BackgroundMethane (CH4) is a potent greenhouse gas (GHG), having a global warming potential 21 times that of carbon dioxide (CO2). Methane emissions from agriculture represent around 40% of the emissions produced by human-related activities, the single largest source being enteric fermentation, mainly in ruminant livestock. Technologies to reduce these emissions are lacking. Ruminant methane is formed by the action of methanogenic archaea typified by Methanobrevibacter ruminantium, which is present in ruminants fed a wide variety of diets worldwide. To gain more insight into the lifestyle of a rumen methanogen, and to identify genes and proteins that can be targeted to reduce methane production, we have sequenced the 2.93 Mb genome of M. ruminantium M1, the first rumen methanogen genome to be completed.Methodology/Principal FindingsThe M1 genome was sequenced, annotated and subjected to comparative genomic and metabolic pathway analyses. Conserved and methanogen-specific gene sets suitable as targets for vaccine development or chemogenomic-based inhibition of rumen methanogens were identified. The feasibility of using a synthetic peptide-directed vaccinology approach to target epitopes of methanogen surface proteins was demonstrated. A prophage genome was described and its lytic enzyme, endoisopeptidase PeiR, was shown to lyse M1 cells in pure culture. A predicted stimulation of M1 growth by alcohols was demonstrated and microarray analyses indicated up-regulation of methanogenesis genes during co-culture with a hydrogen (H2) producing rumen bacterium. We also report the discovery of non-ribosomal peptide synthetases in M. ruminantium M1, the first reported in archaeal species.Conclusions/SignificanceThe M1 genome sequence provides new insights into the lifestyle and cellular processes of this important rumen methanogen. It also defines vaccine and chemogenomic targets for broad inhibition of rumen methanogens and represents a significant contribution to worldwide efforts to mitigate ruminant methane emissions and reduce production of anthropogenic greenhouse gases.

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Development of inhibitors and vaccines that mitigate rumen-derived methane by targeting methanogens relies on knowledge of the methanogens present. We investigated the composition of archaeal communities in the rumens of farmed sheep (Ovis aries), cattle (Bos taurus) and red deer (Cervus elaphus) using denaturing gradient gel electrophoresis (DGGE) to generate fingerprints of archaeal 16S rRNA genes. The total archaeal communities were relatively constant across species and diets, and were less variable and less diverse than bacterial communities. There were diet- and ruminant-species-based differences in archaeal community structure, but the same dominant archaea were present in all rumens. These were members of three coherent clades: species related to Methanobrevibacter ruminantium and Methanobrevibacter olleyae; species related to Methanobrevibacter gottschalkii, Methanobrevibacter thaueri and Methanobrevibacter millerae; and species of the genus Methanosphaera. Members of an archaeal group of unknown physiology, designated rumen cluster C (RCC), were also present. RCC-specific DGGE, clone library analysis and quantitative real-time PCR showed that their 16S rRNA gene sequences were very diverse and made up an average of 26.5% of the total archaea. RCC sequences were not readily detected in the DGGE patterns of total archaeal 16S rRNA genes because no single sequence type was abundant enough to form dominant bands.

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Enzymes of the gluconeogenic/glycolytic pathway (the Embden-Meyerhof-Parnas (EMP) pathway), the reductive tricarboxylic acid cycle, the reductive pentose phosphate cycle and the Entner-Doudoroff pathway are widely distributed and are often considered to be central to the origins of metabolism. In particular, several enzymes of the lower portion of the EMP pathway (the so-called trunk pathway), including triosephosphate isomerase (TPI; EC 5.3.1.1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12/13), phosphoglycerate kinase (PGK; EC 2.7.2.3) and enolase (EC 4.2.1.11), are extremely well conserved and universally distributed among the three domains of life. In this paper, the distribution of enzymes of gluconeogenesis/glycolysis in hyperthermophiles--microorganisms that many believe represent the least evolved organisms on the planet--is reviewed. In addition, the phylogenies of the trunk pathway enzymes (TPIs, GAPDHs, PGKs and enolases) are examined. The enzymes catalyzing each of the six-carbon transformations in the upper portion of the EMP pathway, with the possible exception of aldolase, are all derived from multiple gene sequence families. In contrast, single sequence families can account for the archaeal and hyperthermophilic bacterial enzyme activities of the lower portion of the EMP pathway. The universal distribution of the trunk pathway enzymes, in combination with their phylogenies, supports the notion that the EMP pathway evolved in the direction of gluconeogenesis, i.e., from the bottom up.

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Ruminant-derived methane (CH 4 ), a potent greenhouse gas, is a consequence of microbial fermentation in the digestive tract of livestock. Development of mitigation strategies to reduce CH 4 emissions from farmed animals is currently the subject of both scientific and environmental interest. Methanogens are the sole producers of ruminant CH 4 , and therefore CH 4 abatement strategies can either target the methanogens themselves or target the other members of the rumen microbial community that produce substrates necessary for methanogenesis. Understanding the relationship that methanogens have with other rumen microbes is crucial when considering CH 4 mitigation strategies for ruminant livestock. Genome sequencing of rumen microbes is an important tool to improve our knowledge of the processes that underpin those relationships. Currently, several rumen bacterial and archaeal genome projects are either complete or underway. Genome sequencing is providing information directly applicable to CH 4 mitigation strategies based on vaccine and small molecule inhibitor approaches. In addition, genome sequencing is contributing information relevant to other CH 4 mitigation strategies. These include the selection and breeding of low CH 4 -emitting animals through the interpretation of large-scale DNA and RNA sequencing studies and the modification of other microbial groups within the rumen, thereby changing the dynamics of microbial fermentation.Keywords: genome sequencing, methane mitigation, methanogens, rumen bacteria Implications Development of CH 4 mitigation strategies for ruminants without disrupting normal digestive function and reducing productivity is a major challenge. Genome sequencing is an effective way of gaining information on how rumen methanogens and bacteria interact and contribute to rumen function. This knowledge will be important when considering the design and implementation of ruminant CH 4 abatement strategies. Genomic information is already contributing to vaccine and small molecule inhibitor programmes that are aimed at reducing ruminant CH 4 emissions, but it will also underpin the analysis and comprehension of metagenomic sequence data sets, allowing the generation of testable hypotheses to gain a better understanding of rumen biology. IntroductionRuminants are foregut fermenters and have evolved an efficient digestive system in which microbes ferment plant fibre and provide fermentation end-products and other nutrients for growth of the animal (Clauss et al., 2010). Feed ingested by ruminants is fermented by a complex microbial community, which includes bacteria, ciliate protozoa, anaerobic fungi, archaea and viruses. The rumen is the first and largest foregut compartment and is the main site for microbial fermentation. The microbial ecology of the rumen has been the focus of numerous studies (reviewed by McSweeney and Mackie, 2012), but very little information is available regarding the microflora of the ruminant oral cavity and lower gastrointestinal tract. The rumen provides optimal conditio...