METHANOGEN GENOMICS

Genetics is the cornerstone of modern biological research and recent

advances in methanogen genome sequencing are providing insights
into the processes that underpin their cellular biology and ecological
functions. Methanogens are the rumen microbes responsible for methane
formation. Methanogens belong to an ancient lineage called the Archaea
and share many common features for potential inhibition, allowing them
to be targeted while not harming the other useful microbes in the rumen.
Methanogens occupy various niches in the rumen, some are free-living in
the rumen fluid, while others live on, or inside, rumen protozoa.
Using comparative genomics the unique
features of methanogens are being characterized and has identified a host of
anti-methanogen intervention options.
Methanogen have highly conserved genomes, meaning that many of their genes
are commonly shared by all (and only)
methanogens. Consequently this provides
methanogen-specific opportunities to restrict methanogens without impinging on
the function of other ruminal microbes
essential for the health and productivity
of the ruminant.
The sequencing of the Methanobrevibacter
ruminantium genome has identified a
number of key genes and enzymes and
these have been ranked as potential targets. Most of these targets are involved
in the methanogensis pathway within
the cell. These enzymes are common
between, and unique to, all methanogen
species. Targeting methanogens via the
methanogenesis pathway enzymes is a
very strategic option because, if successful, it will reduce methanogen numbers by
reducing their ability to produce energy
by forming methane and specifically target
the root of the methane problem.
Other potential inhibition targets lie on
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the outside of methanogen cells. Methanogens associate themselves with other

ruminal organisms probably via specific
surface proteins. Disabling these proteins
or preventing their binding could disrupt
these interactions and upset their normal
behaviour. The genome has also identified
additional cell-surface protein targets,
which have been used to design peptide
antigens for testing as possible methanogen vaccines.
The genome shows many potential methanogen-specific enzyme targets, for which
inhibitors may be found using chemi-genomics. This involves computer modeling
of enzyme structures and designing small
chemical molecules that can block the
active site of these enzymes, thereby
impinging on methanogen function.

Methanogen cells fluorescing

Methanobrevibacter ruminantium contigs scaffolded onto the Methanothermobacter thermoautotrophicus genome sequence, also
showing good alignment with Methanoshpaera stadmanae

The genome sequencing programme has

also thrown up unexpected opportunities. One example is the discovery of a
phage sequence that resides within the
M. ruminantium genome. Phage are viruses
that infect microbes and kill them by
breaking open their cells. Some phage
infect and insert a copy of their genes
into the microbes DNA and reside there
until the conditions are right for them to
reproduce, kill the host cell and release
their progeny. Phage use special enzymes
to break open or lyse the microbial cells,
so the enzymes from the M. ruminantium
phage are of potential use as methanogen
control agents.

opportunities for controlling methane

formation. Additionally, information from
this research feeds into other research
programs by providing a range of protein targets enabling the development
of ruminant vaccines that could control
methanogen immunologically.

Overall, the sequencing and analysis of

the M. ruminantium genome is providing
new information on the lifestyle and
methane forming processes in this prominent rumen methanogen. Importantly, this
information could not be obtained using
conventional microbiological methods
and is presenting previously unrecognized
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Methanobrevibacter ruminantium genome

sequencing
Graeme T Attwood, W J Kelly, E H Altermann & S C Leahy
Food, Metabolism and Microbiology Section, Grassland Research Centre, Palmerston North, New Zealand.
Corresponding author: graeme.attwood@agresearch.co.nz

Background
Methane is produced in the foregut (rumen) 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.

Materials and Methods

Methanobrevibacter ruminantium was
grown on BY medium and genomic
DNA was extracted by freezing cell pellets under liquid N2 and grinding. 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. 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 and used to generate a large insert fosmid library. DNA
fragments in the 2 to 4 Kb range were
used to generate a small insert plasmid
library. Clones resulting from both large
and small insert libraries were grown and
sequenced using high throughput sequencing technology. Sufficient clones were
sequenced to give 8 x coverage of the
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M. ruminantium genome. Pyrosequencing

was also performed on randomly sheared
genomic DNA fragments and gave a
final 10 x genome coverage. The DNA
sequences from clones and from pyrosequencing were aligned to find sequence
overlaps and assembled into contiguous
(contig) sequences. Contigs were analysed to find open reading frames (ORFs)
and their predicted amino acid sequences
were compared to public databases via an
automated annotation system. Annotations were subsequently verified manually
and ORFs were categorised by function
using the orthologous proteins database.
Motifs within protein sequences were
determined by Hidden Markov Modeling against public protein databases, and
metabolic pathway reconstructions were
carried out using the Kyoto Encyclopedia
of Genes and Genomes on-line database
using in-house developed software.

Results
Approximately half of the genes identified within the genome have no known
function. However, all of the components
of the methanogenesis pathway have
been identified and comparison of these
gene sequences with those from Methanobrevibacter smithii, Methanobacterium
thermoautotrophicus and Methanosphaera
stadtmanae indicates methanogenesis
gene organisation is conserved within the
Methanobacteriales. The M. ruminantium
genome contains a prophage sequence
(designated mru) with distinct functional

modules encoding phage integration,

DNA replication and packaging, capsid
proteins and lysis functions. A low GC
region of the phage, harbours a putative
DNA restriction/modification system
which might provide additional protection against foreign DNA. The genome
also contains many large surface proteins
with characteristics that indicate that
they may mediate association with other
rumen microbes. Analysis of nucleotide
repeats revealed the presence of at least
two Spacer Interspersed Direct Repeats
(SPIDRs) regions in the genome. SPIDRs
are usually less than 40 nt and are made
up from identical units separated by
heterologous sequences. The biological
function of these SPIDRs is unknown, but
they may be a functional analogue of the
eukaryotic small interfering RNA system.

Conclusions

this organisms lifestyle within the rumen.

Its dependence on simple substrates (H2 +
CO2, formate) and its interaction with the
rumen environment via surface proteins
and exopolysaccharides are potential
targets for inhibition. Similarly, the mru
prophage sequence and SPIDRs hold
promise for both specific inhibition of M.
ruminantium and for future genetic manipulations to assist in determining gene
function. Understanding the metabolism
of this organism and how it interacts with
other microbes will identify conserved
features among methanogens that may be
inactivated to prevent or reduce methane
formation in the rumen.

Related Publications
Attwood et al. 2007. Genome analysis of
Methanobrevibacter ruminantium: understanding
methanogen biology to inhibit their action in the
rumen. Aust J. Exp. Agric.Vol 48 (in press).

Although a significant number of the predicted genes within the M. ruminantium

genome cannot yet be assigned a function,
we are beginning to construct a picture of
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Future directions for methanogen genomics

GraemeT Attwood, W J Kelly, E H Altermann & S C Leahy
Food, Metabolism and Microbiology Section, Grassland Research Centre, Palmerston North, New Zealand.
Corresponding author: graeme.attwood@agresearch.co.nz

Sequencing the M. ruminantium genome

is providing new insights into the biology
of this organism and the information is
being applied in specific methane mitigation objectives targeting methanogen cell
walls and surface proteins via phage lytic
enzymes and immunological approaches.
However, around half of the genes in
the M. ruminantium genome cannot yet
be assigned a function. Furthermore, M.
ruminantium represents only one species
of a larger group methanogens known
to inhabit the rumen. Therefore much
remains to be learned about the role of
M. ruminantium and other methanogens in
methane formation in the rumen.
To begin to assign functions to M. ruminantium genes, we are investigating
whole genome gene expression using
microarrays. Microarrays are being used
to quantify gene expression during coculture with H2-forming rumen microbes
to identify methanogen genes involved
in the transfer and utilization of H2. This
process is central to methanogen growth
in the rumen so understanding the function of the genes involved may identify
new options for methanogen inhibition.
We will also investigate the expression
of mru prophage genes to determine if
the multiple gene insertions in the phage
genome have affected its function.
The M. ruminantium genome encodes 25
very large surface proteins that contain
repeat sequences similar to those found
in proteins from other gut methanogens,
Methanosphaera stadtmanae and Methano40

brevibacter smithii. These proteins are predicted to be membrane-anchored at both

ends with the central portion external to
the cell. Many of these externally exposed
protein regions contain DUF11 (domain
of unknown function) or C-POMP
(chlamydial polymorphic outer membrane protein) repeat domains which are
believed to be involved in cell adhesion
and in some cases act as virulence determinants.This suggests that M. ruminantium
has the capability to attach to surfaces
in the rumen, possibly on protozoa or
bacteria. Interrupting the association of
methanogens with other organisms or
surfaces in the rumen is likely to interfere
with their normal function, therefore
these distinctive cell surface proteins are
candidates as possible vaccine targets.
The genes and corresponding amino acid
sequences of each enzyme involved in
the seven steps of the methanogenesis
pathway have been identified from the genome sequence. We propose to combine
this information with published enzyme
structural data to model the structure
of each enzymatic step in M. ruminantium.
These models will define the enzyme
catalytic sites and will predict the type
of compounds that may interact with
and inhibit each of these enzymatic steps.
Using a chemigenomics approach, we
will screen libraries of small molecules in
bioassays based on these enzymatic steps
to find compounds that specifically inhibit
the methanogenesis pathway.

Fig. 1. Diagram showing position of SPIDR elements found in the M. ruminantium genome

more than 50 genes (glycosyl transferases,

other transferases, epimerases and transporters) involved in the synthesis and
export of exopolysaccharides suggesting
that it decorates its cell surface with
polysaccharides. This confirms previous observations that M. ruminantium
produces a capsule. The relatively large
number of genes devoted to surface
polysaccharide production suggests that
this is an important factor for M. ruminantium survival in the rumen and worthy of
further investigation.

region harbouring a cluster of associated cas-genes (see Fig. 1). Similar repeat
structures have been found in several
methanogen genomes, but their function
is unclear. One hypothesis speculates that
this system is a functional analogue of the
eukaryotic small interfering RNA systems
and represents a defence system against
foreign DNA that operates on the antisense RNA principle. Investigation of this
unusual genetic element is also proposed
as part of future work.

Nucleotide repeat analysis revealed the

presence of at least two elements known
as Spacer Interspersed Direct Repeats
(SPIDRs I and II) which are composed
of short nucleotide repeats separated by
unrelated sequences. SPIDR I has a unique
genetic arrangement which consists of
two repeat structures flanking a 17 Kb

The M. ruminantium genome also encodes

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