Gly Ile
Gly Ala Ala Gln Ala Ala Asn Met Leu Asn Gln 145 150 155 160 Met Lys
Leu Asp Pro Ser Val Gly Ser Asn Leu Asp Arg Ala Lys Leu 165 170 175
Lys Val Ser Glu Met Gly Tyr Ser Leu Asp Ser Thr Lys Gly Lys Ile 180
185 190 Leu Val Leu Gly Thr Ala Ile Gln Thr
Ser Leu Gly Asn Lys Trp Asp 195 200 205 Ser Ile Lys Thr Lys Val Gln
Thr Thr Ala Thr Asn Ile Arg Thr Ala 210 215 220 Leu Gly Asn Ala Leu
Thr Ser Val Lys Ser Lys Val Gln Asn Leu Gly 225 230 235 240 Asn Ala
Phe Ser Gly Leu Gly Gly Ile Ile Ser Ser Ala Ile Gly 245 250 255
60274PRTMethanobrevibacter ruminantium 60Met Ala Ser Val Thr Lys
Tyr Pro Ser Asn Val Ser Gln Thr Thr Gly 1 5 10 15 Gly Lys Phe Val
Ser Phe Ser Asn Leu Ala Asn Ile Lys Asn Asn Ala 20 25 30 Asp Gly
Ala His Ala Val Ser Ser Val Leu Ile Lys Ser Lys Lys Gln 35 40 45
Ser Pro Asn Arg Pro Ser Thr Val Ser Cys Lys Gly Phe Gly Phe Ser 50
55 60 Leu Pro Glu Gly Ala Glu Pro Thr Lys Ile Thr Val Thr Tyr Arg
His 65 70 75 80 Arg Lys Asn Ala Gly Ser Asp Tyr Ser Ser Lys Asn Lys
Thr His Ile 85 90 95 Cys Asn Ile Gly Gly Pro Thr Ile Ser Leu Leu
Gly Val Ser Gly Phe 100 105 110 Ser Ser Lys Gly Ser Gly Cys Thr Thr
Thr Met Thr Thr His Thr Lys 115 120 125 Ala Phe Ser Val His Gly Lys
Leu Ser Arg Ala Gln Val Asn Ser Ala 130 135 140 Asn Phe Gly Val Lys
Leu Asp Tyr Pro Thr Asn Ser Asn Thr Tyr Asn 145 150 155 160 Gly Tyr
Met Arg Ile Ser Tyr Val Arg Val Thr Val Glu Tyr Ile Thr 165 170 175
Ser Gln Tyr Ser Val Ser Val Lys His Val Ser Gly Thr Tyr Glu Asp 180
185 190 Glu Asp Tyr Val Val Ser Leu Gly Ile Ser Asn Lys Asn Leu Thr
Ser 195 200 205 Tyr Asn Pro Thr Cys Thr Leu Thr Val Pro Ala Gly Phe
Thr Tyr Lys 210 215 220 Gly Val Thr Gly Ala Ala Thr Gly Thr Val Thr
Lys Val Asn Asn Arg 225 230 235 240 Thr Phe Ser Trp Asn Pro Gln Leu
Gln Gly Arg Ala Gly Ser Arg Gln 245 250 255 Ile Ser Leu Ala Phe Glu
Pro Asn Val Thr Phe Pro Glu Gly Thr Asp 260 265 270 Ser Tyr
61255PRTMethanobrevibacter ruminantium 61Met Gly Ile Ala Ile Val
Val Met Asp Asn Glu Glu Asn Phe Leu Gln 1 5 10 15 Phe Leu Asp Pro
Asp Leu Cys Thr Ile Asn Glu Thr Ile Glu Glu Leu 20 25 30 Gly Leu
Arg Thr Leu Glu Phe Asn Tyr Lys Phe Gln Asp Tyr Val Glu 35 40 45
Asp Arg Asp Leu Phe Arg Ile Gly Asn Lys Ile Trp Ile Ser Asn Ser 50
55 60 Gln Ser Leu Glu Asp Cys Leu Tyr Val Ile Asn Thr Pro Val Glu
Asn 65 70 75 80 Ser Val Tyr Gln Glu Asn Tyr Phe Ala Cys Glu Ile Glu
Glu Val Leu 85 90 95 Ala Glu Leu Tyr Tyr Ala Pro Leu Phe Ser Gln
Thr Glu Leu Thr Ser 100 105 110 Ala Asn Gly Phe Thr Leu Arg Thr Thr
Asn Gly Glu Gln Thr Val Asp 115 120 125 Val Asp Trp Asn Ala Leu Asn
Tyr Trp Phe Gly Leu Tyr Phe Asn Ile 130 135 140 Gly Val Val Gln Glu
Cys Leu Gly Thr Tyr Ala Asn Arg Ile Thr Val 145 150 155 160 Asn Gly
Thr Met Asn Arg Leu Asn Leu Leu Arg Ser Ile Glu Glu Gln 165 170 175
Thr Gly Asn Arg Phe Val Thr Arg Tyr Glu Lys Asp Leu Leu Asp Asn 180
185 190 Thr Ile His Arg Tyr Leu Asp Phe Leu Asn Pro Val Asn Val Ser
Lys 195 200 205 Asn Trp Lys Leu Asn Ile Glu Tyr Asp Phe Ile Tyr Glu
Asp Asp Gly 210 215 220 Glu Tyr Cys Glu Ala Tyr Thr Ser Asp Gly Asn
Pro Ile Ser Glu Ile 225 230 235 240 Tyr Asp Asp Ile Glu Glu Glu Asp
Asp Ile Val Asp Phe Pro Pro 245 250 255 62255PRTMethanobrevibacter
ruminantium 62Met Val Glu Lys Ile Thr Val Ser Pro Gln Glu Val Arg
Gly Tyr Gly 1 5 10 15 Asn Val Val Asp Glu Lys Glu Leu Glu Asp Tyr
Gly Ser Tyr Arg Cys 20 25 30 Asp Val Ser Glu Ser Ser Glu Val Ile
Lys Gly Val Glu Glu Arg Ile 35 40 45 Phe Ser Val Ser Gly Val Pro
Ala Pro Ala Leu Ser Ile Ala Asn Val 50 55 60 Thr Glu Asp Thr Arg
Arg Gly Arg Cys Ala His Ile Ser Ala Ser Phe 65 70 75 80 Glu Asp Gly
Glu Gly Asp Gly Leu Asp Asp Lys Ala Ile Ser Leu Lys 85 90 95 Ser
Gly Asp Asp Val Leu Ala Thr Ile Thr Thr Gly Ser Gly Glu Asn 100 105
110 Val Phe Asp Val Val Leu Tyr Asp Ser Ala Gln Leu Tyr Ala Val Phe
115 120 125 Asp Gly Asp Asp Tyr Tyr Pro Pro Ala Val Ser Glu Ala Ile
Thr Val 130 135 140 Asn Pro Ala Lys Ser Leu Trp Asp Val Glu Phe Ile
Leu Asp Glu Glu 145 150 155 160 Glu Tyr Glu Val Gly Asp Thr Ala Ile
Leu Ser Gly Thr Val Gly Thr 165 170 175 Ile Val Asp Glu Ile Val Asp
Gly Glu Ile Val Thr Arg Arg Gln Met 180 185 190 Glu Ala Asn Val Thr
Leu Thr Leu Val Thr Asp Leu Gly Ile Arg Arg 195 200 205 Cys Ser Thr
Asn Ala Asn Gly Glu Phe Val Leu Gln Val Pro Asn Ile 210 215 220 Gln
Gln Asn Gln Trp Arg Val Val Ile Ala Ala Thr Ser Thr His Leu 225 230
235 240 Val Phe Asn Gly Leu Ile Asp Val Pro Val His Asp Tyr Ser Leu
245 250 255 63228PRTMethanobrevibacter ruminantium 63Met Val Arg
Phe Ser Arg Asp Met Leu Gln Asp Gly Ala Lys Arg Met 1 5 10 15 Phe
Lys Trp Leu Arg Lys Gly Glu Gly Leu Pro Asn Tyr Leu Ile Met 20 25
30 Tyr Asp Met Asp Arg Asn Lys Glu Tyr Lys Leu Val Pro Lys Glu Tyr
35 40 45 Ala Gly Leu Tyr Glu Ser Arg Asn Ile Phe Trp Ile Lys Asn
Gly Arg 50 55 60 Glu Pro Asn Tyr Val Thr Leu Thr Ser Val Ala Arg
Asn Pro Leu Val 65 70 75 80 Met Asp Tyr Gln Asn Thr Asn Tyr Thr Cys
Cys Pro Thr Ser Leu Ser 85 90 95 Leu Ala Ser Gln Met Leu Tyr His
Tyr Lys Ser Glu Ser Glu Cys Ala 100 105 110 Lys Ala Leu Gly Thr Ser
Lys Gly Ser Gly Thr Ser Pro Ala Gln Leu 115 120 125 Ile Ala Asn Ala
Pro Lys Leu Gly Phe Lys Ile Ile Pro Ile Lys Arg 130 135 140 Asp Ser
Lys Glu Val Lys Lys Tyr Leu Lys Lys Gly Phe Pro Val Ile 145 150 155
160 Cys His Trp Gln Val Asn Gln Ser Arg Asn Cys Lys Gly Asp Tyr Thr
165 170 175 Gly Asn Phe Gly His Tyr Gly Leu Ile Trp Asp Met Thr Ser
Thr His 180 185 190 Tyr Val Val Ala Asp Pro Ala Lys Gly Val Asn Arg
Lys Tyr Lys Phe 195 200 205 Ser Cys Leu Asp Asn Ala Asn Lys Gly Tyr
Arg Gln Asn Tyr Tyr Val 210 215 220 Val Cys Pro Ala 225
64749PRTMethanobrevibacter ruminantium 64Met Lys Lys His Cys Phe
Tyr Phe Leu Gly Asp Ser Phe Ala Asp Ile 1 5 10 15 Cys Asn Glu Ala
Met Phe Cys Glu Lys His Leu Val Glu Gly Asn Tyr 20 25 30 Leu Asp
Ser Ile Ile Arg Ala Gly Lys Ala Ser Glu Ile Ile Thr Val 35 40 45
Asn Ile Cys Glu Leu Glu Gly Gln Asp Gly Leu Ile Ser Ser Gly Gln 50
55 60 Lys Lys Arg Leu Glu Met Leu Gly Tyr Lys Gly Ile Ile Ser Tyr
Asp 65 70 75 80 Ile Tyr Lys Arg Leu Asn His Ile Arg Lys Ile Arg Asn
Lys Ala Val 85 90 95 His Gly His Leu Ser Asp Ile Glu Asp Asn Ala
Asn Ile Leu His Ala 100 105 110 Tyr Leu Tyr Leu Ile Cys Ala Tyr Phe
Tyr Lys Glu Tyr Arg Asp Thr 115 120 125 Asn Phe Ser Ala Glu Asp Tyr
Thr Gly Pro Ile Met Asp Ile Ala Ser 130 135 140 Lys Pro Lys Glu Thr
Ala Ser Glu Thr Ser Glu Asp Asn Glu Asn Ile 145 150 155 160 Gly Glu
Phe Ile Ser Ser Pro Leu Asp Asp Tyr Leu Phe Glu Lys Tyr 165 170 175
Asp Asp Ser Tyr Leu Leu Asn Glu Leu Ser Lys Leu Lys Asp Ser Ser 180
185 190 Lys Glu Ala Val Glu Asp Asp Asn Leu Ser Glu Phe Lys Glu Tyr
Leu 195 200 205 His Ile Asp Arg Ser Ile Gln Glu Asp Phe Leu Lys Ala
Leu Asn Arg 210 215 220 Ala Thr Ser Phe Asn Ser Ser His Leu Ile Met
Leu Cys Gly Ser Val 225 230 235 240 Gly Asp Gly Lys Ser His Leu Ile
Ala Asn Leu Lys Lys Lys Asn Pro 245 250 255 Glu Leu Phe Asn Gln Phe
Ala Ile His Tyr Asp Ala Thr Glu Ser Phe 260 265 270 Asp Pro Glu Lys
Asn Ala Ile Asp Thr Leu Ala Ser Val Leu Glu Pro 275 280 285 Phe Asn
Asp Asn Asn Leu Asn Asn Ser Thr Glu Lys Leu Ile Leu Ala 290 295 300
Ile Asn Leu Gly Val Leu Asn Asn Phe Leu Glu Ser Ser Tyr Ala Asn 305
310 315 320 Glu Asp Tyr Thr Lys Leu Lys Leu Ile Ile Glu Glu Ala Asn
Ile Phe 325 330 335 Glu Ser Asn Glu Val Ser Asp Asn Ile Tyr Gly Asp
Lys Val Ser Phe 340 345 350 Val Thr Phe Ser Asp Tyr Asn Met Phe Glu
Leu Asn Asp Asp Glu Asn 355 360 365 Ser Asn Tyr Thr Ser Ser Lys Tyr
Ile Ser Ser Leu Phe Asn Lys Ile 370 375 380 Thr Gln Lys Glu Asp Thr
Asn Pro Phe Tyr Val Ala Tyr Leu Lys Asp 385 390 395 400 Lys Asp Ser
His Phe Ile Asn Pro Ile Ile Tyr Asn Tyr Glu Met Leu 405 410 415 Met
Asp Glu Glu Val Gln Lys Thr Ile Ile Asp Tyr Leu Ile Lys Ile 420 425
430 Phe Ile Lys Tyr Arg Lys Ile Ile Ser Thr Arg Asp Leu Leu Asn Phe
435 440 445 Ile Tyr Glu Ile Ile Val Pro Pro Glu Phe Leu Lys Ser Glu
Asp Leu 450 455 460 Asp Asn Ile Asn Asp Phe Met Asp Tyr Ser Leu Pro
Asn Leu Leu Phe 465 470 475 480 Gly Tyr Pro Glu Arg Ser Asp Leu Leu
Lys Leu Cys Asn Glu Leu Asp 485 490 495 Pro Thr Leu His Arg Asn Glu
Ser Leu Asp Lys Phe Ile Ile Asp Leu 500 505 510 Asn Ile Asn Asp Asp
Thr Glu Lys Ile Leu Asn Arg Tyr Phe Asp Phe 515 520 525 Thr Arg Phe
Asn Phe Leu Glu Glu Tyr Gly Glu Tyr Leu Val Asp Phe 530 535 540 Arg
Glu Phe Asn Asn Ser Glu Lys Glu Lys Val Thr Asn Ile Leu Ile 545 550
555 560 Arg Phe Ala Val Phe Tyr Gly Lys Ser Ile Ile Lys Asn Asn Phe
Lys 565 570 575 Asp Lys Val Tyr Leu Asn Tyr Leu Lys Tyr Leu Tyr Ala
Tyr Asn Thr 580 585 590 Gln Ser His Lys Asp Tyr Lys Tyr Leu Phe Thr
Glu Val Lys Asp Ala 595 600 605 Ile Phe Asn Trp Lys Gly Ser Tyr Lys
Lys Asn Thr Ile Cys Ile Asp 610 615 620 Thr Leu Asp Ser Phe Lys Val
Tyr Lys Asn Leu Lys Leu Lys Pro Ser 625 630 635 640 Val Asp Lys Phe
Glu Lys Ser Leu Leu Asp Gly Leu Phe Leu Gly Asn 645 650 655 Arg Phe
Lys Thr Asp Ile Lys Ile Tyr Phe Ser Val Glu Ser Asn Lys 660 665 670
Lys Lys Ile Pro Leu Asn Val Asp Phe Ser Leu Tyr Gln Tyr Ile Met 675
680 685 Lys Leu Tyr Asn Gly Phe Lys Pro Asn Gln Ser Asp Lys Asp Asp
Leu 690 695 700 Ile Ile Leu Asp Glu Phe Ile Asn Asn Leu Leu Asp Glu
Asp Thr Asp 705 710 715 720 Asp Asp Leu Tyr Val Ile Ser Leu Glu Thr
Tyr Glu Glu Phe Leu Phe 725 730 735 Glu Ser Asn Asp Phe Gly Thr Phe
Glu Phe Lys Arg Gly 740 745 65456PRTMethanobrevibacter ruminantium
65Met Asp Phe Ser Glu Asn Tyr Asn Ile Leu Leu Lys Gln Met Thr Cys 1
5 10 15 Asp Val Asn Lys Arg Lys Leu Ile His Gln Ile Asn Gln Asn Ser
Pro 20 25 30 Leu Leu Pro Phe Lys Thr Asn Thr Pro Lys Lys Ala Asn
Phe Glu Asn 35 40 45 Gly Phe Asp Ile Ile Leu Gly Glu Leu Ser Arg
Ile Leu Leu Asn Lys 50 55 60 Thr Ile Glu Lys Asn Phe Lys Leu Asp
Asn Ile Val Ser Asn Leu Ile 65 70 75 80 Asp Asn Asn Ile Glu Ile Glu
Asp Gly Thr Lys Glu Tyr Ile Thr Lys 85 90 95 Leu Leu Asn Glu Tyr
Leu Phe Asp Glu Lys Asn Asp Leu Lys Ile Ser 100 105 110 His Pro Asn
Leu Tyr Leu Tyr Ile Pro Leu Ser Asn Asn Lys Ser Ser 115 120 125 Asn
Gly Glu Gln Glu Val Ala Leu Phe Leu Arg Asp Ile Phe Cys Lys 130 135
140 Asn Asn Gln Asn Leu Ile Asn Phe Phe Glu Ser Tyr Asp Ser Asn His
145 150 155 160 Ile Ile Leu Asn Leu Ile Leu Lys Asn Thr Pro Asn Leu
His His Lys 165 170 175 Ile Thr Glu Thr Lys Tyr Val Ile His Phe Glu
Glu Ile Ala Asn Leu 180 185 190 Phe Asn Glu Asp Ile Asn Tyr Ala Ile
Leu Tyr Lys Lys Phe Phe Met 195 200 205 Glu Asn Ile Gly Asn Ile Phe
Ala Tyr Tyr Tyr Phe Phe Tyr Ile Ser 210 215 220 Gln Leu Ile Leu Lys
Ile Ser Lys Gly Phe Asn Asp Asn Asn Glu Phe 225 230 235 240 Glu Lys
Leu Tyr Tyr Leu Leu Asp Trp Glu Ser Ala Ser Lys Asn Arg 245 250 255
Lys Ser Leu Asn Ser Tyr Ser Leu Leu Lys His His Ser Lys Pro Leu 260
265 270 Tyr Ala Lys Met Ala Val Ile Asp Gln Ile Asn Thr Leu Leu Gly
Thr 275 280 285 Asn Asn Leu Leu Glu Lys Asp Ile Ser Glu Tyr Phe Asn
Asn Leu Asp 290 295 300 Ile Asn Ser Lys Asn Asn Phe Leu His Phe Leu
Lys Lys Trp Val Ser 305 310 315 320 Asp Tyr Arg Tyr Val Arg Asn Phe
Asp Asp Lys Glu Leu Pro Asp Asn 325 330 335 Leu Leu Glu Leu Thr Glu
Ile Leu Phe Glu Ser Leu Lys Asn Glu Lys 340 345 350 Leu Gly Val Asp
Gly Ala Val Gln Ser Arg Tyr Ala Leu Asn Leu Glu 355 360 365 Asp Ile
Ala Lys Lys Tyr Leu Leu Lys Arg Arg Gly Ser Tyr Gly Tyr 370 375 380
Val Leu Asn Ile Asn Arg Asp Met Leu Leu Val Leu Thr Ala Leu Cys 385
390 395 400 Val Lys Asp Lys Lys Ile Lys Leu Asn Gln Leu Phe Ile Glu
Phe Glu 405 410 415 Lys Arg Gly Val Tyr Phe Asp Lys Tyr Ser Lys Glu
Glu Val Val Asn 420 425 430 Phe Leu Thr Lys Leu Asn Leu Ile Asp Lys
Lys Ser Asp Ser Gly Asp 435 440 445
Ala Gln Tyr Val Lys Pro Val Leu 450 455 661740PRTMethanobrevibacter
ruminantium 66Met Leu Asn Gln Phe Tyr Asp Tyr Leu Ser Asn Lys Leu
Leu Asn Tyr 1 5 10 15 Phe Asp Asp Thr Lys Ile Leu Ser Gly Glu Lys
Phe Phe Ile Ser Phe 20 25 30 Asp Glu Asp Asp Gln Ile Met Ser Phe
Tyr Asn Ser Leu Arg Ser Ile 35 40 45 Ala Glu Thr Asn Phe Ser Cys
Ser Glu Phe Ile Tyr Val His Thr Ile 50 55 60 Ser Gly Lys Glu Tyr
Asn Thr Tyr Ser Ile Asn Ile Asn Gly Val Lys 65 70 75 80 Phe Val Ile
Ser Glu Ser Leu Thr Ile Asn Val Asp Phe Leu Val Thr 85 90 95 Leu
Arg Asn Gln Val Thr Ser Gln Glu Gly Val Trp Lys Asp Thr Ala 100 105
110 Leu Leu Val Ile Cys Asn Glu Ala Ile Asp Ser Ile Gly Lys Gly Met
115 120 125 Arg Asn Leu Gln Lys Glu Gly Met Pro Leu Asn Val Lys Ser
Ile Ser 130 135 140 Lys Asn Leu Glu Asp Glu Ile Asn Asp Ser Gln Ile
Leu Asn Tyr Ser 145 150 155 160 Asp Lys Gln Ile Ala Lys Phe Ser Leu
Asn Ile Gln Glu Glu Glu Leu 165 170 175 Phe Gln Thr Thr Leu Trp Asp
Tyr Glu Thr Ile Leu Ser Ile Ile Asn 180 185 190 Lys Gly Phe Val Ser
Asp Glu Asp Leu Arg Glu Leu Asn Leu Phe Lys 195 200 205 Asp Asp Gln
Leu Asn Gln Asn Ser Pro Gln Lys Met Leu Lys Arg Leu 210 215 220 Lys
Glu Asn Tyr Asp Thr Phe Asn Glu Val Asn Lys Phe Ser Gln Tyr 225 230
235 240 Gly Asp Lys Lys Glu Gln Leu Lys Asn Met Phe Thr Asp Ser Gly
Val 245 250 255 Ser Ile Leu Ser Lys Asp Asp Trp Tyr Lys Ala Glu Trp
Lys Met Val 260 265 270 Lys Lys Ser Lys Asp Asp Phe Ile Asn Gln Gln
Asn Pro Leu Asn Tyr 275 280 285 Asn Glu Asn Leu Glu Lys Ile Thr Glu
Asn Gly Leu Asn Tyr Trp Glu 290 295 300 Ile Pro Asn Ser Phe Thr Lys
Thr Gly Lys Arg Lys Arg Asn Ile Ile 305 310 315 320 Val Phe Asn Pro
Asn His Ser His Glu Val Ser Leu Lys Phe Ser Phe 325 330 335 Asp Gln
Ile Leu Ser Asn Ser Phe Leu Asn Thr Asn Ser Lys Lys Phe 340 345 350
Thr Ile Ala Arg Gly Lys Ser Leu Ile Val Asn Phe Thr Leu Asp Ser 355
360 365 Ser Glu Pro Ile Phe Lys Thr Ile Lys Tyr Lys His Lys Asn Glu
Asn 370 375 380 Ile Ser Glu Phe Thr Phe Asn Ile Val Val Leu Asn Phe
Glu Pro Glu 385 390 395 400 Ile Phe Asn Ser Ile Lys Ser Arg Phe Ser
Val Asn Val Lys Ser Lys 405 410 415 Gln Ile Ile Val Thr Asn Asp Glu
Asp Ser Phe Asp Ile Val Phe Gly 420 425 430 Thr Gly Ser Lys Glu Ile
Glu Lys Leu Ile Lys Glu Asn Gly Glu Lys 435 440 445 Leu Tyr Leu Tyr
Asp Asp Glu Ser Leu Ile Ile Ser Glu Gln Ser Pro 450 455 460 Ala Trp
Asn Asp Gly Lys Leu Ser Phe Lys Leu Tyr Lys Asp Asn Asn 465 470 475
480 Tyr Ala Pro Phe Leu Ile Lys Glu Lys Ser Lys Lys Thr Leu Pro Val
485 490 495 Asn Ser Tyr Val Ile Trp Asn Leu Lys Arg Arg Asn Met Glu
Asn Phe 500 505 510 Ile Phe Asn Gly Val Lys Ala Val Gln Asp Val Asn
Ser Phe Tyr Leu 515 520 525 Val Glu Glu Phe Lys Glu Phe Leu Lys Met
Glu Arg Glu Ile Ile Lys 530 535 540 Gln Asp Ile Phe Tyr Ala Lys Arg
Asn Ile Asp Gly Ser Leu Glu Lys 545 550 555 560 Ile Glu Val Ser Phe
Ser Asn Glu Leu Glu Thr Ala Tyr Met Asp Ile 565 570 575 Phe Asn Tyr
Tyr Lys Thr Phe Asp Asp Ser Pro Glu Asp Asn Leu Pro 580 585 590 Ser
Leu Val Tyr Leu Asn Asp Asp Leu Lys Glu Leu Tyr Lys Lys Phe 595 600
605 Ile Thr Ile Phe Asn Lys Glu Ile Ser Glu Ile Glu Glu Asn Ser Ile
610 615 620 Leu Ser Asp Phe Lys Tyr Lys Lys Asn Leu Leu Lys Leu Gly
Arg Ile 625 630 635 640 Glu Thr Asp Asn Lys Ile Met Tyr Ser Pro Leu
Ser Pro Leu Asn Ile 645 650 655 Ala Tyr Gln Leu Glu Val Ser Lys Gln
Cys Gly Asn Glu Asp Leu Ser 660 665 670 Val Asn Ile Leu Glu Arg Leu
Val Pro Asn Asn Leu Ile Pro Tyr Ile 675 680 685 Cys Ser Asp Asp Gly
Lys Glu Leu Phe Arg Pro Ile Tyr Gln Glu Glu 690 695 700 Ala His Glu
Trp Leu Ile Tyr Glu Lys Ser Glu Glu Val Ser Ile Gly 705 710 715 720
Thr Thr Asn Val Phe Ile Ser Asn Val Val Thr Glu Lys Leu Asn Gln 725
730 735 Phe Val Lys His Phe Asn Tyr Leu Phe Ser Phe Asn Asn Ser Ser
Pro 740 745 750 Ile Lys Ile Asn Leu Ile Asn Ile Lys Asp Asp Lys Glu
Val Val Lys 755 760 765 Gly Val Phe Asn Phe Ile Arg Ser Arg Leu Pro
Asp Lys Thr Lys Thr 770 775 780 Lys Lys Val Ile Pro Val Glu Ile Asn
Ile Tyr Asn Asp Ala Glu Lys 785 790 795 800 Ser Ser Phe Asp Asn Leu
Phe Asp Cys Gln Ser Glu Ile Gln Leu Leu 805 810 815 Glu Glu Phe Gly
Ile Lys Lys Leu Lys Ser Asp Ile Phe Asp Pro Ile 820 825 830 Asp Ile
Ile His Met Ile Gln Asn Asn Ile Ser Tyr Tyr Lys His Pro 835 840 845
Phe Lys Lys Glu Glu Tyr Glu Tyr Ala His Leu Ser Phe Tyr Lys Val 850
855 860 Lys Ser His Asn Asn Ile Ala Asn Asp Asn Met Asp Lys Ile Glu
Thr 865 870 875 880 Gly Leu Ser Leu Asn Gly Leu Leu Ser Ser Val Thr
Ser Thr Thr Lys 885 890 895 His Ser Glu Tyr Arg Thr Gly Phe Gly Thr
Asn Asn Ile Leu Asn Met 900 905 910 Ser Asn Pro Leu Ile Lys Thr Val
Ile Asn Leu Asn Glu Leu Val Glu 915 920 925 Asn Ser Lys Asn Phe Gly
Lys Asn Thr Tyr Ser Lys Asn Lys Ser Val 930 935 940 Ile Thr Thr Val
Glu Leu Glu Glu Asp Asn Ile Glu Glu Leu Tyr Asp 945 950 955 960 Lys
Ser His Trp Val Thr Phe Ile Glu Pro Thr Phe Gly Ile Glu Tyr 965 970
975 Phe Asp Ser Ser Asp Ser Asn Leu Ile Ile Ile His Tyr Ser Asp Gln
980 985 990 Tyr Ser Ser Ser Ser Lys Tyr Asp Thr Ile Thr Val Thr Asn
Lys Ser 995 1000 1005 Thr Gln Tyr Glu Glu Ile Ile Arg Asp Phe Leu
Gln Ser Lys Tyr 1010 1015 1020 Val Lys Val Thr Asp Glu Glu Leu Tyr
Asp Val Ile Lys Met Phe 1025 1030 1035 Asn Ser Ile Asn Gly Glu Trp
Leu Leu Arg Val Ile Ser Asn Ser 1040 1045 1050 Gly His Tyr Asp Arg
Glu Lys Leu Ser Ile Ile Ser Ala Ile Lys 1055 1060 1065 Tyr Cys Leu
Ser Ile Leu Asp His Lys Asp Ile Val Trp Ile Pro 1070 1075 1080 Val
Ser Met Glu Glu Ile Leu Arg Ile Ala Gly Asn Val Lys Leu 1085 1090
1095 Asp Lys Asn Lys Gly Ile Phe Asp Ser Lys Leu Ile Lys Gly Asn
1100 1105 1110 His Ser Asp Asp Leu Leu Phe Ile Gly Val Lys Phe Asn
Glu Asp 1115 1120 1125 Asn Arg Ile Glu Val Ile Phe Tyr Pro Ile Glu
Val Lys Ile Gly 1130 1135 1140 Leu Asn Asn Ala Ser Thr Ile Lys Lys
Gly Lys Ser Gln Leu Asp 1145 1150 1155 Asn Thr Tyr Lys Leu Leu Lys
Thr Gln Leu Gln Asn Ile Asn Val 1160 1165 1170 Glu Asn Ser Glu Phe
Arg Asn Lys Phe Phe Arg Asn Phe Phe Ile 1175 1180 1185 Gln Ile Leu
Leu Ser Asn Glu Gln Lys Leu Val Thr Asn His Ile 1190 1195 1200 Trp
Asp Glu Lys Gly Leu Asp Arg Ile Glu Glu Phe Lys Ala Glu 1205 1210
1215 Leu Leu Asn Asp Glu Tyr Asp Ile Leu Tyr Gly Leu Glu Glu Tyr
1220 1225 1230 Ile Gly Lys Gly Ser Leu Val Ser Phe Lys Lys Glu Ser
His His 1235 1240 1245 Ile Ser Ile Tyr Met Asp Val Asp Lys Gln Val
Ile Glu Leu Pro 1250 1255 1260 Glu Asp Phe Ala Tyr Tyr Gly Leu Ala
Thr Pro Ile Arg Glu Ile 1265 1270 1275 His Asp Glu Ile Gln Ser Asp
Asn Thr Asp Ile Leu Ala Glu Thr 1280 1285 1290 Leu Leu Ser His Val
Asp Ile Ser Glu Ile Arg Ala Lys Asn Asn 1295 1300 1305 Asp Ile Cys
Asp Ser Asn Glu Asp Met Ser Ile Asp Asp Asp Phe 1310 1315 1320 Asp
Asn Leu Ser Glu Phe Glu Asp Ser Phe Ile Glu Glu Glu Ser 1325 1330
1335 Glu Ile Ser Glu Glu Pro Asp Glu Glu Leu Thr Asp Ala Thr Ser
1340 1345 1350 Ser Ser Asp Asn Glu Ser Ile Glu Asn Ile Gly Glu Ser
Pro Ser 1355 1360 1365 Lys Ile Ser Asn Val Arg Ala Leu Ile Gly Thr
Gln Lys Gly Tyr 1370 1375 1380 Asn His Lys Val Tyr Trp Glu Phe Gly
His Pro Ser Leu Ala Asn 1385 1390 1395 Arg His Met Leu Ile Gln Gly
Lys Ser Gly Gln Gly Lys Thr Tyr 1400 1405 1410 Phe Ile Gln Arg Met
Leu Lys Glu Leu Ser Ile Gln Gly Ile Pro 1415 1420 1425 Ser Ile Ile
Ile Asp Tyr Thr Asp Gly Phe Lys Pro Ser Gln Leu 1430 1435 1440 Glu
Pro Asn Phe Lys Asp Ser Leu Gly Asp Lys Ile Ser Gln Tyr 1445 1450
1455 Phe Val Val Lys Glu Asn Phe Pro Ile Asn Pro Phe Lys Arg Asn
1460 1465 1470 Thr Ile Met Ile Asp Lys Asp Ile Phe Ile Glu Glu Asp
Asn Ser 1475 1480 1485 Thr Ile Ala Ser Arg Phe Lys Ser Ile Ile Asn
Ser Val Tyr Gly 1490 1495 1500 Leu Gly Ile Gln Gln Ser Asn Thr Leu
Tyr Gln Thr Val Leu Asp 1505 1510 1515 Cys Leu Asp Lys Tyr Asp Asp
Asn Phe Asp Leu Asn Ile Leu Lys 1520 1525 1530 Glu Glu Ile Leu Lys
Asp Glu Ser Asn Ser Ala Gln Thr Val Leu 1535 1540 1545 Asn Lys Leu
Asn Glu Leu Leu Asp Lys Asn Pro Phe Ala Ser Thr 1550 1555 1560 Asp
Phe Asp Trp Ser Val Leu Asp Asn Lys Asp Gly Lys Val Tyr 1565 1570
1575 Ile Ile Gln Leu Thr Ala Leu Ser Lys Asp Ile Gln Thr Ile Ile
1580 1585 1590 Thr Glu Phe Ile Leu Trp Asp Leu Trp Asn Tyr Lys Leu
Thr Asn 1595 1600 1605 Gly Ser Glu Asp Asn Pro Phe Ile Val Val Leu
Asp Glu Ala His 1610 1615 1620 Asn Leu Asp Phe Ser Asn Asp Ser Pro
Cys Ser Lys Ile Leu Lys 1625 1630 1635 Glu Gly Arg Lys Phe Gly Trp
Ser Gly Trp Phe Ala Thr Gln Ser 1640 1645 1650 Val Lys Gly Ser Met
Lys Ile Asp Glu Ile Ala Lys Leu Glu Asn 1655 1660 1665 Ala Asp Glu
Lys Ile Tyr Phe His Pro Thr Asp Val Ser Thr Ile 1670 1675 1680 Ala
Lys Asp Leu Ser Lys Asp Asn Glu Asp Lys Lys Ile Tyr Glu 1685 1690
1695 Lys Glu Leu Ser Gln Leu Thr Lys Gly Tyr Cys Ile Val Gln Gly
1700 1705 1710 Ser Ala Ile Asp Ser Ser Gly Asn Leu Tyr Gln Pro Asn
Pro Val 1715 1720 1725 Thr Val Lys Ile Glu Glu Ile Ser Phe Asp Glu
Asn 1730 1735 1740 671054PRTMethanobrevibacter ruminantium 67Met
Glu Ser Lys Asp Arg Ile Glu Asn Thr Ile Phe Asn Asp Lys Arg 1 5 10
15 Met Asp Ala Tyr Ile Asp Lys Tyr Phe Glu Glu Phe Thr Leu Ser Ser
20 25 30 Glu Gln Glu Glu Ala Leu Asn Ile Trp Ile Asp Lys Leu Asn
Asn Asp 35 40 45 Gln Leu Thr Ser Glu Lys Gly Asn Tyr His Asn Phe
Phe Glu Ile Ile 50 55 60 Leu Glu Asp Leu Leu Gly Tyr Lys Arg Ser
Asp Val Lys His Glu Glu 65 70 75 80 Asn Ile Gly Asp Glu Gly His Pro
Val Glu Phe Val Leu Glu Lys Asp 85 90 95 Gly Lys Asp Tyr Val Ile
Ile Glu Leu Lys Gly Thr Thr Tyr Lys Asp 100 105 110 Leu Thr Lys Arg
Arg Pro Gly Gln Gln Ser Pro Val Glu Gln Ala Thr 115 120 125 Asn Tyr
Ala Ser Ala Lys Lys Glu Thr Glu Trp Ala Thr Val Ser Asn 130 135 140
Tyr Asp Glu Phe Arg Phe Phe Asn Pro Thr Ala Arg Asp Asn Tyr Ile 145
150 155 160 Ser Phe Lys Phe Arg Gln Leu Lys Asp Leu Glu Ile Phe Lys
Lys Phe 165 170 175 Leu Leu Val Phe Ser Lys Phe Ser Leu Ile Asp Glu
Asp Ile Pro Lys 180 185 190 Lys Leu Leu Asn Glu Thr Lys Val Ile Glu
Arg Glu Leu Glu Asn Glu 195 200 205 Phe Tyr Gln Leu Tyr Ser Asp Thr
Arg Leu Met Ile Ile Lys Glu Leu 210 215 220 Glu Tyr Ser Ser Glu Asp
Ile Asn Arg Ile Glu Ala Ile Lys Leu Ser 225 230 235 240 Gln Ile Ile
Leu Asn Arg Phe Ile Phe Leu Cys Phe Ala Glu Asp Leu 245 250 255 Ala
Leu Met Glu Glu Glu Thr Thr Ala Asp Val Leu Leu Thr Pro Leu 260 265
270 Lys His Arg Asn Leu Ile Gly Asn Thr Met Trp Asn Arg Leu Asn Glu
275 280 285 Leu Phe Ile Phe Ala Asn Gln Gly Asn Lys His Arg Arg Ile
Pro Ala 290 295 300 Phe Asn Gly Gly Leu Phe Glu Asp Asp Leu Ser Asn
Leu Lys Ile Arg 305 310 315 320 Asp Glu Ile Glu Asp Arg Ser Phe Phe
Glu Asn Trp Asn Leu Lys Glu 325 330 335 Asp Phe Glu Asp Lys Tyr Glu
Asp Ile Ala Lys Leu Ile Gly Val Tyr 340 345 350 Lys Asp Thr Leu Asn
Pro Ile Phe Ile Asn Leu Leu Ile Ile Ser Thr 355 360 365 Tyr Asp Phe
Asp Ser Glu Leu Asp Val Asn Ile Leu Gly His Ile Phe 370 375 380 Glu
Asn Ser Ile Ser Asp Ile Glu Glu Leu Lys Asn Asp Asn Gln Glu 385 390
395 400 Gln Arg Lys Lys Asp Gly Val Tyr Tyr Thr Pro Glu Tyr Ile Thr
Asp 405 410 415 Tyr Ile Cys Arg Asn Thr Ile Ile Pro Tyr Leu Ser Ile
Ser Gly Lys 420 425 430 Ala Ser Thr Val His Glu Leu Leu Tyr Glu Tyr
Glu Ser Ser Asn Ser 435 440 445 Leu Asp Val Leu Asp Ser Lys Leu Thr
Asn Ile Lys Val Leu Asp Pro 450 455 460 Ala Cys Gly Ser Gly Ser Met
Leu Asn Lys Ser Val Asp Ile Leu Phe 465 470 475 480 Glu Ile His Glu
Ala Leu His Ala Ser Lys Tyr Ala Gly Asp Ser Ser 485 490 495 Leu Asp
Arg Phe Phe Asp Ser Leu Glu Lys Arg Lys Glu Ile Ile Ser 500 505
510 Asn Asn Ile Tyr Gly Val Asp Leu Asn Glu Glu Ser Val Glu Ile Thr
515 520 525 Lys Leu Ser Leu Phe Leu Lys Leu Ala Thr Thr Val Gly Leu
Lys Glu 530 535 540 Gly Phe Gln Leu Pro Ser Leu Asp Lys His Ile Lys
Cys Gly Asp Ser 545 550 555 560 Leu Val Asp Asp Glu Ser Ile Ala Gly
Asn Lys Ala Phe Asn Trp Tyr 565 570 575 Glu Ser Phe Ser Glu Val Phe
Glu Ser Gly Gly Phe Asp Ile Ile Val 580 585 590 Gly Asn Pro Pro Tyr
Val Asp Ile Lys Glu Met Asp Glu Lys Thr Ala 595 600 605 Lys Tyr Ile
Phe Asp Asn Tyr Glu Thr Ser Phe Asn Arg Ile Asn Leu 610 615 620 Tyr
Ser Thr Phe Val Glu Lys Ser Tyr Tyr Leu Leu Lys Asn Glu Gly 625 630
635 640 Ile Phe Ser Phe Ile Met Pro Asn Ser Ile Leu Phe Asn Ser Thr
Tyr 645 650 655 Ser Lys Ile Arg Glu Leu Ile Leu Asn Asn Thr Ser Ile
Leu Asn Ile 660 665 670 Val Arg Thr Ser Asp Asp Val Phe Lys Asp Ala
Lys Val Glu Pro Ile 675 680 685 Ile Leu Ile Phe Lys Lys Gly Tyr Asp
Glu Gly Asn Lys Thr Lys Ile 690 695 700 Leu Ile Lys Lys Asp Asp Met
Asp Glu Ile Pro Ile Asn Asn Tyr Ser 705 710 715 720 Glu His Phe Phe
Thr Gln Glu Arg Trp Phe Glu Asn Asn Ser Ile Ile 725 730 735 Asn Ile
Phe Ser Asp Asp Phe Thr Phe Asp Leu Leu Lys Lys Ile Asp 740 745 750
Gly Asn Asn Glu Arg Leu Ile Asp Tyr Cys Asp Phe Ser Leu Gly Leu 755
760 765 Thr Pro Tyr Asp Lys Tyr Lys Gly Met Ser Glu Asp Ile Ile Lys
Asn 770 775 780 Arg Lys Phe His Ser Lys Ile Lys Leu Asp Asp Thr Phe
Lys Glu Leu 785 790 795 800 Leu Asp Gly Ser Asp Ile Thr Arg Tyr Asn
Val Lys Trp Gly Glu Lys 805 810 815 Glu Tyr Ile Lys Tyr Gly Asp Trp
Leu Gly Ala Pro Arg Glu Glu Lys 820 825 830 Phe Phe Lys Asn Pro Arg
Ile Leu Ile Arg Gln Ile Leu Ser Ile Ala 835 840 845 Pro Lys Glu Ser
Arg Lys Arg Ile Phe Ala Ala Tyr Thr Glu Glu Glu 850 855 860 Leu Tyr
Asn Ala Gln Ile Ala Phe Asn Leu Val Leu Lys Glu Gly Phe 865 870 875
880 Asp Asp Lys Asn Leu Leu Lys Tyr Phe Leu Gly Ile Ile Asn Ser Lys
885 890 895 Met Met Thr Trp Tyr Tyr Glu Glu Arg Phe Met Asp Lys Asn
Lys Lys 900 905 910 Asn Phe Ala Lys Ile Leu Ile Glu Asn Ala Lys Asn
Leu Pro Val Ile 915 920 925 Ile Asn Ser Asn Phe Leu Asp Glu Ile Val
Ser Asn Val Asp Ser Ile 930 935 940 Ile Glu Leu Asn Lys Glu Phe Tyr
Ser Val Arg Asn Ala Phe Gln Thr 945 950 955 960 Trp Leu Lys Ile Glu
Phe Glu Ile Glu Lys Leu Ser Lys Lys Leu Glu 965 970 975 Asn Tyr Tyr
Asp Leu Asn Phe Glu Glu Phe Leu Lys Glu Ile Lys Lys 980 985 990 Lys
Lys Val Val Ile Arg Pro Asn Gln Ile Gln Asp Leu Ser Glu Leu 995
1000 1005 Phe Asn Glu Ser Leu Gly Lys Ile Glu Tyr Leu Gln Arg Glu
Ile 1010 1015 1020 Lys Glu Ala Asp Glu Lys Ile Asn Leu Leu Val Tyr
Glu Leu Tyr 1025 1030 1035 Gly Leu Asn His Glu Glu Ile Glu Ile Ile
Glu Asn Ser Phe Asn 1040 1045 1050 Asp 6886PRTMethanobrevibacter
ruminantium 68Met Asp Asp Glu Thr Leu Ile Leu Ile Glu Tyr Ile Arg
Asn Ala Pro 1 5 10 15 Thr Arg Glu Met Val Leu Lys Ser Phe Glu Gly
Val Asp Phe Ile Arg 20 25 30 Pro Ile Gln Ile Ser Arg Lys Thr Gly
Ile His Pro Asn Asn Val Ser 35 40 45 Lys Lys Leu Lys Asp Leu Arg
Glu His Glu Leu Val Tyr Val Ile Asn 50 55 60 Pro Glu Tyr His Val
Pro Lys Leu Tyr Arg Leu Thr Glu Lys Gly Lys 65 70 75 80 Asn Met Leu
Gln Phe Leu 85 69255PRTMethanobrevibacter ruminantium 69Met Asn Lys
Lys Ile Ile Leu Ser Leu Leu Leu Val Leu Leu Val Ala 1 5 10 15 Ile
Ser Val Ser Ala Val Ala Ala Ala Asp Ala Asp Val Thr Tyr Ile 20 25
30 Asn Asp Ala Ala Asp Val Asp Asp Val Ala Asp Glu Lys Val Ala Pro
35 40 45 Leu Thr Ala Ser Ala Asp Ala Gln Asp Ile Gln Thr Lys Leu
Asp Asn 50 55 60 Ala Lys Pro Gly Asp Thr Ile Glu Leu Glu Asn Lys
Thr Tyr Asp Val 65 70 75 80 Asp Thr Thr Phe Asn Val Thr Lys Gln Val
Thr Ile Lys Gly Gln Asp 85 90 95 Thr Val Ile Lys Ala Ser Gly Ala
Ser Gln Gly Gly Ser Gly Ala Leu 100 105 110 Phe Ile Ala Asn Glu Ala
Gly Thr Ala Phe Glu Gly Ile Thr Phe Ile 115 120 125 Asn Thr Asp Gly
His Lys Asn Tyr Gly Glu Gln Val Ser Gly Tyr Ala 130 135 140 Ile Gln
Leu Ala Ile Glu Asn Gly Thr Val Asp Asn Cys Lys Phe Ile 145 150 155
160 Asp Trp Ser Ser Gly Val Tyr Gly Lys Gly Ala Ser Phe Cys Ser Ile
165 170 175 Thr Asn Ser Tyr Phe Asn Gly Ser Ser Glu Gln Val Thr Asn
Gly Gly 180 185 190 Lys Lys Glu Tyr Gly Thr Lys Ala Ile Asn Leu Met
Gly Ser His Asp 195 200 205 Ile Thr Val Thr Gly Cys Thr Phe Glu Gly
Gln Val Leu Asp Ala Ile 210 215 220 Ser Ile Ala Ser Asn Ser Gly Asn
Asn Ile Met Thr Asp Asn Thr Phe 225 230 235 240 Ile Asp Asn Cys Tyr
Ala Ile Tyr Phe Gly Gly Ala Ser Thr Gln 245 250 255
70208PRTMethanobrevibacter marburgensis 70Ser Arg Tyr Asn Arg Phe
Lys Glu Val Asn Gly Arg Glu Pro Arg Val 1 5 10 15 Val Phe Ile Tyr
Ser Gly Gly Gly Pro Ser Val Ser Leu Glu Thr Phe 20 25 30 Lys Asp
Met Cys Lys Arg Tyr Asn Gln Phe Leu Glu Glu Asn Arg Arg 35 40 45
Glu Pro Arg Ile Val Tyr Val Thr Pro Pro Glu Pro Pro Val Pro Glu 50
55 60 Glu Val Arg Glu Met Arg Arg Val Leu Gly Glu Phe Lys Thr Ala
Thr 65 70 75 80 Gln Leu Tyr Thr Leu Val Ser Arg Arg Cys Lys Tyr Lys
Phe Tyr Tyr 85 90 95 Asn Asp Gln Thr Pro Asn Arg Glu Ala Leu Lys
Lys Met Val Thr Asp 100 105 110 Gly Ile Asn Cys Thr Asp Ala Cys Gln
Leu Phe Lys Pro Val Ile Glu 115 120 125 Gly Leu Gly Tyr Ser Val Arg
Ile Glu His Val Lys Val Arg Cys Asn 130 135 140 Asp Asn Lys Trp Tyr
Gly His Tyr Phe Leu Arg Val Ala Gly Lys Glu 145 150 155 160 Leu Ala
Ser Val Ser Leu Pro Ser Glu Arg Trp Thr Val Trp Asp Tyr 165 170 175
Val Ser Ala Thr Lys Thr Gly Arg Pro Leu Gly Ala Pro Cys Cys Ser 180
185 190 Arg Gly Ile Gln His Leu Gly Trp Gly Ile Val Ser Pro Lys His
Asp 195 200 205 71202PRTMethanobrevibacter wolfeii 71Arg Arg Tyr
Glu Asp Phe Val Arg Ile Asn Gly Arg Glu Pro Asn Ile 1 5 10 15 Ile
Tyr Leu Glu Gln Gly Lys Ser Asp His Val Ser Leu Gly Thr Phe 20 25
30 Lys Asp Met Leu Arg Arg Tyr Lys Asp Phe Val Arg Ile Asn Gly Arg
35 40 45 Glu Pro Asn Tyr Ile Ser Ile Gln Pro Gln Pro Ser Leu Lys
Gly His 50 55 60 Trp Thr Thr Lys Val Ile Glu Lys Ile Gly Thr Phe
His Asp Ala Thr 65 70 75 80 Ser Leu Tyr Glu Arg Val Lys Lys Thr Cys
Lys Tyr Lys Tyr Tyr Tyr 85 90 95 Asn Asp Gln Val Pro Asn His Val
Ala Val Met Arg Met Thr Thr Ser 100 105 110 Gly Ile Asn Cys Thr Asp
Ala Cys Gln Leu Phe Ser Lys Val Leu Glu 115 120 125 Glu Met Gly Tyr
Glu Val Lys Ile Glu His Val Arg Val Lys Cys Asn 130 135 140 Asp Gly
Lys Trp Tyr Gly His Tyr Leu Leu Arg Val Gly Gly Phe Glu 145 150 155
160 Leu Lys Asp Gly Thr Ile Trp Asp Tyr Val Ser Ala Thr Lys Thr Gly
165 170 175 Arg Pro Leu Gly Val Pro Cys Cys Thr Ala Gly Phe Gln His
Leu Gly 180 185 190 Trp Gly Ile Val Gly Pro Val Tyr Asp Lys 195 200
72155PRTMethanobrevibacter ruminantium 72Val Ala Arg Asn Pro Leu
Val Met Asp Tyr Gln Asn Thr Asn Tyr Thr 1 5 10 15 Cys Cys Pro Thr
Ser Leu Ser Leu Ala Ser Gln Met Leu Tyr His Tyr 20 25 30 Lys Ser
Glu Ser Glu Cys Ala Lys Ala Leu Gly Thr Ser Lys Gly Ser 35 40 45
Gly Thr Ser Pro Ala Gln Leu Ile Ala Asn Ala Pro Lys Leu Gly Phe 50
55 60 Lys Ile Ile Pro Ile Lys Arg Asp Ser Lys Glu Val Lys Lys Tyr
Leu 65 70 75 80 Lys Lys Gly Phe Pro Val Ile Cys His Trp Gln Val Asn
Gln Ser Arg 85 90 95 Asn Cys Lys Gly Asp Tyr Thr Gly Asn Phe Gly
His Tyr Gly Leu Ile 100 105 110 Trp Asp Met Thr Ser Thr His Tyr Val
Val Ala Asp Pro Ala Lys Gly 115 120 125 Val Asn Arg Lys Tyr Lys Phe
Ser Cys Leu Asp Asn Ala Asn Lys Gly 130 135 140 Tyr Arg Gln Asn Tyr
Tyr Val Val Cys Pro Ala 145 150 155 73212PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
sequence 73Xaa Arg Tyr Xaa Xaa Phe Xaa Xaa Ile Asn Gly Arg Glu Pro
Xaa Val 1 5 10 15 Ile Phe Ile Xaa Asn Gly Xaa Xaa Xaa Xaa Xaa Xaa
Val Ser Leu Xaa 20 25 30 Thr Phe Lys Asp Met Leu Lys Arg Tyr Lys
Xaa Phe Leu Xaa Xaa Asn 35 40 45 Xaa Arg Glu Pro Xaa Xaa Ile Xaa
Ile Xaa Pro Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Glu
Met Xaa Lys Xaa Leu Gly Thr Phe Lys Xaa 65 70 75 80 Ser Ala Thr Ser
Leu Tyr Xaa Leu Val Ala Lys Xaa Cys Lys Tyr Lys 85 90 95 Phe Tyr
Tyr Asn Asp Gln Xaa Pro Asn Xaa Xaa Ala Val Lys Lys Met 100 105 110
Leu Thr Xaa Gly Ile Asn Cys Thr Asp Ala Cys Gln Leu Phe Xaa Xaa 115
120 125 Val Ile Glu Xaa Leu Gly Tyr Ser Val Lys Ile Glu His Val Lys
Val 130 135 140 Lys Cys Asn Asp Xaa Lys Trp Tyr Gly His Tyr Xaa Leu
Arg Val Ala 145 150 155 160 Gly Xaa Glu Leu Xaa Xaa Val Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Thr Val 165 170 175 Trp Asp Tyr Val Ser Ala Thr Lys
Thr Gly Arg Pro Leu Gly Xaa Pro 180 185 190 Cys Cys Ser Xaa Gly Xaa
Gln His Leu Gly Trp Gly Ile Val Xaa Pro 195 200 205 Xaa His Asp Xaa
210 74264DNAMethanobrevibacter ruminantium 74gtgagaaata tgaagaataa
gagtttaata ttaatttctt tattattact gattacaata 60ataagcatag gatctgttgt
tgcaacggat aatgaagaaa ttaatatgga taatataaat 120aatattgata
ataatgagga tatcgctaat attgataatg tcgataatgt cgataactcc
180aatataaaca atccaactga cataagaata gacaattcaa acctaaatag
agaaacagaa 240ctagattcaa atttaaataa atct
26475255DNAMethanobrevibacter ruminantium 75atgaagactt ttagacaaca
gctattggaa gaccctgaat tccagaacta cctgttgcag 60agaccgaacc tgacggagag
cagcctgcag tcatacctca atgccgccac caacttcgtg 120aggttcacag
gggagccgtt ctacaagacc gtgcatgagc tcagaagcca gcagaacgat
180aggattgaga acaacatcat cataaggttc aacccgaacc agtcaaggat
aaacatcatg 240cagtttgagt tcata 25576255DNAMethanobrevibacter
ruminantium 76atgaatacat taaaaataga atgttcaaaa gatgattata
tcataaagac tgcaaaggca 60aattctgaaa atgagttgaa agttgaagtt cctgagaact
ggaactgcga ctatgtgaat 120gctgtcctgt gggaagagga catctgcgaa
gtgcttgaaa agggtgatga aagaatgctt 180ttgattccta tgtgcggtga
actgcttctt gaaggagtgc aggaagatga atacataaag 240tacatttgcc ttcct
25577255DNAMethanobrevibacter ruminantium 77atgggaatga aaaaagttaa
gatcgaaagt aaagcaaaaa ataatatgac aagaaacgaa 60aaactcttct ataaattata
tgacgattta tataatgaag atagattaat tttattctcc 120acatatttta
acatatatga taaatttatt tttaaaaaag atataatcca ttatgtttta
180atgaactact ctgaaaatga aattattgaa gcaatgaaaa aaattgatga
aattcagtca 240gaaggcattg atatt 25578255DNAMethanobrevibacter
ruminantium 78atgaaaacac aagatctaat taatataata aatgatgagg
aatctcctgt attcctcaac 60agggaagtct ttgagatgga ctatgtgccg gacatctaca
aatacaggga cgagcagctg 120gcgaaaatgg cgatgtactg caattcaata
cctgacaaca tagctcccaa gaacctgcaa 180ttgtgcggag gcaatgcgac
aggaaagacc acaacattaa agcagttctt caagatgttg 240aacgaggctt ttcca
25579255DNAMethanobrevibacter ruminantium 79atgtttttgg aaaaatgcga
tggaaaagat acaatcagca tgtctcttca agaaaagatg 60aatcttatat tggaaactat
ggaaagcaaa ggaagcccct tcatatcctg tattcattgt 120aacataccta
taaatgaagc agaagaatgg tacaaaaatg gagaaatagg agatcaggac
180tttataaatt tctatgatga tgtaaacttg attgaagaaa gttttgggtt
tgaaatttat 240aaaaaatcag aatat 25580207DNAMethanobrevibacter
ruminantium 80ttgataattt tccttaaact aaaatttgga cgtgtaatca
tggaaaaatt aatcgaaatt 60gacggagttc aatacacaga agcccaaatc agaagggctt
tagccattga acgagacgtg 120cgctctccta atttcgttga tatgttattg
ggaaagatca aacctagcga acttgcaagc 180agagtttctg aaaaaggtga tgcctaa
20781156DNAMethanobrevibacter ruminantium 81atgtgctatg tgggaaacac
caggacattg gtctatcaca cagaggactg cttctgcaac 60cactggctgt tgaacgagaa
caagaccatt ctagaggaaa agcctgtaga catgaaacct 120tgcagcttct
gcaaaccaca gtttgacact gaatag 15682234DNAMethanobrevibacter
ruminantium 82gtgttggata tggttgcaga gatggttgaa aacatcagga
aaggagaagg agatggatat 60tccatctacc ctcctttttc ctgcattgtt ttcctacaag
gcaaaaagta ttccgaatgc 120tgctgcaagg ctgaggcaag ggatcagaag
tttgcacttg tgaaccttgt aggcttcaga 180agagaggatg tcaaggtcat
agacccaagg accaacgagg agctgttcgt atga 23483162DNAMethanobrevibacter
ruminantium 83atgagcatgc ttgcagactt cgagcctgca aggctccaca
agaggacatg ggctgaaagg 60catgatgtcg aaatccttgc tgtcatatgc cttgccataa
gcattgcaat gctacttctt 120ttctttgcgc ttgcagagcc gactgttgca
ggagtgattt ag 16284255DNAMethanobrevibacter ruminantium
84atgactaagg aatttgagga cttcatgaga aggaacacag gactgcttgt tttcttgaga
60tgggacacag tggcatatct gaaatacctt gagtcccatt atgacgaaag gaagtacgaa
120tgcgcttaca gattgttgga ggcaatcgac aatctctttg acttctacca
gataaccttc 180agcaaaaaga ctgaaaggga aattcctgac cagctttttg
aaaaggacaa gataaacaaa 240ggcttccttt cacac
25585255DNAMethanobrevibacter ruminantium 85atgaatgaga tagttactac
aactaacgag aataatgtac ctgtcgacgt tgactatgcc 60atcgaggaat ggaaggcata
ccagcgattg accagagagc tattggacga aacagattat 120cagacacaca
gaggcaggaa gtacaagacc aagagcgctt ggcagaagta cgcacgtgcc
180ttcaacataa acacacagat aatcgacaag gaaatcgtca agaatgacaa
gggaatcgtc 240attgaggctg agtat
25586231DNAMethanobrevibacter ruminantium 86atgattgagt gcatacagga
agagggagac ttcacggatt gggaggttcc ttcctcctct 60tccgacatca agtacatagt
ttcagtggat gatgagggaa acctgttctg cagctgtcct 120gacttctatt
acaggaagtc caggatgaac cctcacatct caaaccctga atcctattgc
180aaacacatca gacaagtctt ggaggaggac aatagattgc aaatgctcta g
23187255DNAMethanobrevibacter ruminantium 87atggataata taaataagac
aaaaacaagt ttagctaagt ttgaagagtt tttcagcact 60gtatacaagg acgaagtcat
ggaagttttg gaaaagtatc ctgaggaaag gacattggtt 120gtggactatg
agaatttgga aatgtttgat cctgatttgg cagacttgtt gattgaaaag
180cctgacgaag taatcgcagc ttcacagaaa gcaatcaaga acattgaccc
attgatgaaa 240gatccaaagt tggac 25588255DNAMethanobrevibacter
ruminantium 88atggctaaca agattagagt taatctcacg gttgacccta
atttgtggca attagcaaaa 60gacaagttac cttgcagcag aagcgaattc tttgagaatc
agttgaagat gttcttaggg 120attgaagatg acgagtcgga aatcattaag
gacatccaaa ccaaagaaaa tgagattaac 180gcactaagag acaagttatg
tcatgttcgc aaatcaaagc agttgaaatt ggaatcaaac 240aaatctatgg agaag
25589255DNAMethanobrevibacter ruminantium 89atgacaattg gaagtttgga
caattttgga aagtcagatg gagaaaacat gaaccctgaa 60gatttcgact gttctgtttt
ctttgaaatg tacaaggcac tttttgagat attggatgtt 120gaagttggca
gcttcgctga actcttggat gtgtacaaga atgtcgagat ggattacacc
180ttgaagagac atgccctcaa gcagaaggaa atcctttact ggttcaatac
cgactggaag 240gaggaacttg gaaag 25590255DNAMethanobrevibacter
ruminantium 90atgaatgtga aaacagtcat gaatgacctt ataggattgt
caaaggagtt cgagggagtg 60gaatatgaga ttgaatccaa aaattcaatc tacttctatt
cctttccaaa gtacatgaag 120gaaggcattg tgattctgaa atattccgca
atctatgacc tgcacaccat attgaaaggc 180atggatggaa tcatagtgga
cattctggag gttgaggaca atcctggtga tgagaagaag 240gatttgctat atgtg
25591234DNAMethanobrevibacter ruminantium 91atgattagcg acgaatggga
agaggaatac tatgtcaagg tcaacgagga actggagcag 60gtggaagtca ggtttttcag
caaggtcgac aggctcgtct ttgctcaatc ctactcttct 120tcatcttcct
tttcctttga ggaggctgaa atcctgtgcg acaggatatg cgacatactg
180acaaacgatt tgggaaatgc caagtattat ctaggaggaa aagatgaatt atag
23492150DNAMethanobrevibacter ruminantium 92atgaattata gggaatgggt
tgcaagccag ttcgagctga ggatgactga ggacggaagg 60gtttgcttca agacaccatg
cagctactac acattttcaa aggaggactt tgagataata 120agggagatgt
tccttaattt cgagtcctga 15093174DNAMethanobrevibacter ruminantium
93atggatagaa taaaggaatt gaatgtatgc ggaacctgca agcacagcca tttgattcca
60gacatcaacg gagagatagc agtgaacatt tgcaggatag gttcagaggc tgtcaacaag
120gacggaggac ttacatattg cgtagactgg acaccaagga ggaagctgtc atga
17494336DNAMethanobrevibacter ruminantium 94atgttaagta aaaaagaagc
catacagatg acgctggaca atgagaagca ctatcctgtt 60aagtgcaagt actgcggaaa
gccgttcacc aagtctcaca acaggcagat gtactgttca 120gacagttgca
gacggaatgc cttgagggaa cagaaggcaa gataccaggc taaaaggagg
180ctaaaaataa agcagaaagt gctgattgta gatgaataca agaaatacgg
tttgggtagc 240tatggaacaa gtgcgaacgg acacagaaag aacaattttt
cacatgagta catggctatt 300caaaaggaga tgaaaagaat aggattgaag agataa
33695261DNAMethanobrevibacter ruminantium 95atgtatttgg ctaaattttg
tcctaactgt ggaaataaag tagaagaaaa tgataaattc 60tgcatttatt gtggaaataa
actaagagtt ataattcctg aaaaaaaagt gaaaagaagc 120tcaaatagta
ttaatgatga aaaaactagc aaatatgttg aagtcattga tgggctaatg
180agatataaag tttttccatc aagtttacct gtgaaatata ttatttataa
agtgaattat 240ggaacaacat ctgatgaaat a 26196234DNAMethanobrevibacter
ruminantium 96atgatttatg ataaagcgac agttacaagc ctcatagtgg
ctatcctatt gccattgatg 60agcatgttag gaatcggaga attgactcag aactacatat
tggcaatagt gagcggtatg 120atagcccttg tggtttggta ctacaacgag
aagcacaaca gcgatttggt gagcggaacc 180accaagtgcg actgcgagct
ttgctatggg ggagacgatg aagcacttat atga 23497255DNAMethanobrevibacter
ruminantium 97atgaagcact tatatgagat aatcccttac aggaggactg
tttggattac agggttcctg 60aagacaacag tctcctcggc aatgataacc actggcgtag
tgatattgtt caacagcata 120accgagcacc cttacttcat ggagtgggac
gagataggaa tcgttcttgg aatcgtttcc 180atcaccatcg cctgcattta
catagcgatg atagacagat ggaaggaacg caggaagaag 240gaggagcttg acacg
25598255DNAMethanobrevibacter ruminantium 98atgatagaaa tcagcaccat
aaagatcacc gacataaagc ctgccgaata caatccgagg 60ataatgagcc agcttgaaca
cacaaagctc aggaactcca tggagacatt cggagtggtt 120gacccaataa
taatcaacct gaagaacaac cacatcatag gagggcacca aaggtacgag
180gtccttctgg acaagtccat ggaggacaac gagttcataa aggagctcca
cctcatcagg 240cttggagatg taggc 25599330DNAMethanobrevibacter
ruminantium 99atggacgtcg acaacctgct gggatatgac aaggtactgg
aattgaggtc tttattggag 60gaggtcacgg acaaggtgat acccgtatgg cacaagaacc
gcggaatcaa ggacttcaag 120cagatgtgcc aggactacaa tttcgtcagc
ataagcggct ggagaaacga ggacgtcaag 180gatgaccagt tcatccattt
cgtcaggcat gcacacagga acggctgcag gattcacgga 240ttgggattga
cacggagaaa ggttctcgac agggttccct tcgacagcgt cgacagcagc
300agctggctcc agaccatatt gtatgcaagg 330100255DNAMethanobrevibacter
ruminantium 100atgattaatg aaaggataag accttatttg aattttagct
tcaatgacaa gaaagtcatt 60ttcgtcacat tgttcgttgt gagcaatctg ataagcaacc
tgttagccat caaggttttc 120aacttgggat tttggggatt gacaaccgat
tgcggaaatc tcctgttccc gttaggatac 180cttatggcag acgtgattac
agaagtctat ggagagagga cagctcgaag ggtcatattg 240cttgggctct ttgca
255101255DNAMethanobrevibacter ruminantium 101atgcccgaac cttgggaaag
gcaaagggac gagaacggaa agcttgagcc aataaaggca 60ttcgagtact tcaccgagta
cctgacaatg gacaagccac gaagcatgag ggttctgtgc 120gagaggctcg
gcaagaaaga tgggtatatt aggcaacttc atgcctactc atccacatgg
180aactgggtcg aaagggcaga ggcatacgat gaacacataa tattgaagaa
acggttaagg 240aaagagaagt tttac 255102255DNAMethanobrevibacter
ruminantium 102gtgaaggaca ttgtcaatca ttacggctac ctttcaccgt
acaagccgac catcaggtct 60gatagtaagg ctaaaaacaa gttcaagctc aatgagcctt
atcgtggaca gatgttgagt 120gcaggtgctg gcggttcaat catgggatac
ggtgcaggtc tccttattgt cgatgaccct 180atcaagaacg tcgctgaagc
cgaatccaag gtacgtcagg caaagctcaa ggactggtgg 240ggaggaacca tcaag
255103273DNAMethanobrevibacter ruminantium 103atgagtaaga aacaagaaat
gatgagaatt gaaaggttaa aacactatgc atatcaaaca 60ggactaataa tccctatttt
taaaaatcat gatttaatta aaaaaataga aaatggaaaa 120ataactaata
ctgacgaaat aaagatatat attgaggaaa atgaaaagca gataaaaaga
180agaagagaat ttataagtat tatttatgat aattgcaaat attttaaata
cgacagcata 240tgttataaat taatatccaa agtaaataat ttt
273104255DNAMethanobrevibacter ruminantium 104ttgagtaaga atgctaaagc
agacgccttt gttgtgacca ctgaagacgg atcctatgac 60attgtcgatg cagatgtctt
ggagaggtat gcaataaaga gcgaatctga cgagacagga 120agcaagcagt
tgaagactga tggttgggaa tatgatgata cattgcttga gccgttgtat
180gacccattgc agttgtgtga gctattggag ataaacacat atcatgagaa
ctgtgtcgat 240gttgttgcaa gggac 255105252DNAMethanobrevibacter
ruminantium 105atgatggcgc tcaaggtgcg tcattcaaag aggcagattc
agaatatcaa gagagaatac 60agaaggcgtt tgatactcga agagcagtgc agtagggaga
ttgcggactt cttcagaagg 120cttgagagga aaatccacaa ggtcatggat
gagcactggg aaagcgaatt gggtcttttc 180catctgaaca aggtttcaga
catcatacag gacagcaggc aggagtacta tgacatcctg 240ttcaagtact gc
252106255DNAMethanobrevibacter ruminantium 106atgataattg aaattcctgg
aaatgacagg acagaagaaa taaatttgcc gaacggacag 60tttgtactga taacctactt
gcaggaaaat gacatgatgt cattgcctga cgggaagtat 120gtctgcccat
tcagaatgat tcagcttttc actgaagagg gcggtgaact gattggagag
180tgcattgagg aaaatcctca ctacaacact cgcttctaca atactgtctt
tgagcatttg 240gatgaaggaa ttgag 255107255DNAMethanobrevibacter
ruminantium 107atgttaagag tcgtagagag aacctattat caacaagaag
aaatcaagac actagactgt 60cgaattagag aggcaggagt caacacttac tcccttgctc
gtcaaggagc agttgactat 120cctacctacg aatcctacaa cgaggtgaga
gaggaaagga taaaagaggc taaggaaaaa 180tacggtgaat cctactacta
tcactggaga gacgttgaga cctacttcta ttacttagga 240agattcttta gtgat
255108255DNAMethanobrevibacter ruminantium 108atggacgcga taaacgttat
caaccaaaac aagatattag tagatgtttt gtaccgtggg 60actgtaaacc tgattgacat
cgtcatcggt gatgcattgg tctatgacaa tccgacagtt 120gtagtcaagt
gctacaccac agatttggct tttgcaacca cagagattga tgagattgta
180ttagagaacg aagaatttga gttattaata acttacgagg acggagagtt
ctcaattttg 240ttgaagtccc acaat 255109255DNAMethanobrevibacter
ruminantium 109atggtttttg tcgatttgtg cgagaacgaa atagcagaca
tggtcgagag cttctatcgc 60aatggagacg gtagcagcag gatattgacc aacagagcag
ttggagagat agtggagcac 120tactgttcct ttgaagtaga cggtagaatc
accactaatc tccgagattt cctattatat 180agtgttgtct tatatgacac
cattggagag gctgtcgatg acggagtcaa ccttgaggaa 240gtgttcatca tagaa
255110255DNAMethanobrevibacter ruminantium 110gtggtgaatt ggttgaaggc
tattggagat aacttctcag tggattatct ccttttagca 60ttattctcta gtggagattt
gatattggtt gccatcgttc tcaattctta tggtgtcatc 120tcacctgaga
atgtaaggga actggtgatt gactatataa gttacaggaa agttgatatt
180ttttggagac atctaaggag acctagaatg agttttgaag attatgtctt
agacaatttt 240gaagagatgg aaact 255111192DNAMethanobrevibacter
ruminantium 111atgacaatga aagttacttt tgaagatgag aacggtgaga
gaacagtcga gttcggcgat 60gacgtcgatt tcgttttaat cgaaagcgat gatgacggaa
acattgagat tcgcgaagga 120gactgggaat tggacggaga ttccgacgat
gattgggaag agtacgatga ctgggacgag 180gaggagttct ga
192112279DNAMethanobrevibacter ruminantium 112gtggacgaat tcgttgagac
cctgttcgac acctactgga aggtgaatga gaacggagag 60tacatgagcc ttaccgattg
cggtgatttc tacatcgcaa aggttgctcc ctgtgttcgc 120aactggagca
tagtgataga atgcaattgc ttctgttttc attgcaagga atttgtctac
180catgagaacg gagcaatact tgagataggt atggagatta gctctcttta
cctaagccaa 240atggagataa aagatttgaa gatatacatg atagattgt
279113255DNAMethanobrevibacter ruminantium 113atgtttttgg aaggaatatt
tgaacaagac ggagagaatg taagagaaca ggtgatttac 60tggagaaagt ccaatcaggt
acacaactgg ttcgtagtca acgctcagga cggagaggac 120aactgccaac
cacactcagt aagcagagaa cagttagagg aactaagaga cctatgcaga
180gcagtgctcg cagacaatga caaggcagag gaacttctcc caacaagacc
tggtttcttc 240tttggtgcga tagac 255114174DNAMethanobrevibacter
ruminantium 114ttgcagttgg tggtagaagg tgagaatatg gaatgccctt
gcgataattg tgagatgtta 60agagagaacg aaccaatcaa ggtgattgac tggaagtcca
attctccatt tggaaatggg 120atttktattc acttttggag ggaacggctt
caggatgttg gctataatgg gtga 174115255DNAMethanobrevibacter
ruminantium 115atggaactga tgactaggga aatggaaggg aaactcaaat
ccttcccttt ctattcccaa 60gacggaaaag gagatgacgc gatagtggtg atgaagttct
tcaaccctta cggattggga 120acttggtatg tattggaggc agagaaacag
gagaatggtg attacctttt cttcggatat 180gttgaatcac cgataacacc
tgaattcaac gagtacggtt acttctcatt gtctgagttg 240gagaacctta agata
255116225DNAMethanobrevibacter ruminantium 116atgttcggac agaagaaaga
atttgtcaaa aagatgtatc atgtcggaga cgttgtcgaa 60ctggttcata tggacgatgc
acaggctcca cctagtggaa ccagaggaga aatactcttc 120gtagacgata
ttggtcaaat ccatgtgaga tgggagaacg gctcaggtct ggcactaata
180tatggtgagg acaggttcaa agttgtagag aggaaaggag aatag
225117153DNAMethanobrevibacter ruminantium 117atggatttgt tgtatcttta
tgatgacctg actgcaagga gagaagtcta cgattcagtc 60ggattgagtt ttgtggtcaa
gtataaattc tcatctcgta aggaagcaca ggatttcgca 120ttgaagtacg
gtgcagaact gattgaggaa tga 153118189DNAMethanobrevibacter
ruminantium 118atgattgaac ggaggagatt aggaatgaag tacgatattt
ttactatctt ggatgaaatt 60tcaaggaagt tagatgacgg tgaattatcc gatgagcagg
ttgatttcct gttgcagatg 120gagattctag tagaggaagg cactatcaca
gacgagcagg cacaagatgt catgaatgga 180gattattaa
189119168DNAMethanobrevibacter ruminantium 119atgatgaaat taagtttgaa
ggaactcggt gaggaaattg agatgatact tgcagaaggt 60ggattaacct ttgatcaagt
cgattatctg ttgtacttgg agacctgcat tgcagacgga 120agcataacag
aggagcagaa gagagaaatt atctgtaggg acttctaa
168120234DNAMethanobrevibacter ruminantium 120ttggtgagag aacaggagag
attgattatg tatttggaaa ttgacgaggt caagtgcgaa 60aacattgaca gaattgagtt
tgacgatgtc gcaatggaaa tcgtcctgac agacgaaaag 120gtgtacgaga
gaataaagag atggctcaag tccaatgaga ttgactacga ttgcagggaa
180gaccggtatt tcgccaatct catagaatat gtcattagga taacttggtg gtaa
234121216DNAMethanobrevibacter ruminantium 121gtggtaaccg tgagagatgt
cggattcacc attgaggaac ggttcttcct tactgctcag 60gaattggagt attccgaagt
gggagaggaa cacgagagcg tcattgacag agccatcgca 120ttgctgtaca
cgaagctcag aaccagagac ttcgagttca ccgacgaaga acgggaactc
180ttggaggatg cttttgtcat tgtcagcgac caatag
216122255DNAMethanobrevibacter ruminantium 122atggctaaag aaaatgttat
agattataag attgagcgac aaaatgataa cacttggagt 60tatttatatg taactgaaag
agggagagga aatatcattg cttcatcttt tggagaattg 120aggcagaaag
ttttaaaaag aggtttgcct tggaatgata tatctaacct attcactaaa
180aagagttctg attctaatgt tcgtcgcgaa tctgtaaaga atattgatga
tgaatctgtt 240ttggctgatg tcgct 255123255DNAMethanobrevibacter
ruminantium 123atggtagtca aatgtcctaa ttgttttagt ccacgtgttt
caaaatgtga agatactaat 60attaaatggc aatgtgataa atgcaaatgc aaatttaacc
atggtgcttt cgatattaat 120gctgaaatgg agaaagtaga acaattaaca
atagaaaaaa ttgaaagaga acgagaaaga 180actgaacaat ttgaaagagc
tattaaagaa gcaaaggaac aatttgaaag agaaagaact 240gaacaatttg aaaga
255124255DNAMethanobrevibacter ruminantium 124ttgaccgagg gtcagataag
gcagattgca catgagtatc tggcaaatta cagccttgtc 60gacaagaacc atgagttctt
cgagaccaga gaagttatag gagttcctgt ggagtcatat 120ataacaaacg
agcctataag cctcaagggc ttggacggaa cagtcaatga gtatccaaaa
180ggaacctgga tagccaccac aaggataact gatgaagagg agatggaaaa
ggcactcaat 240ggagagtaca ctgga 255125330DNAMethanobrevibacter
ruminantium 125atgggaaacg aagctacttt aaaccaattg gtaaacgagc
aggaaaaggc agtcttcaag 60tccatgagga ctgacatgga gacaggaaag gctgtattga
acgtcgagca gctaggttac 120ttccttagag aagcaacatt agacaataca
attttaagag atgcggactt caagctgatg 180aaatcattca agaagcatct
caacagggta ggaataaacg gaagggtcct cacaaacgga 240tatgacgtga
acggcgagac cgaccctgag attcctgcgg ctgacgtcga cttcggagca
300aacgagttgg acgtcaagaa gctcaaggca 330126255DNAMethanobrevibacter
ruminantium 126atgcggaggc gttgcctgaa ctctccagag cataatggta
tgatttcgca tatcattgtg 60cttctcattt gctttatagg attggttgag gcgatactga
tggcattggt tgattgggag 120gacttggcaa tatccgttcg caagtctcct
agaaagcttt ataatgtttt gaaggatgag 180ttaggtcttc ctgaatggaa
cgaactgtct gtgattgaaa ggagaagcat gaagaaaagg 240tatgctgtca taaga
255127255DNAMethanobrevibacter ruminantium 127atgacttgga ttggtacgga
agatgtcatc gagttcacag gagtcaagcc tcagacattc 60aggttcgaga agggagacac
ctccagcctg gaaacattgc ttgagaagtg gattctgcag 120gcagaaggac
ttataatatc ctactgcaac tacgatttca atgacttgga ggagatacct
180ccagcagttg ttaatgtctg ccttaggctc actgcaaaca tggtcgcatt
ggcacaggca 240aggaaggaca ctcct 255128258DNAMethanobrevibacter
ruminantium 128ttggtgaaat tgcagattga cgttgaggaa ctcaagccat
tggagccaag gttcaaaaag 60gttgccaaaa ggacagttgt gttgactgca aatgaattgc
agagaaacct caagaagttg 120agtcctgtgg atcatggaag gcttcagggc
tcttgggtaa tcttccagac aggagaattg 180gaaaggactg tgaaaagcag
tgcaaagtat gccattttcg tgaatgacgg aacaggactg 240tacggtcctt tgggtcat
258129333DNAMethanobrevibacter ruminantium 129atgagattcg taaatactgc
ttcgcttgtt cctcagactg tcaaggcata tcttgaaagg 60gaaatctgcg aaggagggtt
gcttgaggat gtagagacac tcattccgtc cgtgaacagc 120gacgttcctg
ttgacccacc tgcgatatgg atagtccagc accccactac cagatggtct
180ggcagtcagc caaatctctc aaacaagata gctatgtcag tccctttcga
gttcgtatgc 240gtggaataca gcgatgactt ggaagaggct gagatattgg
gaataagcct agccagcagg 300gttggctcaa gcctgatgaa gaacttcaac aag
333130255DNAMethanobrevibacter ruminantium 130atgggaattc gtgttgtagg
aatgaaggaa gaggcaaggt atggagttgc ggagtcagcg 60ccggacttcc atcaggaagt
tagcaaggca aaggcttcct tgaactccac tccgaacaca 120aagtcaagcg
gctcaaggat gaagaagaag gcacgtgcag gcgtgtacaa gcctactgcc
180aacatcgaag gtgaagttga cttgaagagg ataggacatt atctcaaggc
tttcctggac 240aattaccatt ttact 255131255DNAMethanobrevibacter
ruminantium 131atggttgtag ttaagaaatc cgatatttta aagggcgtaa
aaaagattga aaaggtgaag 60attgaggctt tggacggaga cgagatgtac ttgagaccgt
tgtcccaagc cgagatcaac 120gaggtcgacg agattgaggc aaaggctatg
ggaatcttcg agaccaacga gaccgcacac 180aggggaagaa ggcagaagcc
taagagtgtg gttgagagca aaggaaagat aaatctcgaa 240ttgcagcaga aggca
255132255DNAMethanobrevibacter ruminantium 132ttgccgtcaa gcaatgtaat
gaacataata gtcaaggcag aggacatggc atcatctgtc 60gcccaaaagg ttgaaaacag
cttcaggaaa ttgggaaaca caatagacag caccttcacc 120acatctcttt
caaacaccaa gttcaatcag gaactcactt cttttggaac agacttggac
180aaggtaaccc aaaggctgaa gcaggtaggc gtgaacggtc agtccagctt
caaccagctt 240acaaatgctg aaagg 255133312DNAMethanobrevibacter
ruminantium 133atggcaagtg ttacaaaata tccgagcaac gtttcacaga
ccactggagg aaagttcgtg 60tccttcagca atctggcaaa cataaagaac aatgctgacg
gagcgcatgc cgtgagcagt 120gttcttatca aaagcaagaa gcagtctcca
aacagaccgt ccacagtctc atgcaaaggc 180ttcggattca gccttcctga
aggtgctgaa cctactaaaa tcacagtaac ttataggcat 240aggaagaatg
ctggaagcga ctacagctca aagaacaaga ctcacatatg caacatcgga
300ggacctacaa tc 312134255DNAMethanobrevibacter ruminantium
134atgggcatag ctattgttgt tatggacaat gaggagaact tcctgcaatt
ccttgaccct 60gatttatgca ccatcaatga gaccatagag gaattgggct tgaggacttt
ggagttcaac 120tacaagtttc aggactatgt tgaggacagg gatcttttca
ggataggtaa caagatctgg 180attagcaatt cccagagctt ggaggactgc
ctttatgtga taaacactcc tgtggagaac 240agcgtctatc aggag
255135255DNAMethanobrevibacter ruminantium 135atggtcgaga agattacagt
gagtcctcag gaagtcagag gatacggaaa tgttgttgat 60gagaaggaat tggaagatta
cggaagctac aggtgtgatg tgagcgagag ctcggaggtg 120atcaagggag
ttgaggaaag gatattcagc gtttctggtg ttcctgctcc tgctctgagc
180attgccaatg ttactgagga cactcgcagg ggcaggtgcg cccacatctc
cgcctcattt 240gaggatgggg aaggc 255136381DNAMethanobrevibacter
ruminantium 136atggttagat tcagcagaga catgctccag gacggagcga
agagaatgtt caagtggcta 60agaaagggcg aagggttgcc taactacttg ataatgtatg
acatggacag gaataaggag 120tataagttgg ttccaaagga atatgcagga
ctgtatgagt ccagaaacat attctggatt 180aagaacggaa gggagcctaa
ctatgttaca ctgacttccg ttgcaaggaa tcctcttgtg 240atggactacc
agaacaccaa ttacacctgt tgcccaacca gtttgtccct tgcctcacaa
300atgctatatc actataagtc agaaagtgaa tgcgctaagg ctttgggaac
tagcaagggc 360agtggaacaa gccctgcaca g
3811372250DNAMethanobrevibacter ruminantium 137atgaaaaaac
attgctttta ttttttaggg gacagttttg ccgatatatg caatgaagcc 60atgttttgtg
aaaaacattt agtagaagga aactatttgg attcaattat ccgtgcagga
120aaggcttcag agataataac tgtaaacatt tgtgaacttg aaggtcaaga
tggcttaata 180agctctggtc agaagaaaag attggaaatg cttggatata
agggtatcat ttcttatgat 240atttataaaa gattaaacca tattcgtaaa
attagaaata aggctgtcca tgggcattta 300agtgatattg aagacaatgc
aaatattctg catgcctatc tgtatctaat ttgcgcatat 360ttctataagg
aatatagaga cactaatttt tcagcggaag attatacggg ccctattatg
420gacattgcct ctaagcctaa agagactgct tcagagactt cagaggataa
tgaaaatatt 480ggagagttta tttcaagtcc attggatgat tatctttttg
aaaagtatga tgacagttac 540ctgttaaatg aactgtctaa acttaaggac
tcttcaaagg aagctgttga agacgataac 600ttaagcgaat ttaaggaata
tcttcatatt gacagatcta ttcaagaaga ttttttaaag 660gcattgaaca
gagccactag ttttaattca tctcatctga ttatgctttg cggtagtgtt
720ggagatggga aatcacactt gattgcaaat ttaaaaaaga aaaaccctga
actctttaat 780caatttgcta tccattatga tgctacagag agttttgatc
ctgaaaagaa tgcaatagac 840actttagcct cagtattgga acctttcaat
gacaataatc ttaacaattc gacagaaaaa 900cttattttag ccattaactt
gggtgtattg aacaatttcc tggaatcatc ttatgctaat 960gaagattaca
ctaaactcaa gttaattata gaagaagcga acatatttga atctaatgag
1020gtttcagata acatttatgg agataaggtt agttttgtca ctttcagtga
ttacaacatg 1080tttgaattga atgatgatga aaattcaaac tacacatcat
caaagtacat ctcttcccta 1140tttaataaga taacccaaaa ggaggataca
aacccttttt atgttgcata tctcaaggat 1200aaggactctc actttatcaa
tcctataatc tataattatg agatgctgat ggatgaggag 1260gtccagaaga
caatcattga ttatttaatc aagattttca taaaatacag aaaaatcatt
1320tcaactagag acttattgaa cttcatttat gagataatag ttcctccgga
attcctgaaa 1380agtgaggatt tggacaatat caatgatttc atggactatt
cattgcctaa tttactcttc 1440ggatatccag aaaggtcaga tttattgaag
ctatgcaatg aattggatcc gacattgcat 1500cgtaatgaat ctttagataa
gttcataata gacttgaaca ttaatgacga tactgaaaag 1560atattaaatc
gttattttga tttcactcgt tttaatttcc ttgaagagta tggagagtat
1620ctggttgatt ttagggagtt taacaattct gaaaaagaga aggtcaccaa
tattctaatt 1680aggtttgcag tattctatgg aaagagcatt atcaagaata
atttcaagga taaggtttat 1740ttaaattatt taaaatatct gtatgcctat
aatacacagt ctcataagga ttacaaatat 1800ctgttcactg aagttaagga
cgctattttt aattggaaag gttcctataa gaagaatact 1860atatgtattg
acactttgga ttcatttaaa gtgtataaaa atttaaaatt aaaaccatct
1920gttgataaat ttgaaaaaag tttattagat ggtctctttt taggaaatag
atttaaaaca 1980gatataaaaa tttatttttc agttgaatct aacaaaaaga
aaataccttt aaatgttgat 2040ttttcattat atcaatacat aatgaaatta
tataatggtt ttaagcctaa tcaatcagat 2100aaagacgatt taataatttt
agatgaattt attaataatt tattagatga agatacagat 2160gatgatttat
atgtaattag tttagaaact tatgaagaat ttttatttga gtcaaatgat
2220tttggcactt ttgaatttaa gaggggttaa
22501381372DNAMethanobrevibacter ruminantium 138atggattttt
cagaaaatta taatatatta ttaaaacaaa tgacttgtga tgtaaataaa 60aggaaattaa
tacatcagat taatcaaaat tctccattgt taccttttaa aactaatact
120cctaaaaaag ctaattttga aaatggtttt gatataatat tgggtgaatt
atccagaatc 180ttattaaata aaactattga aaaaaatttt aaattagata
atattgtttc aaatttaatt 240gataataata ttgaaattga agatggaact
aaggagtata taacaaaatt gctaaatgaa 300tatttattcg atgaaaaaaa
tgatttaaaa atatctcatc cgaatttgta tttatatatt 360cctctttcaa
ataataaaag ttctaatgga gaacaggaag tagcattgtt tttaagagat
420attttttgta agaataatca aaatcttatt aacttttttg aatcatatga
ttcaaatcac 480attatcttaa atttaatttt aaaaaatact cctaatttac
atcataaaat aactgaaact 540aagtatgtaa ttcattttga ggaaattgca
aatttattta atgaagatat caattatgca 600atactttata agaaattttt
tatggaaaac attggtaata tttttgctta ttactatttc 660ttttatatct
ctcaattaat tctaaaaatt tctaaaggct ttaatgataa taatgaattt
720gaaaaattgt actatttatt ggactgggaa tctgcaagta aaaatcgtaa
atcattaaat 780agttatagtt tactaaaaca tcattctaaa ccattatatg
caaaaatggc agtcatagat 840caaataaata cactattagg tacaaataat
ttattggaaa aagatatttc agaatatttt 900aataatttgg acatcaattc
taaaaataat tttttacatt ttcttaaaaa atgggtttca 960gattataggt
atgtaaggaa ttttgatgat aaagaattac cagataattt attagaatta
1020actgaaattc tttttgaaag tttaaaaaat gaaaaactgg gtgtggatgg
agctgtacaa 1080tctaggtacg ctttaaatct tgaagatata gctaaaaaat
atttgttaaa gcgtagaggt 1140tcgtatggtt atgtattaaa tattaataga
gatatgttgc ttgttttaac tgcattatgt 1200gttaaagata agaaaattaa
gttaaatcaa ctatttattg aatttgagaa aagaggagtt 1260tattttgaca
aatattcaaa agaagaagta gtaaattttt taactaaatt aaatttaatt
1320gacaagaaaa gtgatagtgg agatgctcaa tatgttaaac cagttttatg at
13721395223DNAMethanobrevibacter ruminantium 139atgttaaacc
agttttatga ttatctatct aataaattac taaattattt tgatgatact 60aagattttaa
gtggtgaaaa atttttcatt agttttgatg aagatgacca aataatgtct
120ttttataata gtttaagaag tattgcagaa actaattttt cttgttctga
attcatatat 180gtccatacta tttcaggaaa ggaatataat acttattcca
taaacattaa tggagtcaaa 240tttgtcattt ctgaaagttt aacaattaac
gtagatttct tagttacttt aagaaatcaa 300gttacttctc aagagggtgt
ttggaaggat actgcattat tagttatctg taatgaagca 360atagatagta
ttggaaaagg tatgagaaat ttacagaaag aaggaatgcc attaaatgta
420aaatctattt caaaaaattt agaggatgaa attaacgatt ctcaaattct
taactattcc 480gataagcaaa tcgctaagtt ttctttgaat attcaagaag
aagaattgtt tcaaacaaca 540ttatgggatt atgaaactat cttatcaata
atcaacaagg gttttgttag tgatgaagat 600ttaagagagt tgaatctttt
taaagatgac caactaaatc aaaattctcc tcaaaagatg 660ttaaaaagat
taaaagaaaa ttatgacaca tttaatgaag taaataaatt ttcacaatat
720ggagataaaa aagaacagct taaaaatatg tttactgatt ctggagtttc
tattttaagt 780aaagatgact ggtataaagc agaatggaaa atggttaaaa
aatcaaagga tgactttatt 840aatcaacaaa atcctttaaa ttacaatgaa
aatcttgaaa aaatcacaga aaatggctta 900aattattggg aaattcctaa
ctcatttact aaaactggaa aaagaaaacg aaatatcatt 960gtttttaatc
caaatcattc tcatgaagtt agtttaaagt tcagttttga tcagattctt
1020agtaattcat tccttaatac aaattcaaaa aaattcacta ttgcaagagg
caaatcatta 1080atagtcaatt ttactctgga ttctagtgaa ccaattttca
aaactataaa atataaacat 1140aaaaatgaaa atatctctga atttactttt
aatattgttg ttttgaattt tgagcctgaa 1200atttttaatt ctattaaatc
tcgttttagt gtcaatgtca aatctaaaca gattattgta 1260acaaatgatg
aggatagttt tgatattgtt tttggtactg gttcaaaaga aatagagaaa
1320ttaattaaag aaaatggtga aaagttatat ctttatgatg atgaatcttt
aataatttct 1380gaacaatctc ctgcttggaa tgatggaaaa ttaagtttta
aattatataa agacaataat 1440tatgccccat ttttgattaa agaaaaatct
aagaaaacac tgccagtaaa ttcctatgta 1500atttggaatt tgaaaagaag
aaatatggaa aactttattt ttaatggagt aaaagctgtt 1560caagatgtaa
acagtttcta tctcgtagaa gaatttaaag aatttctaaa aatggaacga
1620gaaatcatta aacaggacat attttatgct aaaaggaata ttgatggttc
tttagaaaaa 1680attgaagttt cattcagtaa cgagttagaa actgcttaca
tggacatatt taattattat 1740aagacttttg atgattctcc agaagacaat
cttccaagtt tagtttattt aaatgatgat 1800ttaaaagagt tatataagaa
atttataact atcttcaata aggaaatatc agaaattgaa 1860gagaattcta
ttttgtctga ttttaaatat aaaaagaatt tgcttaagtt aggtcgtatt
1920gaaactgata acaaaataat gtattctcca ttatctccac taaatatcgc
ttatcaatta 1980gaagtttcta aacaatgcgg aaatgaggat ttatccgtaa
atatattaga acggttagtt 2040ccaaataact taattcctta tatttgttca
gatgatggaa aagaattgtt tagacctatt 2100tatcaagaag aagctcatga
atggttgatt tatgagaaaa gtgaagaagt atcaattgga 2160acaacaaatg
tatttatttc taatgttgtt actgaaaaac ttaatcaatt tgtcaaacat
2220tttaattact tatttagttt taacaactcc tcaccaatta aaattaactt
aatcaatata 2280aaagatgata aagaagttgt gaaaggagta tttaatttta
ttagatctag attgcctgat 2340aaaactaaaa caaaaaaagt tattcctgtt
gaaatcaata tttacaacga cgctgaaaag 2400agttcttttg acaatttatt
tgattgtcag tctgaaattc agttactgga agaatttgga 2460attaaaaaat
taaaatcaga tatttttgat cctatagata ttatccatat gattcaaaat
2520aatatttcat attataaaca tccatttaaa aaagaagaat atgaatatgc
tcatctttct 2580ttctataaag tcaaatcaca taacaatatt gcaaatgaca
atatggataa aattgaaact 2640ggattatctt taaacggttt attatcttca
gttacatcaa ctactaaaca ttcagaatat 2700agaacgggtt tcggtacaaa
caatatttta aatatgagta atcctcttat taaaacagtg 2760attaacttga
acgaacttgt tgagaatagc aagaactttg gaaaaaatac ttattcaaaa
2820aataaatcag ttatcactac tgttgaatta gaagaagaca atattgaaga
attgtacgac 2880aaatctcatt gggtaacatt tattgaacct acatttggta
tcgaatactt tgacagttct 2940gatagtaatt tgatcattat tcattacagc
gatcaatata gttcctctag caagtatgat 3000actattactg taactaataa
atcaacacaa tatgaggaaa ttataagaga tttcctccaa 3060tctaaatatg
taaaagttac agatgaagaa ttgtatgatg taataaagat gtttaattca
3120attaatggtg aatggttact tagagtaatt tctaattccg gccattatga
tagggaaaaa 3180ttaagtatta tttctgctat taaatattgc ttatccattt
tggaccataa agatattgtt 3240tggattccag tttctatgga agaaatctta
agaattgcag gaaatgttaa attagataaa 3300aataaaggaa tttttgactc
taagttaata aaaggaaatc atagtgatga tctgctattc 3360attggtgtaa
aattcaatga agataataga atcgaagtta tattctaccc aatcgaagtt
3420aaaataggtt tgaataatgc ttcaactatt aaaaaaggta aaagtcaatt
agataatact 3480tataaacttc ttaaaactca actacaaaat attaatgtag
agaattctga atttagaaat 3540aaattcttta gaaatttctt catccaaata
ttattgtcca atgagcaaaa attagtaact 3600aatcatattt gggatgaaaa
aggattggat agaattgaag aattcaaagc agaactatta 3660aatgatgaat
atgatatttt atatggttta gaagagtaca ttggtaaagg ttctttggtt
3720tcatttaaaa aagaatctca tcacatctct atctatatgg atgttgataa
gcaagtaatt 3780gagttaccag aagattttgc ttattacggt ttagccactc
ctataagaga aatccatgat 3840gagattcagt cagacaatac agatattctt
gcagaaacat tgttatcaca tgtagatata 3900agtgaaatta gagcaaagaa
taatgatata tgcgattcaa atgaggatat gtcaatagat 3960gatgattttg
ataatttaag tgaatttgag gatagtttta ttgaagaaga atcagaaatt
4020tctgaagagc ctgatgaaga attaactgat gcaacttcaa gcagtgataa
tgaatcaatt 4080gagaatattg gagaatctcc ttctaaaatt tctaatgtca
gagcattaat tggaactcag 4140aagggttata atcataaagt ttattgggaa
tttggacatc cttctttagc aaataggcat 4200atgttaattc aaggaaaatc
tggacaaggt aaaacatatt tcattcaacg aatgttaaaa 4260gagttgtcta
ttcaaggcat tcctagcata attattgatt atactgatgg attcaagcct
4320tctcaattag aacctaattt taaagactct ttaggagata aaataagcca
atattttgtt 4380gtaaaagaaa atttcccaat aaatccattt aaacgaaaca
ctatcatgat tgataaggac 4440atatttattg aagaagacaa cagcactatt
gcgagccgat ttaaatctat cattaattct 4500gtttatgggc taggtattca
acagtccaat actttatatc aaacagtttt agattgcttg 4560gataagtatg
atgataattt tgatttgaat atcctaaaag aagaaattct aaaagacgaa
4620tcaaatagtg cacaaacagt cttgaataaa ttaaatgagt tattggataa
aaatccgttc 4680gcatccactg atttcgactg gtctgtctta gataataaag
acggtaaagt ttacattatc 4740caattaactg cactttctaa ggatatacag
acaattataa ctgaattcat cctttgggat 4800ttatggaatt acaaattaac
aaatggtagt gaagataatc ctttcattgt tgttttagat 4860gaagctcata
atcttgattt ttccaatgac tctccatgta gtaaaattct taaagaaggc
4920aggaaatttg gatggtctgg gtggtttgca acacaatctg ttaaaggatc
tatgaaaatt 4980gatgaaatag ctaaattaga gaatgcagat gagaagatat
atttccatcc aacggatgtt 5040tcaacaatag ctaaagattt gtcaaaagac
aatgaagata aaaaaatata tgaaaaagag 5100ttatctcaat tgacaaaagg
atattgtatt gttcaagggt ctgcaataga ttcaagtggc 5160aatttgtatc
agccaaatcc tgtaactgta aaaattgagg agataagttt tgatgaaaat 5220taa
52231403165DNAMethanobrevibacter ruminantium 140atggaaagta
aagacagaat agagaatact attttcaatg ataaaagaat ggatgcttat 60atagacaaat
attttgaaga gtttactctg agttctgaac aagaagaggc actgaatatt
120tggatagata aattaaataa cgatcaatta acaagtgaaa aagggaatta
tcataatttt 180tttgaaatta ttcttgaaga tttacttggt tataaacgtt
ctgatgttaa acatgaagag 240aatattggtg atgaaggcca tcctgtagag
tttgtattag aaaaagatgg aaaagattat 300gtgattatag aactaaaagg
aacaacttat aaagacctca ctaaaagaag acctggacag 360caatcaccag
tagagcaagc tacaaattat gctagtgcta aaaaagaaac tgaatgggct
420acagtttcaa attatgatga atttagattt tttaatccaa cagcaagaga
taattatatt 480tcattcaagt ttaggcaatt gaaagatttg gaaatcttta
aaaaattttt attagtattc 540agtaaatttt cacttatcga tgaagacata
cctaaaaaat tacttaatga aacaaaagtc 600attgaaagag aattagaaaa
tgaattttat caattgtata gtgacactag attaatgatt 660attaaggaat
tggaatattc ttcagaagat attaatagga ttgaagcaat aaaattatct
720caaataatat taaatagatt tattttcctt tgttttgcag aagatttagc
gcttatggag 780gaagagacaa ctgctgatgt attattaaca cctttaaagc
atagaaattt aatcggaaat 840acaatgtgga ataggttaaa tgaattattt
atttttgcaa atcaaggaaa taaacatagg 900agaatacctg catttaatgg
aggattattt gaggatgatt tatctaattt gaaaataagg 960gatgaaattg
aagataggtc tttctttgaa aattggaatt taaaagaaga ttttgaagac
1020aaatatgagg atattgctaa actaattgga gtttataaag atactcttaa
tccaattttc 1080attaatttat taataatttc tacatatgac tttgattcag
aacttgatgt aaatatctta 1140ggacatattt ttgaaaacag tattagcgat
attgaagagt taaaaaatga taatcaagag 1200caaagaaaaa aagatggagt
gtattacact ccagaatata ttacagatta tatttgtaga 1260aatacaatta
ttccatattt aagtatttct ggtaaagcca gcacagttca tgaattatta
1320tacgaatatg aatcatctaa ttcattggat gttttagatt caaaattaac
taacatcaaa 1380gttctagatc ctgcctgtgg tagtgggagc atgttaaata
aatcagtaga tatattattt 1440gaaattcatg aagcattaca tgcaagtaaa
tatgctggag attcatcttt agatagattt 1500tttgatagtt tagaaaaacg
aaaggaaatt ataagtaata atatttatgg ggttgatttg 1560aacgaggagt
ccgttgaaat tactaaatta tccttattcc taaaattagc aactactgtt
1620ggacttaaag aagggtttca acttcctagt ttagataaac atattaaatg
tggggattca 1680ttagtggatg atgagtcaat tgccggaaat aaagcattta
actggtatga atcattttca 1740gaagtttttg aaagtggcgg atttgatatt
attgtaggaa atcctccgta tgtggatatt 1800aaagaaatgg atgaaaaaac
agcaaaatac atatttgata attatgaaac ttcttttaac 1860aggataaatt
tatattctac atttgtggaa aaaagttatt atttgctaaa aaatgaaggg
1920attttttcat ttatcatgcc taattccatt ttatttaatt ctacttactc
taaaatcaga 1980gaactaattc ttaataatac atccatatta aatattgtaa
gaacttctga tgatgttttt 2040aaagatgcta aagtggaacc tattatttta
atcttcaaaa aaggatatga tgaaggaaat 2100aagactaaga tacttataaa
aaaagatgat atggatgaaa ttccaataaa taattattca 2160gaacatttct
ttacacaaga aagatggttt gaaaataatt caataattaa tattttttca
2220gatgatttta cttttgattt attaaagaaa attgatggaa ataatgaaag
attaattgat 2280tattgtgatt ttagtttagg tttaactcct tatgataaat
ataaaggtat gagtgaggat 2340attattaaaa atagaaaatt ccattcaaaa
ataaaacttg acgatacttt taaagaatta 2400ttggatgggt ccgatattac
aagatataat gtaaaatggg gcgaaaaaga atatattaaa 2460tatggggact
ggttaggagc acctcgggaa gaaaaattct ttaaaaatcc tagaatttta
2520ataagacaga ttttatctat agctcctaag gaatctcgaa aaaggatttt
tgccgcttac 2580acagaagaag aattatataa cgctcaaatt gcttttaatt
tagtattgaa agaaggcttt 2640gatgataaaa atttattgaa atatttttta
ggaataatta attctaaaat gatgacttgg 2700tattatgaag aaagatttat
ggataaaaat aaaaagaatt ttgcaaaaat tttgattgaa 2760aatgctaaaa
atcttccagt cattattaat tcaaattttt tagatgagat agtatctaat
2820gtggattcaa taatagagtt aaataaagag ttttatagtg taagaaatgc
atttcaaaca 2880tggcttaaaa tagaatttga aattgaaaaa ctctctaaga
aactagaaaa ctattatgat 2940ttaaactttg aagaattctt aaaagagata
aaaaagaaaa aagtagtcat tagaccaaat 3000caaatacagg acttgtccga
attatttaat gaaagtttag gaaaaataga atatctgcaa 3060agagagatta
aagaagcaga cgaaaaaatt aacctactgg tctatgaatt atatggttta
3120aatcatgaag aaatagaaat tatagaaaat agctttaatg attaa
3165141261PRTMethanobrevibacter ruminantium 141Ala Thr Gly Gly Ala
Cys Gly Ala Thr Gly Ala Ala Ala Cys Ala Thr 1 5 10 15 Thr Ala Ala
Thr Cys Cys Thr Ala Ala Thr Thr Gly Ala Ala Thr Ala 20 25 30 Cys
Ala Thr Cys Ala Gly Ala Ala Ala Cys Gly Cys Gly Cys Cys Ala 35 40
45 Ala Cys Ala Ala Gly Ala Gly Ala Ala Ala Thr Gly Gly Thr Gly Cys
50 55 60 Thr Thr Ala Ala Gly Thr Cys Ala Thr Thr Thr Gly Ala Ala
Gly Gly 65 70 75 80 Ala Gly Thr Gly Gly Ala Cys Thr Thr Thr Ala Thr
Ala Ala Gly Ala 85 90 95 Cys Cys Ala Ala Thr Cys Cys Ala Ala Ala
Thr Thr Thr Cys Thr Ala 100 105 110 Gly Gly Ala Ala Ala Ala Cys Ala
Gly Gly Cys Ala Thr Thr Cys Ala 115 120 125 Cys Cys Cys Ala Ala Ala
Cys Ala Ala Thr Gly Thr Cys Ala Gly Cys 130 135 140 Ala Ala Gly Ala
Ala Ala Thr Thr Ala Ala Ala Gly Gly Ala Thr Cys 145 150 155 160 Thr
Thr Cys Gly Cys Gly Ala Ala Cys Ala Thr Gly Ala Ala Cys Thr 165 170
175 Gly Gly Thr Cys Thr Ala Thr Gly Thr Cys Ala Thr Ala Ala Ala Thr
180 185 190 Cys Cys Thr Gly Ala Gly Thr Ala Cys Cys Ala Thr Gly Thr
Thr Cys 195 200 205 Cys Thr Ala Ala Ala Cys Thr Thr Thr Ala Cys Ala
Gly Gly Cys Thr 210 215 220 Thr Ala Cys Thr Gly Ala Ala Ala Ala Gly
Gly Gly Thr Ala Ala Ala 225 230 235 240 Ala Ala Thr Ala Thr Gly Cys
Thr Thr Cys Ala Ala Thr Thr Cys Thr 245 250 255 Thr Gly Thr Ala Gly
260 142255DNAMethanobrevibacter ruminantium 142atgaataaaa
aaattatctt atccctcctt ttagtattat tagtagctat ttctgtctct 60gcagttgcag
cagcagatgc tgatgtcaca tatataaacg atgctgcaga tgtagacgat
120gttgcagacg aaaaagttgc tcctcttaca gctagtgctg atgcacaaga
catccaaact 180aagcttgata atgctaaacc tggagacaca attgaattag
aaaacaagac atatgacgtt 240gatacaacat ttaat
25514340DNAMethanobrevibacter ruminantium 143tttttaattg attgtaataa
ttaattattc tgggtctgac 4014480DNAMethanobrevibacter ruminantium
144aaacctttat ggaagtcgag taagtattgg tacgtattat aagtataaat
acgtatccta 60tacttacttt attaaaattt 8014542DNAMethanobrevibacter
ruminantium 145cagaataagg ataataatta attatattgt ttttattttt tt
4214625DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 146caaagagaga ttaaagaagc agacg
2514725DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 147agtagtgttg gaatcagtga aaagg
2514825DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 148caccatggtt agattcagca gagac
2514925DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 149tcatgcagga cagacaacat agtag 251506PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
150His His His His His His 1 5 15139PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
sequence 151Xaa Lys Lys Lys Lys Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Ala 35
* * * * *