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Butyrivibrio hungatei MB2003 was isolated from the plant-adherent fraction of rumen contents from a pasturegrazed New Zealand dairy cow, and was selected for genome sequencing in order to examine its ability to degrade plant polysaccharides. The genome of MB2003 is 3.39 Mb and consists of four replicons; a chromosome, a secondary chromosome or chromid, a megaplasmid and a small plasmid. The genome has an average G + C content of 39.7%, and encodes 2983 putative protein-coding genes. MB2003 is able to use a variety of monosaccharide substrates for growth, with acetate, butyrate and formate as the principal fermentation endproducts, and the genes encoding these metabolic pathways have been identified. MB2003 is predicted to encode an extensive repertoire of CAZymes with 78 GHs, 7 CEs, 1 PL and 78 GTs. MB2003 is unable to grow on xylan or pectin, and its role in the rumen appears to be as a utilizer of monosaccharides, disaccharides and oligosaccharides made available by the degradative activities of other bacterial species. [ABSTRACT FROM AUTHOR]

Background: Ruminants are important contributors to global methane emissions via microbial fermentation in their reticulo-rumens. This study is part of a larger program, characterising the rumen microbiomes of sheep which vary naturally in methane yield (g CH4/kg DM/day) and aims to define differences in microbial communities, and in gene and transcript abundances that can explain the animal methane phenotype. Methods: Rumen microbiome metagenomic and metatranscriptomic data were analysed by Gene Set Enrichment, sparse partial least squares regression and the Wilcoxon Rank Sum test to estimate correlations between specific KEGG bacterial pathways/genes and high methane yield in sheep. KEGG genes enriched in high methane yield sheep were reassembled from raw reads and existing contigs and analysed by MEGAN to predict their phylogenetic origin. Protein coding sequences from Succinivibrio dextrinosolvens strains were analysed using Effective DB to predict bacterial type III secreted proteins. The effect of S. dextrinosolvens strain H5 growth on methane formation by rumen methanogens was explored using co-cultures. Results: Detailed analysis of the rumen microbiomes of high methane yield sheep shows that gene and transcript abundances of bacterial type III secretion system genes are positively correlated with methane yield in sheep. Most of the bacterial type III secretion system genes could not be assigned to a particular bacterial group, but several genes were affiliated with the genus Succinivibrio, and searches of bacterial genome sequences found that strains of S. dextrinosolvens were part of a small group of rumen bacteria that encode this type of secretion system. In co-culture experiments, S. dextrinosolvens strain H5 showed a growth-enhancing effect on a methanogen belonging to the order Methanomassiliicoccales, and inhibition of a representative of the Methanobrevibacter gottschalkii clade. Conclusions: This is the first report of bacterial type III secretion system genes being associated with high methane emissions in ruminants, and identifies these secretions systems as potential new targets for methane mitigation research. The effects of S. dextrinosolvens on the growth of rumen methanogens in co-cultures indicate that bacteria-methanogen interactions are important modulators of methane production in ruminant animals. [ABSTRACT FROM AUTHOR]

Methane emissions from agriculture represent around 9% of global anthropogenic greenhouse emissions. The single largest source of this methane is animal enteric fermentation, predominantly from ruminant livestock where it is produced mainly in their fermentative forestomach (or reticulo-rumen) by a group of archaea known as methanogens. In order to reduce methane emissions from ruminants, it is necessary to understand the role of methanogenic archaea in the rumen, and to identify their distinguishing characteristics that can be used to develop methane mitigation technologies. To gain insights into the role of methylotrophic methanogens in the rumen environment, the genome of a methanogenic archaeon has been sequenced. This isolate, strain ISO4-H5, was isolated from the ovine rumen and belongs to the order Methanomassiliicoccales. Genomic analysis suggests ISO4-H5 is an obligate hydrogen-dependent methylotrophic methanogen, able to use methanol and methylamines as substrates for methanogenesis. Like other organisms within this order, ISO4-H5 does not possess genes required for the first six steps of hydrogenotrophic methanogenesis. Comparison between the genomes of different members of the order Methanomassiliicoccales revealed strong conservation in energy metabolism, particularly in genes of the methylotrophic methanogenesis pathway, as well as in the biosynthesis and use of pyrrolysine. Unlike members of Methanomassiliicoccales from human sources, ISO4-H5 does not contain the genes required for production of coenzyme M, and so likely requires external coenzyme M to survive. [ABSTRACT FROM AUTHOR]

Methanobrevibacter millerae SM9 was isolated from the rumen of a sheep maintained on a fresh forage diet, and its genome has been sequenced to provide information on the phylogenetic diversity of rumen methanogens with a view to developing technologies for methane mitigation. It is the first rumen isolate from the Methanobrevibacter gottschalkii clade to have its genome sequence completed. The 2.54 Mb SM9 chromosome has an average G + C content of 31.8%, encodes 2269 protein-coding genes, and harbors a single prophage. The overall gene content is comparable to that of Methanobrevibacter ruminantium M1 and the type strain of M. millerae (ZA-10T) suggesting that the basic metabolism of these two hydrogenotrophic rumen methanogen species is similar. However, M. millerae has a larger complement of genes involved in methanogenesis including genes for methyl coenzyme M reductase II (mrtAGDB) which are not found in M1. Unusual features of the M. millerae genomes include the presence of a tannase gene which shows high sequence similarity with the tannase from Lactobacillus plantarum, and large non-ribosomal peptide synthase genes. The M. millerae sequences indicate that methane mitigation strategies based on the M. ruminantium M1 genome sequence are also likely to be applicable to members of the M. gottschalkii clade. [ABSTRACT FROM AUTHOR]

Methanosarcina species are the most metabolically versatile of the methanogenic Archaea and can obtain energy for growth by producing methane via the hydrogenotrophic, acetoclastic or methylotrophic pathways. Methanosarcina barkeri CM1 was isolated from the rumen of a New Zealand Friesian cow grazing a ryegrass/clover pasture, and its genome has been sequenced to provide information on the phylogenetic diversity of rumen methanogens with a view to developing technologies for methane mitigation. The 4.5 Mb chromosome has an average G + C content of 39%, and encodes 3523 protein-coding genes, but has no plasmid or prophage sequences. The gene content is very similar to that of M. barkeri Fusaro which was isolated from freshwater sediment. CM1 has a full complement of genes for all three methanogenesis pathways, but its genome shows many differences from those of other sequenced rumen methanogens. Consequently strategies to mitigate ruminant methane need to include information on the different methanogens that occur in the rumen. [ABSTRACT FROM AUTHOR]

Background In silico, secretome proteins can be predicted from completely sequenced genomes using various available algorithms that identify membrane-targeting sequences. For metasecretome (collection of surface, secreted and transmembrane proteins from environmental microbial communities) this approach is impractical, considering that the metasecretome open reading frames (ORFs) comprise only 10% to 30% of total metagenome, and are poorly represented in the dataset due to overall low coverage of metagenomic gene pool, even in large-scale projects. Results By combining secretome-selective phage display and next-generation sequencing, we focused the sequence analysis of complex rumen microbial community on the metasecretome component of the metagenome. This approach achieved high enrichment (29 fold) of secreted fibrolytic enzymes from the plant-adherent microbial community of the bovine rumen. In particular, we identified hundreds of heretofore rare modules belonging to cellulosomes, cellsurface complexes specialised for recognition and degradation of the plant fibre. Conclusions As a method, metasecretome phage display combined with next-generation sequencing has a power to sample the diversity of low-abundance surface and secreted proteins that would otherwise require exceptionally large metagenomic sequencing projects. As a resource, metasecretome display library backed by the dataset obtained by next-generation sequencing is ready for i) affinity selection by standard phage display methodology and ii) easy purification of displayed proteins as part of the virion for individual functional analysis. [ABSTRACT FROM AUTHOR]

Background: Enteric fermentation by farmed ruminant animals is a major source of methane and constitutes the second largest anthropogenic contributor to global warming. Reducing methane emissions from ruminants is needed to ensure sustainable animal production in the future. Methane yield varies naturally in sheep and is a heritable trait that can be used to select animals that yield less methane per unit of feed eaten. We previously demonstrated elevated expression of hydrogenotrophic methanogenesis pathway genes of methanogenic archaea in the rumens of high methane yield (HMY) sheep compared to their low methane yield (LMY) counterparts. Methane production in the rumen is strongly connected to microbial hydrogen production through fermentation processes. In this study, we investigate the contribution that rumen bacteria make to methane yield phenotypes in sheep., Results: Using deep sequence metagenome and metatranscriptome datasets in combination with 16S rRNA gene amplicon sequencing from HMY and LMY sheep, we show enrichment of lactate-producing Sharpea spp. in LMY sheep bacterial communities. Increased gene and transcript abundances for sugar import and utilisation and production of lactate, propionate and butyrate were also observed in LMY animals. Sharpea azabuensis and Megasphaera spp. act as important drivers of lactate production and utilisation according to phylogenetic analysis and read mappings., Conclusions: Our findings show that the rumen microbiome in LMY animals supports a rapid heterofermentative growth, leading to lactate production. We postulate that lactate is subsequently metabolised mainly to butyrate in LMY animals, producing 2 mol of hydrogen and 0.5 mol of methane per mol hexose, which represents 24 % less than the 0.66 mol of methane formed from the 2.66 mol of hydrogen produced if hexose fermentation was directly to acetate and butyrate. These findings are consistent with the theory that a smaller rumen size with a higher turnover rate, where rapid heterofermentative growth would be an advantage, results in lower hydrogen production and lower methane formation. Together with previous methanogen gene expression data, this builds a strong concept of how animal traits and microbial communities shape the methane phenotype in sheep.

Methanobacterium formicicum BRM9 was isolated from the rumen of a New Zealand Friesan cow grazing a ryegrass/clover pasture, and its genome has been sequenced to provide information on the phylogenetic diversity of rumen methanogens with a view to developing technologies for methane mitigation. The 2.45 Mb BRM9 chromosome has an average G + C content of 41%, and encodes 2,352 protein-coding genes. The genes involved in methanogenesis are comparable to those found in other members of the Methanobacteriaceae with the exception that there is no [Fe]-hydrogenase dehydrogenase (Hmd) which links the methenyl-H4MPT reduction directly with the oxidation of H2. Compared to the rumen Methanobrevibacter strains, BRM9 has a much larger complement of genes involved in determining oxidative stress response, signal transduction and nitrogen fixation. BRM9 also has genes for the biosynthesis of the compatible solute ectoine that has not been reported to be produced by methanogens. The BRM9 genome has a prophage and two CRISPR repeat regions. Comparison to the genomes of other Methanobacterium strains shows a core genome of ~1,350 coding sequences and 190 strain-specific genes in BRM9, most of which are hypothetical proteins or prophage related.