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The rumen of dairy cattle can be thought of as a large, stable fermentation vat and as such it houses a large and diverse community of microorganisms. The bacterium Butyrivibrio proteoclasticus is a representative of a significant component of this microbial community. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fibre-degrading enzymes. This suite of enzymes is essential for the organism to utilize the plant material within the rumen as a fuel source, facilitating its survival in this competitive environment. Xsa43E, a GH43 enzyme from B. proteoclasticus, has been structurally and functionally characterized. Here, the structure of selenomethionine-derived Xsa43E determined to 1.3 Å resolution using single-wavelength anomalous diffraction is reported. Xsa43E possesses the characteristic five-bladed β-propeller domain seen in all GH43 enzymes. GH43 enzymes can have a range of functions, and the functional characterization of Xsa43E shows it to be an arabinofuranosidase capable of cleaving arabinose side chains from short segments of xylan. Full functional and structural characterization of xylan-degrading enzymes will aid in creating an enzyme cocktail that can be used to completely degrade plant material into simple sugars. These molecules have a range of applications as starting materials for many industrial processes, including renewable alternatives to fossil fuels. [ABSTRACT FROM AUTHOR]

Condensed tannins in forage legumes improve the nutrition of sheep by reducing ruminal degradation of plant protein and increasing crude protein flow to the intestine. However, the effects of condensed tannins in forage legumes on rumen bacterial populations in vivo are poorly understood. The aim of this study was to investigate the specific effects of condensed tannins from Lotus corniculatus on four proteolytic rumen bacteria in sheep during and after transition from a ryegrass (Lolium perenne) – white clover (Trifolium repens) diet (i.e., low condensed tannins) to a Lotus corniculatus diet (i.e., higher condensed tannins). The bacterial populations were quantified using a competitive polymerase chain reaction. Lotus corniculatus was fed with or without ruminal infusions of polyethylene glycol (PEG), which binds to and inactivates condensed tannins, enabling the effect of condensed tannins on bacterial populations to be examined. When sheep fed on ryegrass – white clover, populations of Clostridium proteoclasticum B316[sup T] , Butyrivibrio fibrisolvens C211a, Eubacterium sp. C12b, and Streptococcus bovis B315 were 1.5 × 10[sup 8] , 1.1 × 10[sup 6] , 4.6 × 10[sup 8] , and 7.1 × 10[sup 6] mL[sup –1] , respectively. When the diet was changed to Lotus corniculatus, the average populations (after 8–120 h) of C. proteoclasticum, B. fibrisolvens, Eubacterium sp., and S. bovis decreased (P < 0.001) to 2.4 × 10[sup 7] , 1.1 × 10[sup 5] , 1.1 × 10[sup 8] , and 2.5 × 10[sup 5] mL[sup –1] , respectively. When PEG was infused into the rumen of sheep fed Lotus corniculatus, the populations of C. proteoclasticum, B. fibrisolvens, Eubacterium sp., and S. bovis were higher (P < 0.01–0.001) than in sheep fed Lotus corniculatus without the PEG infusion, with average populations (after 8–120 h) of 4.9 × 10[sup 7] , 3.8 × 10[sup 5] , 1.9 ×10[sup 8] , and 1.0 ×10[sup 6] , respectively. Sheep fed the Lotus corniculatus diet had lower rumen proteinase activity, ammonia, and soluble nitrogen (P < 0.05–0.001) than sheep that were fed Lotus corniculatus plus PEG. The Lotus corniculatus diet reduced rumen nitrogen digestibility (P < 0.05) and ammonia pool size and increased the flow of undegraded feed nitrogen to the abomasum. The nitrogen intake, rumen non-ammonia nitrogen pool size, rumen microbial non-ammonia nitrogen pool size, and abomasal microbial non-ammonia nitrogen fluxes were similar both in sheep fed only Lotus corniculatus and in sheep fed Lotus corniculatus plus PEG, but nonmicrobial non-ammonia nitrogen flux to the abomasum was higher (P < 0.01) for the sheep fed only Lotus corniculatus. Although condensed tannins in Lotus corniculatus reduced the populations of some proteolytic bacteria, total ruminal microbial protein and microbial protein outflow to the abomasum were unchanged, suggesting a species-specific effect of condensed tannins on bacteria in the rumen. Key words: condensed, tannin, rumen, bacteria, PCR.Les tannins condensés de légumineuses fourragères améliorent la nutrition des moutons en diminuant la dégradation de protéines végétales dans le rumen et en augmentant le débit de protéines brutes vers les intestins. Toutefois, l'influence des tannins condensés dans les légumineuses fourragères sur les populations bactériennes du rumen in vivo est peu connue. Le but de cette étude fut d'analyser les effets spécifiques des tannins condensés de Lotus corniculatus sur quatre bactéries ruminales protéolytiques du mouton pendant et après une transition d'un régime à base de fromental (Lolium perenne) – trèfle blanc (Trifolium repense) renfermant peu de tannins condensés à un régime à base de Lotus corniculatus contenant des tannins condensés. Les populations bactériennes ont été quantifiées par réaction de polymérase en chaîne compétitive. Le Lotus corniculatus a été donné avec ou sans infusions ruminales de polyéthylène glycol (PEG), qui se lie et inactive les tannins condensés, permettant ainsi d'évaluer l'impact des tannins condensés sur les populations bactériennes. Lorsque le fromental – trèfle blanc ont été donnés à manger, les populations de Clostridium proteoclasticum B316[sup T] , Butyrivibrio fibrisolvens C211a, Eubacterium sp. C12b, et Streptococcus bovis B315 étaient de 1,5 × 10[sup 8] , 1,1 × 10[sup 6] , 4,6 × 10[sup 8] , et 7,1 × 10[sup 6] mL[sup –1] respectivement. Lorsque le régime est passé à Lotus corniculatus, les populations moyennes (8–120 h) de C. proteoclasticum, B. fibrisolvens, Eubacterium sp., et S. bovis ont baissé (P < 0,001) jusqu'à 2,4 × 10[sup 7] , 1,1 × 10[sup 5] , 1,1 × 10[sup 8] , et 2,5 × 10[sup 5] mL[sup –1] respectivement. Lorsque du PEG a été infusé dans le rumen de moutons auxquels on avait donné du Lotus corniculatus, les populations de C. proteoclasticum, B. fibrisolvens, Eubacterium sp., et S. bovis étaient supérieures (P < 0,01–0,001) à celles du groupe nourri au Lotus corniculatus, avec des populations moyennes (8–120 h) de 4,9 × 10[sup 7] , 3,8 × 10[sup 5] , 1,9 ×10[sup 8] et 1,0 ×10[sup 6] respectivement. Les moutons qui ont été nourris au Lotus corniculatus démontraient une plus faible activité protéinase du rumen inférieur ainsi que des taux d'ammoniac et d'azote solubles inférieurs (P < 0,05–0,001) au groupe ayant reçu du Lotus corniculatus + PEG. Le régime à base de Lotus corniculatus a diminué la digestibilité de l'azote du rumen (P < 0,05) et la taille de la masse commune d'ammoniac, et a augmenté le débit d'azote absorbé et non dégradé vers la caillette. L'apport en azote, la masse ruminal commune d'azote sans ammoniac, la masse microbienne commune d'azote ruminal sans ammoniac et les flux d'azote microbien abomasal sans ammoniac étaient semblables entre les moutons nourris au Lotus corniculatus et ceux nourris auLotus corniculatus + PEG. Cependant, le flux d'azote non-microbien sans ammoniac vers la caillete était plus élevé (P < 0,01) chez les moutons nourris au Lotus corniculatus. Bien que les tannins condensés de Lotus corniculatus ont fait baissé les populations de certaines bactéries protéolytiques, les quantités de protéines ruminales totales et le débit de protéines microbiennes vers la caillette sont demeurés inchangés, ce qui indique que les tannins condensés ont un effet spécifique à l'espèce chez les bactérie du rumen. Mots clés : condensés, tannins, rumen, bactéries, PCR. [Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

Competitive Polymerase Chain Reaction primers were designed for Streptococcus, B. fibrisolvens, P. bryantii, Eubacterium sp., Prevotella, and a universal primer for the eubacteria. DNA was extracted from rumen contents collected from eight dairy cows fed four diets: adequate nitrogen, adequate nitrogen plus carbohydrate, low nitrogen, and low nitrogen plus carbohydrate. B. fibrisolvens was significantly higher on the adequate nitrogen plus carbohydrate and the low nitrogen plus carbohydrate diets compared with the other diets, while P. bryantii was significantly higher on the low nitrogen plus carbohydrate diet compared with the adequate nitrogen diet. The population of Eubacterium sp. was significantly lower on both the adequate nitrogen plus carbohydrate and low nitrogen plus carbohydrate diets. Streptococcus populations were significantly lower on the low nitrogen plus carbohydrate diet compared with all three other diets, whereas there were no significant differences in populations of Prevotella or total eubacteria on any of the diets. [ABSTRACT FROM AUTHOR]

Ruminant-derived methane (CH4), a potent greenhouse gas, is a consequence of microbial fermentation in the digestive tract of livestock. Development of mitigation strategies to reduce CH4 emissions from farmed animals is currently the subject of both scientific and environmental interest. Methanogens are the sole producers of ruminant CH4, and therefore CH4 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 CH4 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 CH4 mitigation strategies based on vaccine and small molecule inhibitor approaches. In addition, genome sequencing is contributing information relevant to other CH4 mitigation strategies. These include the selection and breeding of low CH4-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.

Meat and milk produced by ruminants are important agricultural products and are major sources of protein for humans. Ruminant production is of considerable economic value and underpins food security in many regions of the world. However, the sector faces major challenges because of diminishing natural resources and ensuing increases in production costs, and also because of the increased awareness of the environmental impact of farming ruminants. The digestion of feed and the production of enteric methane are key functions that could be manipulated by having a thorough understanding of the rumen microbiome. Advances in DNA sequencing technologies and bioinformatics are transforming our understanding of complex microbial ecosystems, including the gastrointestinal tract of mammals. The application of these techniques to the rumen ecosystem has allowed the study of the microbial diversity under different dietary and production conditions. Furthermore, the sequencing of genomes from several cultured rumen bacterial and archaeal species is providing detailed information about their physiology. More recently, metagenomics, mainly aimed at understanding the enzymatic machinery involved in the degradation of plant structural polysaccharides, is starting to produce new insights by allowing access to the total community and sidestepping the limitations imposed by cultivation. These advances highlight the promise of these approaches for characterising the rumen microbial community structure and linking this with the functions of the rumen microbiota. Initial results using high-throughput culture-independent technologies have also shown that the rumen microbiome is far more complex and diverse than the human caecum. Therefore, cataloguing its genes will require a considerable sequencing and bioinformatic effort. Nevertheless, the construction of a rumen microbial gene catalogue through metagenomics and genomic sequencing of key populations is an attainable goal. A rumen microbial gene catalogue is necessary to understand the function of the microbiome and its interaction with the host animal and feeds, and it will provide a basis for integrative microbiome-host models and inform strategies promoting less-polluting, more robust and efficient ruminants.

Aims: To serotype a subset of Shiga toxin-producing Escherichia coli (STEC) isolates from cattle and sheep to determine whether any corresponding serotypes have been implicated in human diarrhoeal disease, both in New Zealand and worldwide, and to examine the distribution of STEC and enteropathogenic Escherichia coli (EPEC) amongst cattle (calves, heifers and dairy) and sheep (lambs, rams and ewes), to assess whether carriage of identified bacterial genotypes may be associated with a particular age of animal., Methods: Recto-anal mucosal swabs (RAMS) were taken from 91 calves, 24 heifers and 72 dairy cattle, and 46 lambs, 50 ewes and 36 rams, from four sites in the Manawatu and Rangitikei regions of New Zealand. Strains of E. coli selected from primary isolation plates were subjected to a multiplex polymerase chain reaction (PCR), to determine the presence of Shiga toxin genes (stx1 and stx2) and the E. coli attaching and effacing gene (eae)., Results: Overall, 186/319 (58.3%) animals sampled were positive for stx1, stx2, or eae isolates. More sheep (43.9%) were stx1-positive than cattle (2.7%; p = 0.036), and amongst sheep more lambs and ewes were stx1-positive than rams (p = 0.036). Amongst cattle, more calves and heifers were eae-positive than dairy cows (p = 0.030). Two or more different STEC were isolated from at least 28 (9%) animals (three cattle and 25 sheep), based on their stx/eae genotype. Enterohaemolysin genes were found in 39/51 (76%) isolates serotyped. Twenty-one different serotypes were detected, including O5:H-, O9:H51, O26:H11, O84:H-/H2 and O149:H8 from cattle, and O26:H11, O65:H-, O75:H8, O84:H-, O91:H-, O128:H2 and O174:H8 from sheep; O84:H-, O26:H11, O5:H-, O91:H- and O128:H2 serotypes have been associated with human disease., Conclusions: If nationally representative, this study confirms that cattle and sheep in New Zealand may be a major reservoir of STEC serotypes that have been recognised as causative agents of diarrhoeal disease in humans. Distribution of STEC and EPEC in cattle and sheep indicates that direct contact with, in particular, calves or their faeces, or exposure to environments cross contaminated with ruminant faeces, may represent an increased risk factor for human disease in New Zealand.

Pasture-grazed dairy cows, deer, and sheep were tested for the presence of ammonia-hyperproducing (HAP) bacteria in roll tubes containing a medium in which tryptone and Casamino Acids were the sole nitrogen and energy sources. Colonies able to grow on this medium represented 5.2, 1.3, and 11.6% of the total bacterial counts of dairy cows, deer, and sheep, respectively. A total of 14 morphologically distinct colonies were purified and studied further. Restriction fragment length polymorphisms of 16S rRNA genes indicated that all isolates differed from the previously described HAP bacteria, Clostridium aminophilum, Clostridium sticklandii, and Peptostreptococcus anaerobius. Carbon source utilization experiments showed that five isolates (C2, D1, D4, D5, and S1) were unable to use any, or very few, of the carbon sources tested. Biochemical tests and phylogenetic analyses of 16S ribosomal DNA sequences indicated that all isolates were monensin sensitive; that D1 and S1 belonged to the genus Peptostreptococcus, that D4 and D5 belonged to the family Bacteroidaceae, where D4 was similar to Fusobacterium necrophorum; and that C2 was most similar to an unidentified species from the genus Eubacterium. Growth on liquid medium containing tryptone and Casamino Acids as the sole nitrogen and energy source showed that D1, D4, and S1 grew rapidly (specific growth rates of 0.40, 0.35, and 0.29 h-1, respectively), while C2 and D5 were slow growers (0.25 and 0.10 h-1, respectively). Ammonia production rates were highest in D1 and D4, which produced 945.5 and 748.3 nmol/min per mg of protein, respectively. Tests of individual nitrogen sources indicated that D1 and D4 grew best on tryptone, S1 grew equally well on Casamino Acids or tryptone, and C2 and D5 grew poorly on all nitrogen sources. The intact proteins casein and gelatin did not support significant growth of any of the isolates. These isolates extend the diversity of known HAP rumen bacteria and indicate the presence of significant HAP bacterial populations in pasture-grazed New Zealand ruminants.

A genomic library of Ruminococcus albus 8 DNA was constructed in Escherichia coli using bacteriophage lambda ZapII. This library was screened for cellulase components and several Ostazin brilliant red/carboxymethyl cellulose positive clones were isolated. All of these clones contained a common 3.4-kb insert, which was recovered as a plasmid by helper phage excision. The carboxymethyl cellulase coding region was localized to a 1.4-kb region of DNA by nested deletions, and a clone containing the entire celA gene was sequenced. Analysis of the sequence revealed a 1231-bp open reading frame, coding for a protein of 411 amino acids with a predicted molecular weight of 45 747. This protein, designated CelA, showed extensive homology with family 5 endoglucanases by both primary amino acid sequence alignment and hydrophobic cluster analysis. Cell-free extracts of E. coli containing the celA clone demonstrated activity against carboxymethyl cellulose and acid swollen cellulose but not against any of the p-nitrophenol glycosides tested, indicating an endo-beta-1,4-glucanase type of activity. In vitro transcription-translation experiments showed that three proteins of 48,000, 44,000, and 23,000 molecular weight were produced by clones containing the celA gene. Northern analysis of RNA extracted from R. albus 8 grown on cellulose indicated a celA transcript of approximately 2700 bases, whereas when R. albus 8 was grown on cellobiose, celA transcripts of approximately 3000 and 600 bases were detected. Primer extension analysis of these RNAs revealed different transcription initiation sites for the celA gene when cells were grown with cellulose or cellobiose as the carbon source. These two sites differed by 370 bases in distance. A model, based on transcription and sequence data, is proposed for celA regulation.

The protease activities of 212 strains of rumen bacteria isolated from New Zealand cattle grazing pasture were measured. Thirty-seven per cent of strains had activity greater than or equal to the proteolytic rumen bacterium Prevotella ruminicola and 43 of these isolates were identified by morphology, carbon source utilization, Gram stain, biochemical tests and fermentation end-product analysis. Hierarchical Cluster Analysis showed that the strains formed four clusters: cluster A contained 26 strains and clustered with a reference strain of Streptococcus bovis; cluster C contained three strains and clustered with a reference strain of Butyrivibrio fibrisolvens, while clusters B (10 strains) and D (three strains) did not cluster with any of the remaining rumen bacterial type strains. Further tests identified strains of cluster B as Eubacterium budayi, while cluster D strains most closely resembled B. fibrisolvens and were described as B. fibrisolvens-like. An unclustered strain, C21a, was identified as P. ruminicola. The significance of these proteolytic bacterial populations is discussed in relation to protein breakdown in New Zealand ruminants.

An endoglucanase gene, which was shown to be identical to the previously sequenced engB gene [Attwood et al. (1993) Abstr. Ann. Meet. Am. Soc. Microbiol.], was isolated from a Clostridium cellulovorans genomic library. Because of the lack of transcriptional information concerning engB we examined its expression in C. cellulovorans and in the heterologous hosts Escherichia coli and C. acetobutylicum following transformation of engB. Northern analysis suggested that both E. coli and C. acetobutylicum produced several transcripts of various sizes. C. cellulovarans produced a single transcript of 1600 bp with the relative amount of engB mRNA from cellulose-grown cells being much greater than that from cellobiose-grown cells. Primer extensions showed that engB was transcribed from a single transcription initiation site in C. cellulovorans preceded by sequences similar to promoter sequences found in Gram-positive bacteria. Primer extensions from both E. coli and C. acetobutylicum strains containing the engB gene showed multiple transcription initiation sites, none of which corresponded to the site determined in C. cellulovorans. We conclude that transcriptional control of the engB gene is less stringent in heterologous backgrounds and postulate that expression of the engB gene in C. cellulovorans is increased in the presence of cellulose.

A plasmid from Selenomonas ruminantium subspecies lactilytica has been subcloned in Escherichia coli K-12 and completely sequenced. Three open reading frames (ORFs) of 909, 801, and 549 bp were identified and the complete sequence was analyzed by comparison with DNA and protein databases. No significant deoxynucleotide or amino acid sequence homology with other published genes or proteins was detected. The plasmid was shown to replicate independently in E. coli K-12 by a DNA polymerase I-dependent mechanism and deletion analysis defined the DNA sequence responsible for this phenotype.