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.

No abstract

Antigens from methanogens are immunogenic in ruminants, and antisera from sheep vaccinated with fractions of methanogens have a significant impact on these organisms, inducing cell agglutination, and decreasing growth of methanogens and production of methane. Only antisera to selected methanogen fractions were able to achieve these effects. The results demonstrate the feasibility of a vaccination strategy to mitigate emission of methane.

Background: An enigma in the bioenergetics of methanogens is how the generation of proton and sodium gradients are used to synthesize ATP. Results: Purified methanogen ATP synthase was stimulated by sodium ions that also provided pH-dependent protection against DCCD. Conclusion:Methanobrevibacter ruminantium harbors an A-type enzyme with the ability to switch between sodium ions and protons. Significance: ATP synthesis by methanogens depends on the environmental conditions that prevail.

Background: Archaea synthesize glycerol-based membrane lipids of unique stereochemistry, utilizing distinct enzymology. Results: The structure of sn-glycerol-1-phosphate dehydrogenase (G1PDH), the first step in archaeal lipid synthesis, was determined. Conclusion: G1PDH is a member of the iron-dependent alcohol dehydrogenase and dehydroquinate synthase superfamily. Significance: The data contribute to our understanding of the origins of cellular lipids at the divergence of the Archaea and Bacteria.

Pseudomurein endoisopeptidases cause lysis of the cell walls of methanogens by cleaving the isopeptide bond Ala-ε-Lys in the peptide chain of pseudomurein. PeiW and PeiP are two thermostable pseudomurein endoisopeptidases encoded by phage ΨM100 of Methanothermobacter wolfei and phages ΨM1 and ΨM2 of Methanothermobacter marburgensis, respectively. A continuous assay using synthetic peptide substrates was developed and used in the biochemical characterisation of recombinant PeiW and PeiP. The advantages of these synthetic peptide substrates over natural substrates are sensitivity, high purity, and characterisation and the fact that they are more easily obtained than natural substrates. In the presence of a reducing agent, purified PeiW and PeiP each showed similar activity under aerobic and anaerobic conditions. Both enzymes required a divalent metal for activity and showed greater thermostability in the presence of Ca2+. PeiW and PeiP involve a cysteine residue in catalysis and have a monomeric native conformation. The kinetic parameters, K M and k cat, were determined, and the ε-isopeptide bond between alanine and lysine was confirmed as the bond lysed by these enzymes in pseudomurein. The new assay may have wider applications for the general study of peptidases and the identification of specific methanogens susceptible to lysis by specific pseudomurein endoisopeptidases.

Rotavirus-induced diarrhea is a common infection that results in the death of nearly 500,000 children annually. Currently, no large-scale preventative treatments or vaccines exist. Because some whey protein concentrates (WPC) were shown to contain bioactive ingredients that may activate immune cells and/or prevent infection, the current study was conducted to assess whether the proprietary WPC IMUCARE (WPC-IC) could protect against rotavirus. Suckling BALB/c mice were treated by gavage once daily with WPC-IC or with the control protein bovine serum albumin from the age of 9 to 17 d, and were infected with murine rotavirus at the age of 11 d. Disease symptoms were graded as mild, moderate, or severe, and viral shedding was measured in fecal samples during the postinfection period. Severe diarrhea occurred in 63% of control mice; this was significantly reduced to 36% in WPC-IC-fed mice. Severe diarrhea occurred for a 4-d period in the control group but only for a 2-d period in the WPC-IC group. Although the mean viral load per mouse did not differ between the groups, the proportion of mice shedding high levels of the virus in the feces postinfection was significantly lower in the WPC-IC group on d 13, 16, and 17, and significantly higher on d 14. Rotavirus-specific antibody levels in serum and gut fluid did not differ between groups. Thus, prophylactic treatment with WPC-IC may reduce rotaviral disease by decreasing the prevalence of severe diarrhea and by decreasing the time period during which severe symptoms and high viral shedding occur.

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H 2 and CO 2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a highthroughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.KEYWORDS methanogen, greenhouse gas, Methanococcus maripaludis, highthroughput, rumen, Methanobrevibacter M ethane emissions from ruminants are a significant contributor to global greenhouse gas emissions (1). In countries such as New Zealand, with a large pasturebased livestock sector, greenhouse gas emissions from agriculture represent approxi-