RUMINANT NUTRITION SYMPOSIUM: Use of genomics and transcriptomics to identify strategies to lower ruminal methanogenesis.

Globally, methane (CH4) emissions account for 40% to 45% of greenhouse gas emissions from ruminant livestock, with over 90% of these emissions arising from enteric fermentation. Reduction of carbon dioxide to CH4 is critical for efficient ruminal fermentation because it prevents the accumulation of reducing equivalents in the rumen. Methanogens exist in a symbiotic relationship with rumen protozoa and fungi and within biofilms associated with feed and the rumen wall. Genomics and transcriptomics are playing an increasingly important role in defining the ecology of ruminal methanogenesis and identifying avenues for its mitigation. Metagenomic approaches have provided information on changes in abundances as well as the species composition of the methanogen community among ruminants that vary naturally in their CH4 emissions, their feed efficiency, and their response to CH4 mitigators. Sequencing the genomes of rumen methanogens has provided insight into surface proteins that may prove useful in the development of vaccines and has allowed assembly of biochemical pathways for use in chemogenomic approaches to lowering ruminal CH4 emissions. Metagenomics and metatranscriptomic analysis of entire rumen microbial communities are providing new perspectives on how methanogens interact with other members of this ecosystem and how these relationships may be altered to reduce methanogenesis. Identification of community members that produce antimethanogen agents that either inhibit or kill methanogens could lead to the identification of new mitigation approaches. Discovery of a lytic archaeophage that specifically lyses methanogens is 1 such example. Efforts in using genomic data to alter methanogenesis have been hampered by a lack of sequence information that is specific to the microbial community of the rumen. Programs such as Hungate1000 and the Global Rumen Census are increasing the breadth and depth of our understanding of global ruminal microbial communities, steps that are key to using these tools to further define the science of ruminal methanogenesis.

Structural analysis of the GH43 enzyme Xsa43E from Butyrivibrio proteoclasticus.

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 Šresolution using single-wavelength anomalous diffraction is reported. Xsa43E possesses the characteristic five-bladed ß-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.

The Complete Genome Sequence of Methanobrevibacter sp. AbM4.

Methanobrevibacter sp. AbM4 was originally isolated from the abomasal contents of a sheep and was chosen as a representative of the Methanobrevibacter wolinii clade for genome sequencing. The AbM4 genome is smaller than that of the rumen methanogen M. ruminantium M1 (2.0 Mb versus 2.93 Mb), encodes fewer open reading frames (ORFs) (1,671 versus 2,217) and has a lower G+C percentage (29% versus 33%). Overall, the composition of the AbM4 genome is very similar to that of M1 suggesting that the methanogenesis pathway and central metabolism of these strains are highly similar, and both organisms are likely to be amenable to inhibition by small molecule inhibitors and vaccine-based methane mitigation technologies targeting these conserved features. The main differences compared to M1 are that AbM4 has a complete coenzyme M biosynthesis pathway and does not contain a prophage or non-ribosomal peptide synthase genes. However, AbM4 has a large CRISPR region and several type I and type II restriction-modification system components. Unusually, DNA-directed RNA polymerase B' and B'' subunits of AbM4 are joined, a feature only previously observed in some thermophilic archaea. AbM4 has a much reduced complement of genes encoding adhesin-like proteins which suggests it occupies a ruminal niche different from that of M1.

Genome sequencing of rumen bacteria and archaea and its application to methane mitigation strategies.

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.

Rumen microbial (meta)genomics and its application to ruminant production.

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.

Serotypes and analysis of distribution of Shiga toxin producing Escherichia coli from cattle and sheep in the lower North Island, New Zealand.

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.

The prevalence of Shiga toxin-producing Escherichia coli in cattle and sheep in the lower North Island, New Zealand.

AIM: To genotype Escherichia coli cultured from the faeces of healthy cattle and sheep in the lower North Island, in order to investigate the possible role of ruminants as a reservoir for Shiga toxin-producing E. coli (STEC) in New Zealand. METHODS: A total of 952 strains of E. coli were isolated on selective media, from faecal swabs from 319 animals (187 cattle and 132 sheep) from four sites in the Manawatu and Rangitikei regions of New Zealand. A multiplex polymerase chain reaction (PCR) was used to genotype the E. coli isolates, using amplification of Shiga toxin genes (stx1 and stx2) and the E. coli attaching and effacing gene (eae). RESULTS: Isolates of E. coli were cultured from swabs from 178/187 (95.2%) cattle and all 132 (100%) sheep. Ninety-nine (10.4%) of the isolates were stx1 only, 83 (8.7%) stx2 only, 33 (3.5%) stx1 and stx2, 23 (2.4%) stx1 and eae, one (0.1%) stx2 and eae, and 115 (12.1%) were eae only. Overall, 51 (27.3%) cattle and 87 (65.9%) sheep were stx-positive, whereas 69 (36.9%) cattle and 36 (27.3%) sheep were eae-positive. CONCLUSIONS: Both healthy cattle and sheep are asymptomatic reservoirs of STEC in New Zealand. Direct contact with cattle and sheep or consumption of water or foodstuffs contaminated with cattle of sheep faeces may represent a significant source of infection for humans.

Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets.

AIM: To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. METHODS AND RESULTS: Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 10(7) cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 10(6) CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 10(8) CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. CONCLUSION: Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7-10 days earlier than in uninoculated cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR.

Lotus corniculatus condensed tannins decrease in vivo populations of proteolytic bacteria and affect nitrogen metabolism in the rumen of sheep.

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 B316T, Butyrivibrio fibrisolvens C211a, Eubacterium sp. C12b, and Streptococcus bovis B315 were 1.5 x 10(8), 1.1 x 10(6), 4.6 x 10(8), and 7.1 x 10(6) mL(-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 x 10(7), 1.1 x 10(5), 1.1 x 10(8), and 2.5 x 10(5) mL(-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 x 10(7), 3.8 x 10(5), 1.9 x 10(8), and 1.0 x 10(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.

Design and use of 16S ribosomal DNA-directed primers in competitive PCRs to enumerate proteolytic bacteria in the rumen.

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.

The effect of condensed tannins from Lotus pedunculatus and Lotus corniculatus on the growth of proteolytic rumen bacteria in vitro and their possible mode of action.

Five strains of proteolytic rumen bacteria were treated with condensed tannins (CT) purified from Lotus pedunculatus and Lotus corniculatus to investigate their effect on the growth of these bacteria in vitro. Streptococcus bovis NCFB 2476, Eubacterium sp. C124b, Prevotella bryantii B14, Butyrivibrio fibrisolvens H17c, and Clostridium proteoclasticum B316T were tested against 200, 400, and 600 microg CT x mL(-1) extracted from L. pedunculatus and L. corniculatus. In the absence of CT, all bacterial strains showed typical growth and reached maximum optical density (OD) after 6-8 h of incubation in a plant protein medium. Growth of Eubacterium sp., P. bryantii, and B. fibrisolvens was inhibited (P < 0.01-0.001) more by the CT from L. pedunculatus than by the CT from L. corniculatus. All strains continued to grow in the presence of 200 microg x mL(-1) of the CT from L. pedunculatus, but attained significantly (P < 0.05-0.01) lower maximum OD600 values than (minus CT) controls, except for S. bovis. At 400 and 600 microg x mL(-1), the addition of CT from L. pedunculatus inhibited (P < 0.05-0.001) the growth of all bacterial strains tested compared with controls. The growth of Eubacterium sp. and P. bryantii was stimulated for the first 4-6 h of incubation (P < 0.001) by 200 microg x mL(-1) of CT from L. corniculatus, but then declined leading to a significant difference in OD values compared with the controls. At 400 microg x mL(-1), the CT from L. corniculatus reduced (P < 0.05-0.01) the growth of all strains except S. bovis, while 600 microg x mL(-1) inhibited (P < 0.01-0.001) the growth of all strains. To study the mechanism of CT action, the degradation of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; Fraction 1 Leaf protein) was followed after bacterial cells or Rubisco were preincubated with CT extracted from L. corniculatus and L. pedunculatus. Both preincubations decreased LSU degradation, but they differed in their response to polyethylene glycol (PEG) addition. Addition of PEG to CT-Rubisco preincubations negated the effects of CT, while PEG addition to CT-bacteria preincubations did not. This implies that the CT-bacterial interaction is stronger than the CT-Rubisco interaction or the interaction is of a different type. Also, L. pedunculatus CT reduced the degradation of the LSU to a greater extent than the CT from L. corniculatus when preincubated with bacteria.

Ammonia-hyperproducing bacteria from New Zealand ruminants.

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.

Detection of Clostridium proteoclasticum and closely related strains in the rumen by competitive PCR.

A competitive PCR technique was used to enumerate the proteolytic bacterium Clostridium proteoclasticum from the rumen. A PCR primer, which circumscribes this organism and several closely related strains, was designed for a variable region within their 16S rRNA genes and was used in conjunction with a universal forward primer. This primer pair was tested for specificity against 85 ruminal bacterial strains. An internal control DNA was constructed for use in competitive PCRs and was shown to amplify under the same reaction conditions and with the same amplification efficiency as the target DNA. DNA from a known number of C. proteoclasticum cells was coamplified with the internal control to construct a standard curve. Rumen samples were collected from eight dairy cows fed four diets in rotation: high nitrogen, high nitrogen supplemented with carbohydrate, low nitrogen, and low nitrogen supplemented with carbohydrate. DNA extracted from these and spiked with internal control DNA was amplified with the C. proteoclasticum primer pair. The relative intensities of the PCR products were used to quantitate the numbers of C. proteoclasticum cell equivalents from the rumen samples. The numbers ranged from 2.01 x 10(6) ml-1 to 3.12 x 10(7) ml-1. There was no significant effect on the numbers of C. proteoclasticum detected in rumen samples among cows fed the four diets. The utility of the competitive PCR approach for quantifying ruminal bacterial populations in vivo and the occurrence of C. proteoclasticum in forage-fed dairy cows are discussed.

Characterization of proteolytic activities of rumen bacterial isolates from forage-fed cattle.

The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates. The Streptococcus bovis strains had predominantly cell-bound activity, which included a mixture of serine and cysteine-type proteinases which had high activity against leucine p-nitroanilide (LPNA). The Eubacterium strains had a mainly cell-associated activity with serine and metallo-type proteinases which showed high activity against the chymotrypsin substrate, N-succinyl alanine alanine phenylalanine proline p-nitroanilide (NSAAPPPNA) and some LPNA activity. A Butyrivibrio strain, C211, had a cell-bound mixture of cysteine and metallo-proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio-like strain, B316, had a cell-bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA. A Prevotella-like strain, C21a, had a mixture of cysteine, serine and metallo-proteinase activities which were cell-bound and hydrolysed LPNA. The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N-succinyl phenylalanine p-nitroanilide. The contribution of these strains to proteolysis in the rumen is discussed.

Clostridium proteoclasticum sp. nov., a novel proteolytic bacterium from the bovine rumen.

A novel proteolytic bacterium was isolated from rumen contents of New Zealand cattle grazing fresh forage and was designated strain B316(T) (T = type strain). Strain B316(T) cells were straight to slightly curved rods (width, 0.4 to 0.6 microns; length, 1.3 to 3.0 microns) that were gram-positive and possessed a single subterminal flagellum. This isolate did not produce catalase, indole, ammonia, lipase, or lecithinase, or reduce nitrate, but it did produce a curd reaction with milk. Strain B316(T) was proteolytic, hydrolyzing casein and fraction I leaf protein. The crude proteinase was predominantly the serine type, but some cysteine proteinase and metallo-proteinase activities were also detected. The DNA base composition of strain B316(T) was 28 mol% G+C. A 16S ribosomal DNA sequence analysis of strain B316(T) indicated that it was most closely related to a member of clostridial cluster XIVa, viz., Clostridium aminophilum, an amino acid-fermenting organism isolated from the rumen; the similarity value was 92.2%. The results of the phenotypic characterization analysis, G+C content analysis, and phylogenetic analysis of the 16S ribosomal DNA sequence set strain B316(T) apart from all of the members of cluster XIVa. We propose that strain B316(T) should be designated a new species of the genus Clostridium, Clostridium proteoclasticum. Strain B316 is the type strain and has been deposited in the American Type Culture Collection as strain ATCC 51982.

An endo-beta-1,4-glucanase gene (celA) from the rumen anaerobe Ruminococcus albus 8: cloning, sequencing, and transcriptional analysis.

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.

Identification of proteolytic rumen bacteria isolated from New Zealand cattle.

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.

Transcriptional analysis of the Clostridium cellulovorans endoglucanase gene, engB.

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.