Search

The complex microbiota that resides within the rumen is responsible for the break-down of plant fibre. The bacteria that attach to ingested plant matter within the rumen are thought to be responsible for initial fibre degradation. Most studies examining the ecology of this important microbiome only offer a ‘snapshot’ in time. We monitored the diversity of rumen bacteria in four New Zealand dairy cows, grazing a rye-grass and clover pasture over five consecutive seasons, using high throughput pyrosequencing of bacterial 16S rRNA genes. We chose to focus on the digesta-adherent bacterial community to learn more about the stability of this community over time. 16S rRNA gene sequencing showed a high level of bacterial diversity, totalling 1539 operational taxonomic units (OTUs, grouped at 96% sequence similarity) across all samples, and ranging from 653 to 926 OTUs per individual sample. The nutritive composition of the pasture changed with the seasons as did the production phase of the animals. Sequence analysis showed that, overall, the bacterial communities were broadly similar between the individual animals. The adherent bacterial community was strongly dominated by members of Firmicutes (82.1%), followed by Bacteroidetes (11.8%). This community differed between the seasons, returning to close to that observed in the same season one year later. These seasonal differences were only small, but were statistically significant (p < 0.001), and were probably due to the seasonal differences in the diet. These results demonstrate a general invariability of the ruminal bacterial community structure in these grazing dairy cattle. [ABSTRACT FROM AUTHOR]

Examines the isolation of the ammonia-hyperproducing (HAP) bacteria from the rumen of various New Zealand pasture-grazed animals. Representation of the colonies able to grow on the medium containing tryptone casamino acids; Purification of the 14 morphologically distinct colonies; What the carbon source utilization experiments showed.

Disposal of electrons generated during the fermentation of ingested feed is a fundamental feature of anaerobic microbial gut ecosystems. Here, we focus on the well-studied rumen environment to highlight how electrons are transferred through anaerobic fermentation pathways and how manipulating this electron flow is important to reducing methane emissions from ruminants. Priorities for research that can accelerate understanding in this area are highlighted. [ABSTRACT FROM AUTHOR]

Hydrogen (H2) is the primary electron donor for methane formation in ruminants, but the H2-producing organisms involved are largely uncharacterized. This work integrated studies of microbial physiology and genomics to characterize rumen bacterial isolate NK3A20 of the family Lachnospiraceae. Isolate NK3A20 was the first recognized isolate of the NK3A20 group, which is among the ten most abundant bacterial genera in 16S rRNA gene surveys of rumen microbiota. NK3A20 produced acetate, butyrate, H2, and formate from glucose. The end product ratios varied when grown with different substrates and at different H2 partial pressures. NK3A20 produced butyrate as a major product using glucose or under high H2 partial pressures and switched to mainly acetate in the presence of galacturonic acid (an oxidized sugar) or in coculture with a methanogen. Growth with galacturonic acid was faster at elevated H2 concentrations, while elevated H2 slowed growth with glucose. Genome analyses revealed the presence of multiple hydrogenases including a membrane-bound Ech hydrogenase, an electron bifurcating butyryl-CoA dehydrogenase (Bcd-Etf), and an Rnf complex that may be involved in modulating the observed metabolic pathway changes, providing insight into H2 formation in the rumen. [ABSTRACT FROM AUTHOR]

Pectin is a complex polysaccharide that forms a substantial proportion of the plant's middle lamella of forage ingested by grazing ruminants. Methanol in the rumen is derived mainly from methoxy groups released from pectin by the action of pectin methylesterase (PME) and is subsequently used by rumen methylotrophic methanogens that reduce methanol to produce methane (CH4). Members of the genus Butyrivibrio are key pectin-degrading rumen bacteria that contribute to methanol formation and have important roles in fibre breakdown, protein digestion, and the biohydrogenation of fatty acids. Therefore, methanol release from pectin degradation in the rumen is a potential target for CH4 mitigation technologies. Here, we present the crystal structures of PMEs belonging to the carbohydrate esterase family 8 (CE8) from Butyrivibrio proteoclasticus and Butyrivibrio fibrisolvens, determined to a resolution of 2.30 Å. These enzymes, like other PMEs, are right-handed β-helical proteins with a well-defined catalytic site and reaction mechanisms previously defined in insect, plant, and other bacterial pectin methylesterases. Potential substrate binding domains are also defined for the enzymes. [ABSTRACT FROM AUTHOR]

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B12, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents ∼75% of the genus-level bacterial and archaeal taxa present in the rumen. [ABSTRACT FROM AUTHOR]

Methane emissions from livestock are a significant contributor to greenhouse gas emissions and have become a focus of research activities, especially in countries where agriculture is a major economic sector. Understanding the complexity of the rumen microbiota, including methane-producing Archaea, is in its infancy. There are currently no robust, reproducible and economically viable methods for reducing methane emissions from ruminants grazing on pasture and novel innovative strategies to diminish methane output from livestock are required. In this review, current approaches towards mitigation of methane in pastoral farming are summarised. Research strategies based on vaccination, enzyme inhibitors, phage, homoacetogens, defaunation, feed supplements, and animal selection are reviewed. Many approaches are currently being investigated, and it is likely that more than one strategy will be required to enable pastoral farming to lower its emissions of methane significantly. Different strategies may be suitable for different farming practices and systems. [ABSTRACT FROM AUTHOR]

Abstract: Clostridium proteoclasticum is commonly associated with the rumen microflora of pasture-fed cattle and sheep and has significant hemicellulose degradation abilities. Genes involved in plant fibre breakdown are commonly identified by producing site-specific mutations. However, the genetics of C. proteoclasticum and other closely-related Butyrivibrio/Pseudobutyrivibrio species is not well-established. Therefore random transposon mutants of C. proteoclasticum were generated by conjugation with Enterococcus faecalis containing Tn916. A new counter-selection agar medium was developed containing L-arabinose and D-raffinose as carbon sources, both of which are utilized by C. proteoclasticum only, and also ciprofloxacin, an antibiotic that suppresses the growth of E. faecalis. With this new medium the enhanced growth and more rapid separation of C. proteoclasticum transposon mutants from the background of E. faecalis cells was made, thereby facilitating the selection of transposon mutants and the identification of their Tn916 insertion sites. [Copyright &y& Elsevier]

Bacterial species belonging to the genus Pseudobutyrivibrio are important members of the rumen microbiome contributing to the degradation of complex plant polysaccharides. Pseudobutyrivibrio xylanivorans MA3014 was selected for genome sequencing to examine its ability to breakdown and utilize plant polysaccharides. The complete genome sequence of MA3014 is 3.58 Mb, consists of three replicons (a chromosome, chromid, and plasmid), has an overall G + C content of 39.6%, and encodes 3,265 putative protein-coding genes (CDS). Comparative pan-genomic analysis of all cultivated and currently available P. xylanivorans genomes has revealed a strong correlation of orthologous genes within this rumen bacterial species. MA3014 is metabolically versatile and capable of growing on a range of simple mono- or oligosaccharides derived from complex plant polysaccharides such as pectins, mannans, starch, and hemicelluloses, with lactate, butyrate, and formate as the principal fermentation end products. The genes encoding these metabolic pathways have been identified and MA3014 is predicted to encode an extensive range of Carbohydrate-Active enZYmes with 78 glycoside hydrolases, 13 carbohydrate esterases, and 54 glycosyl transferases, suggesting an important role in solubilization of plant matter in the rumen. [ABSTRACT FROM AUTHOR]

Plant polysaccharide breakdown by microbes in the rumen is fundamental to digestion in ruminant livestock. Bacterial species belonging to the rumen genera Butyrivibrio and Pseudobutyrivibrio are important degraders and utilizers of lignocellulosic plant material. These bacteria degrade polysaccharides and ferment the released monosaccharides to yield short-chain fatty acids that are used by the ruminant for growth and the production of meat, milk, and fiber products. Although rumen Butyrivibrio and Pseudobutyrivibrio species are regarded as common rumen inhabitants, their polysaccharide-degrading and carbohydrate-utilizing enzymes are not well understood. In this study, we analyzed the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains isolated from the plant-adherent fraction of New Zealand dairy cows to explore the polysaccharide-degrading potential of these important rumen bacteria. Comparative genome analyses combined with phylogenetic analysis of their 16S rRNA genes and short-chain fatty acid production patterns provide insight into the genomic diversity and physiology of these bacteria and divide Butyrivibrio into 3 species clusters. Rumen Butyrivibrio bacteria were found to encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) and binding proteins. In total, 4,421 glycoside hydrolases (GHs), 1,283 carbohydrate esterases (CEs), 110 polysaccharide lyases (PLs), 3,605 glycosyltransferases (GTs), and 1,706 carbohydrate-binding protein modules (CBM) with predicted activities involved in the depolymerization and transport of the insoluble plant polysaccharides were identified. Butyrivibrio genomes had similar patterns of CAZyme families but varied greatly in the number of genes within each category in the Carbohydrate-Active Enzymes database (CAZy), suggesting some level of functional redundancy. These results suggest that rumen Butyrivibrio species occupy similar niches but apply different degradation strategies to be able to coexist in the rumen. [ABSTRACT FROM AUTHOR]

Rumen bacterial species belonging to the genus Butyrivibrio are important degraders of plant polysaccharides, particularly hemicelluloses (arabinoxylans) and pectin. Currently, four species are recognized which have very similar substrate utilization profiles, but little is known about how these microorganisms are able to co-exist in the rumen. To investigate this question, Butyrivibrio hungatei MB2003 and Butyrivibrio proteoclasticus B316T were grown alone or in co-culture on xylan or pectin, and their growth, release of sugars, fermentation end products and transcriptomes were examined. In mono-cultures, B316T was able to grow well on xylan and pectin, while MB2003 was unable to utilize either of these insoluble substrates to support significant growth. Co-cultures of B316T grown with MB2003 revealed that MB2003 showed almost equivalent growth to B316T when either xylan or pectin were supplied as substrates. The effect of co-culture on the transcriptomes of B316T and MB2003 was assessed; B316T transcription was largely unaffected by the presence of MB2003, but MB2003 expressed a wide range of genes encoding carbohydrate degradation, central metabolism, oligosaccharide transport and substrate assimilation in order to compete with B316T for the released sugars. These results suggest that B316T has a role as an initiator of primary solubilization of xylan and pectin, while MB2003 competes effectively for the released soluble sugars to enable its growth and maintenance in the rumen. [ABSTRACT FROM AUTHOR]

Abstract Sharpea and Kandleria are associated with rumen samples from low-methane-emitting sheep. Four strains of each genus were studied in culture, and the genomes of nine strains were analysed, to understand the physiology of these bacteria. All eight cultures grew equally well with d -glucose, d -fructose, d -galactose, cellobiose, and sucrose supplementation. d -Lactate was the major end product, with small amounts of the mixed acid fermentation products formate, acetate and ethanol. Genes encoding the enzymes necessary for this fermentation pattern were found in the genomes of four strains of Sharpea and five of Kandleria. Strains of Sharpea produced traces of hydrogen gas in pure culture, but strains of Kandleria did not. This was consistent with finding that Sharpea , but not Kandleria , genomes contained genes coding for hydrogenases. It was speculated that, in co-culture with a methanogen, Sharpea and Kandleria might change their fermentation pattern from a predominately homolactic to a predominately mixed acid fermentation, which would result in a decrease in lactate production and an increase in formation of acetate and perhaps ethanol. However, Sharpea and Kandleria did not change their fermentation products when co-cultured with Methanobrevibacter olleyae , a methanogen that can use both hydrogen and formate, and lactate remained the major end product. The results of this study therefore support a hypothesis that explains the link between lower methane yields and larger populations of Sharpea and Kandleria in the rumens of sheep. Highlights • Sharpea and Kandleria produce mainly lactate, and some acetate, ethanol and formate. • Genome analysis found all genes needed for the products detected in culture studies. • Products were unchanged in co-cultures with a hydrogen and formate-using methanogen. • This supports a previous hypothesis explaining low methane emissions by sheep. [ABSTRACT FROM AUTHOR]

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]

Symbiotic associations are ubiquitous in the microbial world and have a major role in shaping the evolution of both partners. One of the most interesting mutualistic relationships exists between protozoa and methanogenic archaea in the fermentative forestomach (rumen) of ruminant animals. Methanogens reside within and on the surface of protozoa as symbionts, and interspecies hydrogen transfer is speculated to be the main driver for physical associations observed between the two groups. In silico analyses of several rumen methanogen genomes have previously shown that up to 5% of genes encode adhesin-like proteins, which may be central to rumen interspecies attachment. We hypothesized that adhesin-like proteins on methanogen cell surfaces facilitate attachment to protozoal hosts. Using phage display technology, we have identified a protein ( Mru_1499) from M ethanobrevibacter ruminantium M1 as an adhesin that binds to a broad range of rumen protozoa (including the genera E pidinium and E ntodinium). This unique adhesin also binds the cell surface of the bacterium B utyrivibrio proteoclasticus, suggesting a broad adhesion spectrum for this protein. [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]

Murrah and Nili-Ravi are water buffalo breeds widely used as dairy animals in Asian countries. In this study, we investigated the diversity of ruminal microbes in six Murrah and six Nili-Ravi water buffaloes consuming diets typical of those used in Southern China which had forage to concentrate ratios of 3.2 and 1.6. After feeding the diets for 25 days, ruminal fluid was sampled by stomach tube before the morning feeding. Bacterial and archaeal 16S rRNA genes and the ciliate protozoal 18S rRNA genes were PCR-amplified from DNA extracted from rumen samples, sequenced using 454 Titanium pyrosequencing, and analyzed using the QIIME software package. Our results showed that, at the phylum level, Bacteroidetes was the predominant bacterial group, accounting for 42–72% of total bacteria, followed by Firmicutes, Fibrobacteres, Proteobacteria and Spirochaetes. At genus level, Prevotella dominated, accounting for 22–58% of total bacteria, followed by Fibrobacter , Paludibacter , and Ruminococcus . While there were differences between the bacterial community compositions of different animals, there was no obvious correlation of bacterial community composition at the phylum or genus level with the diets or with buffalo breeds. Methanobrevibacter -related organisms were the dominant archaeal group, accounting for around 80% of the total, followed by Methanomassiliicoccales (Rumen Cluster C (RCC), 15%) and Methanosphaera (3%). Similar to the bacterial community, there was no obvious correlation of archaeal community profiles with diet or buffalo breed. The ciliate protozoal communities differed between the samples analyzed, and Entodinium was the most abundant group of ciliates in every sample, accounting for more than 40% of total protozoa. The second largest ciliate group varied in different samples, with Isotricha , Polyplastron or Dasytricha being the most dominant genera after Entodinium . [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]

Complex microbial communities typically contain a large number of low abundance species, which collectively, comprise a considerable proportion of the community. This 'rare biosphere' has been speculated to contain keystone species and act as a repository of genomic diversity to facilitate community adaptation. Many environmental microbes are currently resistant to cultivation, and can only be accessed via culture-independent approaches. To enhance our understanding of the role of the rare biosphere, we aimed to improve their metagenomic representation using DNA normalization methods, and assess normalization success via shotgun DNA sequencing. A synthetic metagenome was constructed from the genomic DNA of five bacterial species, pooled in a defined ratio spanning three orders of magnitude. The synthetic metagenome was fractionated and thermally renatured, allowing the most abundant sequences to hybridize. Double-stranded DNA was removed either by hydroxyapatite chromatography, or by a duplex-specific nuclease ( DSN). The chromatographic method failed to enrich for the genomes present in low starting abundance, whereas the DSN method resulted in all genomes reaching near equimolar abundance. The representation of the rarest member was increased by approximately 450-fold. De novo assembly of the normalized metagenome enabled up to 18.0% of genes from the rarest organism to be assembled, in contrast to the un-normalized sample, where genes were not able to be assembled at the same sequencing depth. This study has demonstrated that the application of normalization methods to metagenomic samples is a powerful tool to enrich for sequences from rare taxa, which will shed further light on their ecological niches. [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]

Abstract: The research was aimed at finding which membrane proteins of the rumen bacterium Butyrivibrio proteoclasticus are involved in the uptake of carbohydrates resulting from extracellular enzymatic degradation of hemicellulose and fructan. The proteomic analysis of cells grown with fructose or xylan as the sole substrate identified 13 membrane proteins predicted to function as carbohydrate transporters. One protein detected was the membrane component of a fructose-specific phosphoenolpyruvate:sugar phosphotransferase system believed to be involved in the fructose uptake following extracellular fructan breakdown. The other 12 proteins were all ABC transport system substrate-binding proteins, nine of which belong to functional category COG1653 that includes proteins predicted to transport oligosaccharides. Four of the SBPs were significantly upregulated in xylan grown cells, and three of these were found in polysaccharide utilisation loci where they are clustered with other genes involved in hemicellulose breakdown and metabolism. It is possible that the carbon source available regulates a wider network of genes. The information on the mechanisms used by rumen bacteria to take up carbohydrates from their environment may improve our understanding of the ruminant digestion and facilitate strategies for improved pasture and stored feed utilisation. [Copyright &y& Elsevier]

An unresolved question in the bioenergetics of methanogenic archaea is how the generation of proton-motive and sodium-motive forces during methane production is used to synthesize ATP by the membrane-bound A1Ao-ATP synthase, with both proton- and sodium-coupled enzymes being reported in methanogens. To address this question, we investigated the biochemical characteristics of the A1Ao-ATP synthase (MbbrA1Ao) of Methanobrevibacter ruminantium M1, a predominant methanogen in the rumen. Growth of M. ruminantium M1 was inhibited by protonophores and sodium ionophores, demonstrating that both ion gradients were essential for growth. To study the role of these ions in ATP synthesis, the ahaHIKECFABD operon encoding the MbbrA1Ao was expressed in Escherichia coli strain DK8 (Δatp) and purified yielding a 9-subunit protein with an SDS-stable c oligomer. Analysis of the c subunit amino acid sequence revealed that it consisted of four transmembrane helices, and each hairpin displayed a complete Na+-binding signature made up of identical amino acid residues. The purified MbbrA1Ao was stimulated by sodium ions, and Na+ provided pH-dependent protection against inhibition by dicyclohexylcarbodiimide but not tributyltin chloride. ATP synthesis in inverted membrane vesicles lacking sodium ions was driven by a membrane potential that was sensitive to cyanide m-chlorophenylhydrazone but not to monensin. ATP synthesis could not be driven by a chemical gradient of sodium ions unless a membrane potential was imposed. ATP synthesis under these conditions was sensitive to monensin but not cyanide m-chlorophenylhydrazone. These data suggest that the M. ruminantium M1 A1Ao-ATP synthase exhibits all the properties of a sodium-coupled enzyme, but it is also able to use protons to drive ATP synthesis under conditions that favor proton coupling, such as low pH and low levels of sodium ions. [ABSTRACT FROM AUTHOR]

Abstract: The genome of Butyrivibrio proteoclasticus B316T contains three large episomes including a 302kb chromid (BPc2) and two large plasmids of 361 (pCY360) and 186kb (pCY186). The two plasmids are largely cryptic and it is therefore difficult to gauge their contributions or importance to the biology of B. proteoclasticus. Here, we provide evidence that at least BPc2 and pCY360 are essential as neither could be cured using several previously described curing techniques. We show that BPc2 exists at a copy number of 1, while pCY360 and pCY186 exist at copy numbers of 4 and 0.9, respectively. Yet the transcriptional activities of each episome are much less than that of the 3.5Mb chromosome. Codon usage analyses did not support the hypothesis that the genes of all three episomes were acquired horizontally. Instead our analyses suggest that the vast majority of genes on each episome were transferred from the 3.5Mb B. proteoclasticus chromosome. Analysis of their replication origins, however, suggests the plasmid backbones share an evolutionary lineage with the smaller Butyrivibrio specific plasmids, pRJF1 and pRJF2. A survey of 13 species of the Butyrivibrio/Pseudobutyrivibrio assemblage identified similar large episomes in nine strains. DNA hybridization experiments revealed none contained an rRNA operon and only a 145kb episome from Pseudobutyrivibrio ruminis possessed an ortholog of the pCY360 plasmid replication initiation protein. The size and distribution of episomes within the nine strains of Butyrivibrio/Pseudobutyrivibrio showed no correlation with 16S rRNA based phylogeny, leading to a hypothesis that the large episomes of Butyrivibrio spp., have arisen through intragenomic gene transfer events from the chromosome to small horizontally acquired elements. [Copyright &y& Elsevier]

The rumen bacterium Butyrivibrio proteoclasticus B316 has a 4.4-Mb genome composed of four replicons (approximately 3.55 Mb, 361, 302 and 186 kb). Mutagenesis of B316 was performed with the broad host-range conjugative transposon Tn 916 to screen for functionally important characteristics. The insertion sites of 123 mutants containing a single copy of Tn 916 were identified and corresponded to 53 different insertion points, of which 18 (34.0%), representing 39 mutants (31.7%), were in ORFs and 12 were where transposition occurred in both directions (top and bottom DNA strand). Up to eight mutants from several independent conjugation experiments were found to have the same integration site. Although transposition occurred in all four replicons, the number of specific insertion sites, transposition frequency and the average intertransposon distance between insertions varied between the four replicons. In silico analysis of the 53 insertion sites was used to model a target consensus sequence for Tn 916 integration into B316. A search of the B316 genome using the modelled target consensus sequence (up to two mismatches) identified 39 theoretical Tn 916 insertion sites (19 coding, 20 noncoding), of which nine corresponded to Tn 916 insertions identified in B316 mutants during our conjugation experiments. [ABSTRACT FROM AUTHOR]

Determining the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals. Butyrivibrio proteoclasticus B316T is a Grampositive, butyrate-forming rumen bacterium with a key role in plant polysaccharide degradation. The 4.4Mb genome consists of 4 replicons; a chromosome, a chromid and two megaplasmids. The chromid is the smallest reported for all bacteria, and the first identified from the phylum Firmicutes. B316 devotes a large proportion of its genome to the breakdown and reassembly of complex polysaccharides and has a highly developed glycobiome when compared to other sequenced bacteria. The secretion of a range of polysaccharide-degrading enzymes which initiate the breakdown of pectin, starch and xylan, a subtilisin family protease active against plant proteins, and diverse intracellular enzymes to break down oligosaccharides constitute the degradative capability of this organism. A prominent feature of the genome is the presence of multiple gene clusters predicted to be involved in polysaccharide biosynthesis. Metabolic reconstruction reveals the absence of an identifiable gene for enolase, a conserved enzyme of the glycolytic pathway. To our knowledge this is the first report of an organism lacking an enolase. Our analysis of the B316 genome shows how one organism can contribute to the multi-organism complex that rapidly breaks down plant material in the rumen. It can be concluded that B316, and similar organisms with broad polysaccharide-degrading capability, are well suited to being early colonizers and degraders of plant polysaccharides in the rumen environment. [ABSTRACT FROM AUTHOR]

Virulence gene profiles of atypical enteropathogenic Escherichia coli (aEPEC) and Shiga toxin-producing E. coli (STEC) from cattle, sheep, and humans were examined to determine the relationship between pathotypes. Shared virulence factors (intimin, EHEC hemolysin, serine protease, and a type II secretion system) were identified, suggesting a dynamic evolutionary relationship between aEPEC and STEC. [ABSTRACT FROM AUTHOR]

Background: Methane (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 Findings: The 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 peptidedirected 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/Significance: The 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. [ABSTRACT FROM AUTHOR]

Atypical enteropathogenic Escherichia coli (aEPEC) and Shiga toxin-producing E. coli (STEC) were examined to determine the prevalence and sequence of espP, which encodes a serine protease. These analyses indicated shared espP sequence types between the two E. coli pathotypes and thus provide further insights into the evolution of aEPEC and STEC. [ABSTRACT FROM AUTHOR]

Abstract: The development of high-throughput DNA sequencing techniques has enabled the sequencing of several hundred bacterial genomes. However, the major step towards understanding the molecular basis of an organism will be the determination of all gene functions in its genome. Current gene assignments by sequence homology generate numerous hints to putative or unknown functions. Even hits with good homology are often not specific enough to describe the in vivo biochemical functions and the underlying biological roles. In this work we applied metabolic footprinting analysis to characterize Tn916-inserted mutants of a hemicellulose-degrading rumen bacterium grown on complex culture medium. Interestingly, the most distinctive phenotypic difference was observed in a mutant with a transposon insertion in a non-coding region of the genome, while disruption of a gene with high homology to a known α-glucosidase/xylosidase showed no distinctive phenotypic effect. Our results demonstrate that extracellular metabolomics data coupled to genome information is a powerful and low-cost approach to rapidly screen and characterize microbial mutants with single gene deletions. However, metabolomics as a stand-alone technique is unlikely to give a complete answer to define gene functions, and, therefore, is an approach to be used to generate hypotheses and direct new experiments to confirm gene function. [Copyright &y& Elsevier]

Analyses of the distribution of virulence factors among different Eseherichia coli pathotypes, including Shiga toxin-producing E. coli (STEC), may provide some insight into the mechanisms by which different E. coli strains cause disease and the evolution of distinct E. coli types. The aim of this study was to examine the DNA sequence of the gene for enterohemolysin, a plasmid-encoded toxin that readily causes the hemolysis of washed sheep erythrocytes, and to assess the distribution of enterohemolysin subtypes among E. coli isolates from various human and animal sources. The 2,997-bp ehxA gene was amplified from 227 (63.8%) of 356 stx- and/or eae-positive E. coli strains isolated from cattle and sheep and from 24 (96.0%) of 25 STEC strains isolated from humans with diarrheal disease. By using PCR and restriction fragment length polymorphism (RFLP) analysis of ehxA, six distinct PCR-RFLP types (A to F) were observed, with strains of subtypes A and C constituting 91.6% of all the ehxA-positive strains. Subtype A was associated mainly with ovine strains with stx only (P < 0.001), and subtype C was associated with bovine eae-positive strains (P < 0.001). Eleven ehxA alleles were fully sequenced, and the phylogenetic analysis indicated the presence of three closely related (>95.0%) ehxA sequence groups, one including eae-positive strains (subtypes B, C, E, and F) and the other two including mainly eae-negative STEC strains (subtypes A and D). In addition to being widespread among STEC strains, stx-negative, eae-positive strains (atypical enteropathogenic E. coli strains) isolated from cattle and sheep have similar ehxA subtypes and hemolytic activities. [ABSTRACT FROM AUTHOR]

The outer membrane protein, intimin ( eae), which mediates bacterial attachment to epithelial cells, is associated with enteropathogenic Escherichia coli and some Shiga toxin-producing E. coli. The eae subtype of E. coli strains isolated from healthy cattle and sheep was identified using a rapid PCR-restriction fragment length polymorphism (RFLP) method to produce profiles that were compared with those generated in silico. The 139 eae-positive E. coli strains were separated into 11 different PCR-RFLP profiles. The most common eae PCR-RFLP type was β (23.7%), followed by ζ (20.1%), θ (16.5%), ι (12.2%), κ (8.6%), ℇ (7.2%), γ (2.9%), ν and β2 (2.2%) and ι2 (1.4%). Four isolates did not yield a PCR-RFLP amplification product but complete sequencing of the eae gene matched subtype ρ. Two different eae variants were isolated from the same swab from 18 different animals and subtype ι was the most ‘promiscuous’, being isolated with four other eae subtypes from seven separate animals. None of the eae-positive STEC were subtype γ, which is associated with STEC serogroup O157. This method allowed the rapid identification of eae subtypes and indicates that forage-fed animals possessed a wide diversity of bacterial eae subtypes with a low frequency of eae subtype γ. [ABSTRACT FROM AUTHOR]

Abstract: Inhibitory effects of condensed tannins (CT) on methane, hydrogen, total gas and volatile fatty acid (VFA) formation were explored for the forage legumes Lotus pedunculatus (also known as L. uliginosus; CT 0.10 of DM) and Medicago sativa (lucerne; CT&#60;0.001 of DM) in the presence and absence of polyethylene glycol (PEG; a tannin binding reagent) and 2-bromo-ethylsulfonic acid (BES; a methanogen inhibitor). Treatment×forage interactions were explored using a factorial design for in vitro batch incubations with rumen fluid and minced forages over a 12h period. The accumulated methane volume (ml/0.5g DM) at 12h for M. sativa (12.5ml) was higher than L. pedunculatus (8.8ml; P &#60;0.05). PEG increased methane production for L. pedunculatus (17%, P &#60;0.001) but not for M. sativa. BES inhibited methane (>80%) and increased accumulated hydrogen for all treatments. For L. pedunculatus, addition of PEG in the presence of BES increased hydrogen accumulation (70%, P &#60;0.001) compared to L. pedunculatus and relative production (49%, P &#60;0.01), with no effect of PEG in combination with BES for M. sativa. The VFA (i.e., C2–C5) concentrations were determined at 12h for the batch incubations of rumen fluid with forage. Addition of PEG increased total VFA production (P &#60;0.01) for L. pedunculatus (16.9%) and M. sativa (1.8%). Addition of PEG increased C2–C5 concentrations for L. pedunculatus (5.9, 30, 22, 53%, respectively) with M. sativa having an increase only for C2 (13%). Branched chain VFA (bC4 and bC5) increased with PEG addition for L. pedunculatus (67 and 59%, respectively), but not for M. sativa. Inhibitory effects of extractable condensed tannin fractions from L. pedunculatus on the common rumen methanogens Methanobrevibacter ruminantium, strains YLM-1 and DSM1093, were evaluated. Oligomeric CT fractions were inactive against both strains in broth culture, as determined by methane production measurements. A polymeric fraction completely inhibited methane production. Inhibitory effects in broth culture for strain YLM1 were bacteriostatic, while strain DSM1093 did not recover growth, as indicated by methane production, even upon prolonged incubation. In a plate assay, the zone of inhibition with the polymeric fraction remained after a further week of incubation. This is the first demonstration of inhibition of growth of a rumen methanogen by a polymeric CT fraction. Results indicate that CT action on methanogenesis can be attributed to indirect effects via reduced hydrogen production (and presumably reduced forage digestibility) and via direct inhibitory effects on methanogens. [Copyright &y& Elsevier]

PCR primers were designed to amplify the gene that encodes bovicin 255 from Streptococcus gallolyticus LRC0255 and the bacteriocin genes from Butyrivibrio fibrisolvens strains AR10 and OR79A (bviD and bvi79A) in order to screen for their incidence in rumen and caecal B. fibrisolvens and Streptococcus bovis-like isolates from New Zealand and North American ruminants. None of the B. fibrisolvens-like strains (n=34) isolated from New Zealand or North America had the genes encoding for butyrivibriocins AR10 (bviD) or OR79 (bvi79A). However, seven S. bovis isolates from New Zealand ruminants and three from North American animals had the bovicin 255 gene. Sequence comparison of cloned bovicin 255 PCR products indicated a 92.9–95.7% similarity to that of the corresponding bovicin 255 gene sequence of S. gallolyticus. Four of the New Zealand bovicin 255 positive S. bovis isolates were from the caecal contents of the same sheep and had identical PFGE profiles. Two other S. bovis isolates sharing the same PFGE profile were isolated from a separate animal from the same flock. PFGE analysis of the North American strains indicated that all three were closely related as two of three had identical PFGE profiles with the remaining isolate differing only by a single band position. The 16S rRNA gene sequences of the 10 isolates were at least 99.8% identical to S. bovis. All 10 S. bovis isolates having the gene for bovicin 255 produced bacteriocin activity that inhibited the growth of Peptostreptococcus anaerobius D1 in a deferred antagonism plating (DAP) assay. Certain S. bovis isolates obtained from ruminants have bacteriocin activity associated with a distinct bovicin 255 gene sequence but it appears that bacteriocin production by the rumen anaerobe B. fibrisolvens may be uncommon in strains isolated from cattle and sheep in New Zealand. [Copyright &y& Elsevier]

The degradation of plant cell walls by ruminants is of major economic importance in the developed as well as developing world. Rumen fermentation is unique in that efficient plant cell wall degradation relies on the cooperation between microorganisms that produce fibrolytic enzymes and the host animal that provides an anaerobic fermentation chamber. Increasing the efficiency with which the rumen microbiota degrades fiber has been the subject of extensive research for at least the last 100 years. Fiber digestion in the rumen is not optimal, as is supported by the fact that fiber recovered from feces is fermentable. This view is confirmed by the knowledge that mechanical and chemical pretreatments improve fiber degradation, as well as more recent research, which has demonstrated increased fiber digestion by rumen microorganisms when plant lignin composition is modified by genetic manipulation. Rumen microbiologists have sought to improve fiber digestion by genetic and ecological manipulation of rumen fermentation. This has been difficult and a number of constraints have limited progress, including: (a) a lack of reliable transformation systems for major fibrolytic rumen bacteria, (b) a poor understanding of ecological factors that govern persistence of fibrolytic bacteria and fungi in the rumen, (c) a poor understanding of which glycolyl hydrolases need to be manipulated, and (d) a lack of knowledge of the functional genomic framework within which fiber degradation operates. In this review the major fibrolytic organisms are briefly discussed. A more extensive discussion of the enzymes involved in fiber degradation is included. We also discuss the use of plant genetic manipulation, application of free-living lignolytic fungi and the use of exogenous enzymes. Lastly, we will discuss how newer technologies such as genomic and metagenomic approaches can be used to improve our knowledge of the functional genomic framework of plant cell wall degradation in the rumen. [Copyright &y& Elsevier]