Molecular profiling and identification of methanogenic archaeal species from rabbit caecum

During a comparison of 16S rDNA PCR-denaturant gradient gel electrophoresis (DGGE) profiles of methanogenic archaea from rumen fluid, rabbit caecum and pig feces, a unique band common to all rabbit caecum samples was observed. DGGE profiling also showed that the methanogen community from the New Zealand White adult rabbits is different and less complex than the methanogen communities from the rumen and pig feces. Small subunit ribosomal gene sequences of methanogenic archaea were subsequently retrieved from the constructed rabbit caecum 16S rDNA gene library. Results of the phylogenetic analysis indicated that rabbit caecum is inhabited by members of the genus Methanobrevibacter and is possibly one-species dominated, because all the retrieved sequences exhibited similarity values of 99% or higher. This species may well be a novel species of the genus Methanobrevibacter. It belongs to a distinct phylogenetic group containing Methanobrevibacter woesei, Methanobrevibacter thaueri and Methanobrevibacter gottschalkii strains isolated from animal feces, and Methanobrevibacter smithii from the predominating methanogen population of the human large bowel.     

Diversity of Phytases in the Rumen

Examples of a new class of phytase related to protein tyrosine phosphatases (PTP) were recently isolated from several anaerobic bacteria from the rumen of cattle. In this study, the diversity of PTP-like phytase gene sequences in the rumen was surveyed by using the polymerase chain reaction (PCR). Two sets of degenerate primers were used to amplify sequences from rumen fluid total community DNA and genomic DNA from nine bacterial isolates. Four novel PTP-like phytase sequences were retrieved from rumen fluid, whereas all nine of the anaerobic bacterial isolates investigated in this work contained PTP-like phytase sequences. One isolate, Selenomonas lacticifex, contained two distinct PTP-like phytase sequences, suggesting that multiple phytate hydrolyzing enzymes are present in this bacterium. The degenerate primer and PCR conditions described here, as well as novel sequences obtained in this study, will provide a valuable resource for future studies on this new class of phytase. The observed diversity of microbial phytases in the rumen may account for the ability of ruminants to derive a significant proportion of their phosphorus requirements from phytate.     

Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) estimated by 16S rDNA homology analyses

Prokaryote diversity in the rumen of yak (Bos grunniens) and Jinnan cattle (Bos taurus) was estimated by 16S rDNA homology analysis. Two rumen 16S rDNA libraries were constructed. Of the 194 clones in the library of yak rumen, the sequences were mainly clustered to two phyla, low G+C Gram-positive bacteria (LGCGPB, 54.12% total clones) and Bacteroidetes (30.93%), respectively. While in the 197 clone-library of the cattle rumen, the sequences were mainly related to three phyla, Bacteroidetes (39.59%), gamma-Proteobacteria (26.9%) and LGCGPB (22.34%), respectively. The sequence analysis indicated that more than half of the species harbored in yak rumen belonged to the not-yet-cultured groups at <90% 16S rDNA similarity levels with cultured species, while 36% 16S rDNA sequences amplified from the rumen of Jinnan cattle fell in these catalogues. By comparing the uncultured sequences in yak rumen with those in Jinnan cattle and cow, the former formed distinct clusters loosely related to the later, implying that yak rumen could harbor some special prokaryote phyla. 10.8% sequences retrieved in yak rumen were related to the known rumen fibrolytic bacterial species; however none was related to the known amylolysis species. While 4% and 17.8% sequences retrieved from Jinnan cattle rumen were related to cultured fibrolytic and amylolysis species, respectively. The bacterial structures seemed to be in accordance with the feed of the two kinds of animals. In both rumens, retrieved methanogenic Archaea-related 16S rDNA sequences were at an unreasonable low level; in addition, none sequence was related to Ruminococcus albus, a classical rumen fibrolytic species. The reason can be due to the experimental biases.     

High genetic diversity and different distributions of glycosyl hydrolase family 10 and 11 xylanases in the goat rumen

Background

The rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment.

Methodology/Principal Findings

We explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11) in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95%) were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family) were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific.

Conclusion/Significance

The numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen microenvironment.     

晋南牛瘤胃中古菌分子多样性的研究

采用3对古菌特异性引物扩增瘤胃古菌16S rRNA基因分别建立克隆库来研究晋南牛瘤胃古菌的多样性.每个克隆库随机挑选100个克隆.引物Arch f364/1386建立的克隆库中,克隆分为四类,分别与四种甲烷短杆菌1Y(61%)、SM9(23%)、NT7(14%)和AK-87(2%)相似.引物1Af/1100Ar建立的克隆库中,克隆分为两类,分别与Methanobacterium aarhusense(72%)和Methanosphaera stadtmanae DSM 3091(28%)相似.引物Met86F/Met1340R建立的克隆库反映的古菌种类较为全面,除以上4种甲烷短杆菌(所占比例分别为47%、26%、11%和3%)外,还有Methanomicrobium mobile(2%)、以及类似Methanobacterium aarhusense(1%)和Methanosphaera stadtmanae(3%)的序列,还有7%的未匹配序列.系统进化分析表明,这些克隆属于Methanobrevibacter、Methanobacterium、Methanosphaera、Methanomicrobium,和未知广域古菌等5个分支.有25类属于广域古菌的未知序列,提示瘤胃中存在大量的未知产甲烷菌.     

Diversity of bacteria associated with the coral Pocillopora damicornis from the Great Barrier Reef

The microbial community associated with the reef building coral Pocillopora damicornis located on the Great Barrier Reef was investigated using culture-independent molecular microbial techniques. The microbial communities of three separate coral colonies were assessed using clone library construction alongside restriction fragment length polymorphism and phylogenetic analysis. Diversity was also investigated spatially across six replicate samples within each single coral colony using 16S rDNA and rpoB-DGGE analysis. Clone libraries demonstrated that the majority of retrieved sequences from coral tissue slurry libraries affiliated with gamma-Proteobacteria. This contrasted with clone libraries of seawater and coral mucus, which were dominated by alpha-Proteobacteria. A number of retrieved clone sequences were conserved between coral colonies; a result consistent with previous studies suggesting a specific microbe-coral association. rpoB-DGGE patterns of replicate tissue slurry samples underestimated microbial diversity, but demonstrated that fingerprints were identical within the same coral. These fingerprints were also conserved across coral colonies. The 16S rDNA-DGGE patterns of replicate tissue slurry samples were more complex, although non-metric multidimensional scaling (nMDS) analysis showed groupings of these banding patterns indicating that some bacterial diversity was uniform within a coral colony. Sequence data retrieved from DGGE analysis support clone library data in that the majority of affiliations were within the gamma-Proteobacteria. Many sequences retrieved also affiliated closely with sequences derived from previous studies of microbial diversity of healthy corals in the Caribbean. Clones showing high 16S rDNA sequence identity to both Vibrio shiloi and Vibrio coralliilyticus were retrieved, suggesting that these may be opportunist pathogens. Comparisons of retrieved microbial diversity between two different sampling methods, a syringe extracted coral mucus sample and an airbrushed coral tissue slurry sample were also investigated. Non-metric multidimensional scaling of clone library data highlighted that clone diversity retrieved from a coral mucus library more closely reflected the diversity of surrounding seawater than a corresponding coral tissue clone library.     

采用未培养技术对荷斯坦奶牛瘤胃细菌多样性进行初步分析

采用未培养(Culture independent)技术直接从荷斯坦奶牛瘤胃液中提取瘤胃细菌微生物混合DNA(也叫元基因组DNA),利用细菌16SrDNA通用引物27F与1492R,扩增瘤胃混合微生物的16SrDNA,根据16SrDNA序列对瘤胃细菌多样性进行初步分析。通过16SrDNA序列同源性分析,发现有多于一半以上的序列与可培养的菌株的同源性小于90%,属于不可培养的菌株。选用45条测得序列与已知序列构建系统发育树,分析结果表明,它们分属于两大类LGCGPB(the lowG CGram positivebac teria)和CFB(Cytophaga_Flexibacter $CBacteroides group),剩下的克隆尚难确定其分类地位,可能是代表新属和种的序列,这些序列已向GenBank提交并得到序列号(AY986777_AY986791)。     

Isolation and biochemical characterization of two lipases from a metagenomic library of China Holstein cow rumen

Two novel lipase genes RlipE1 and RlipE2 which encoded 361- and 265-amino acid peptides, respectively, were recovered from a metagenomic library of the rumen microbiota of Chinese Holstein cows. A BLAST search revealed a high similarity (90%) between RlipE2 and a carboxylesterase from Thermosinus carboxydivorans Nor1, while there was a low similarity (below 50%) between RlipE1 and other lipases. Phylogenetic analysis indicated that RlipE2 clustered with the lipolytic enzymes from family V while RlipE1 clustered with six other putative bacterial lipases which might constitute a new subfamily. The recombinant lipases were thermally unstable and retained 60% activity over a pH range of 6.5-8.5. Substrate specificity assay indicated that both enzymes had higher hydrolytic activity toward laurate (C₁₂), palmitate (C₁₆) and stearate (C₁₈). The novel phylogenetic affiliation and high specificity of both enzymes for long-chain fatty acid make them interesting targets for manipulation of rumen lipid metabolism.     

16S rDNA library-based analysis of ruminal bacterial diversity

Bacterial 16S rDNA sequence data, incorporating sequences > 1 kb, were retrieved from published rumen library studies and public databases, then were combined and analysed to assess the diversity of the rumen microbial ecosystem as indicated by the pooled data. Low G+C Gram positive bacteria (54%) and the Cytophaga-Flexibacter-Bacteroides (40%) phyla were most abundantly represented. The diversity inferred by combining the datasets was much wider than inferred by individual studies, most likely due to different diets enriching for bacteria with different fermentative activities. A total of 341 operational taxonomic units (OTU) was predicted by the Chao1 non-parametric estimator approach. Phylogenetic and database analysis demonstrated that 89% of the diversity had greatest similarity to organisms which had not been cultivated, and that several sequences are likely to represent novel taxonomic groupings. Furthermore, of the 11% of the diversity represented by cultured isolates (> 95% 16S rDNA identity), not all of the bacteria were of ruminal origin. This study therefore reinforces the need to reconcile classical culture-based rumen microbiology with molecular ecological studies to determine the metabolic role of uncultivated species.     

Investigation of the FeFe-hydrogenase gene diversity combined with phylogenetic microbial community analysis of an anaerobic domestic sewage sludge

Biological hydrogen production through the anaerobic digestion is an environmental friendly alternative for satisfying future hydrogen demands. Microorganisms residing into waste water treatment plants are far from being exhaustively characterized and surveys on hydrogen production through FeFe-hydrogenase in such ecosystems are scarce. This study combined the analysis of 16S rRNA and [FeFe]-hydrogenase (hydA) genes with statistical tools to estimate richness and diversity of the microbial community of a domestic sewage treatment plant at the phylogenetic and functional levels. Archaeal groups were represented by 69 % of sequences assigned to Methanosarcinales and the remaining belonged to Methanomicrobiales. Within the bacterial library, 136 operational taxonomic units (OTUs) were distributed into 9 phyla, being 86 OTUs related to uncultivated bacteria. From these, 25 OTUs represented potential novel taxa within Synergistetes. Proteobacteria was the most predominant (36 % of the OTUs) and diversified phylogenetic group in the bacterial library, most of them assigned to the class Betaproteobacteria. Twenty-two putative hydA sequences were recovered into four distinct clusters and most of them were more closely related to each other than with sequences retrieved from databases, indicating they are hitherto undetected [Fe–Fe]-hydrogenase gene sequences. The richness estimates revealed that the number of sampled sequences was enough for full coverage of the archaeal diversity but not sufficient to cover both bacterial and hydA gene diversities. The results confirmed a great richness and diversity of bacterial and hydA sequences retrieved from the sewage sludge sample, suggesting such environment as a potential reservoir of new hydrogenase genes for biotechnological exploration.     

Molecular identification of methanogenic archaea from sheep in Queensland, Australia reveal more uncultured novel archaea

Molecular diversity of rumen methanogens in sheep in Queensland, Australia was investigated using 16S rRNA gene libraries prepared from pooled rumen contents from nine merino sheep. A total of 78 clones were identified revealing 26 different sequences. Of these 26 sequences, eight sequences (15 clones) were 95-100% similar to cultivated methanogens belonging to the orders Methanobacteriales and Methanomicrobiales, and the remaining 18 phylotypes (63 clones) were 72-75% similar to Thermoplasma acidophilum and Thermoplasma volcanium. These unique sequences clustered within a distinct and strongly supported (100% bootstrap support) phylogenetic group, exclusively composed of sequences from uncharacterized archaea from very diverse anaerobic environments. Members of this unique group that were previously considered atypical for the rumen environment were the predominant clones.     

Metagenomic analysis of Surti buffalo (Bubalus bubalis) rumen: a preliminary study

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial proteins, short chain fatty acids and gases. In this study, metagenomic approaches were used to study the microbial populations and metabolic potential of the microbial community. DNA was extracted from Surti Buffalo rumen samples (four treatments diet) and sequenced separately using a 454 GS FLX Titanium system. We used comparative metagenomics to examine metabolic potential and phylogenetic composition from pyrosequence data generated in four samples, considering phylogenetic composition and metabolic potentials in the rumen may remarkably be different with respect to nutrient utilization. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of fermentation of carbohydrates in a high roughage diet. The distribution of phylotypes and environmental gene tags (EGTs) detected within each rumen sample were dominated by Bacteroidetes/Chlorobi, Firmicutes and Proteobacteria in all the samples. The results of this study could help to determine the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals.     

Microbial and Carbohydrate Active Enzyme profile of buffalo rumen metagenome and their alteration in response to variation in the diet

Rumen microbiome represents rich source of enzymes degrading complex plant polysaccharides. We describe here analysis of Carbohydrate Active Enzymes (CAZymes) from 3.5 gigabase sequences of metagenomic data from rumen samples of Mehsani buffaloes fed on different proportions of green or dry roughages to concentrate ration. A total of 2597 contigs encoding putative CAZymes were identified by CAZyme Analysis Toolkit (CAT). The phylogenetic analysis of these contigs by MG-RAST revealed predominance of Bacteroidetes, followed by Firmicutes, Proteobacteria, and Actinobacteria phyla. Moreover, a higher abundance of oligosaccharide degrading and debranching enzymes in buffalo rumen metagenome and that of cellulases and hemicellulases in termite hindgut was observed when we compared glycoside hydrolase (GH) profile of buffalo rumen metagenome with cow rumen, termite hindgut and chicken caecum metagenome. Further, comparison of microbial profile of green or dry roughage fed animals showed significantly higher abundance (p-value < 0.05) of various polysaccharide degrading bacterial genera like Fibrobacter, Prevotella, Bacteroides, Clostridium and Ruminococcus in green roughage fed animals. In addition, we found a significantly higher abundance (p-value < 0.05) of enzymes associated with pectin digestion such as pectin lyase (PL) 1, PL10 and GH28 in green roughage fed animals. Our study outlines CAZyme profile of buffalo rumen metagenome and provides a scope to study the role of abundant enzyme families (oligosaccharide degrading and debranching enzymes) in digestion of coarse feed.     

Variability of Actinobacteria, a minor component of rumen microflora

Actinobacteria (Actinomycetes) are a significant and interesting group of gram-positive bacteria. They are regular, though infrequent, members of the microbial life in the rumen and represent up to 3 % of total rumen bacteria; there is considerable lack of information about ecology and biology of rumen actinobacteria. During the characterization of variability of rumen treponemas using non-cultivation approach, we also noted the variability of rumen actinobacteria. By using Treponema-specific primers a specific 16S rRNA gene library was prepared from cow and sheep rumen total DNA. About 10 % of recombinant clones contained actinobacteria-like sequences. Phylogenetic analyses of 11 clones obtained showed the high variability of actinobacteria in the ruminant digestive system. While some sequences are nearly identical to known sequences of actinobacteria, we detected completely new clusters of actinobacteria-like sequences, representing probably new, as yet undiscovered, group of rumen Actinobacteria. Further research will be necessary for understanding their nature and functions in the rumen.     

牦牛瘤胃元基因组文库中木聚糖酶基因的分析

【目的】了解牦牛瘤胃微生物木聚糖酶多样性及其降解特征,为木聚糖降解提供新的基因资源。【方法】根据对已构建的瘤胃微生物元基因组细菌人工染色体(BAC)克隆文库高通量测序结果的注释,筛选其中编码木聚糖酶的基因并进行多样性分析;对其中一个木聚糖酶基因及其连锁的木糖苷酶基因进行克隆表达和酶学性质表征,分析其协同作用。【结果】共筛选到14个木聚糖酶基因,均编码GH10家族木聚糖酶,其氨基酸序列之间的相似性为20.5%-91.3%;其中7个木聚糖酶基因所在的不同的DNA片段(contig)上存在木糖苷酶基因,编码的木糖苷酶属于GH43或GH3糖苷水解酶家族。将其中一对连锁的木聚糖酶(Xyn32)和木糖苷酶基因(Xyl33)分别克隆、表达和纯化。纯化后的木聚糖酶比活为1.98 IU/mg,但不具有阿魏酸酯酶活性;木糖苷酶比活为0.07 U/mg,且具有α-阿拉伯呋喃糖苷酶活性。体外实验证明,木糖苷酶Xyl33对与之连锁的木聚糖酶Xyn32的木聚糖降解具有协同作用。     

Detection of methanogens and proteobacteria from a single cell of rumen ciliate protozoa

Rumen ciliate-associated bacteria and methanogenic archaea were analyzed by a 16S rRNA gene retrieved from a single cell of Polyplastron multivesiculatum, Isotricha intestinalis, and Ophryoscolex purkynjei. Rumen fluid was taken from a ruminally fistulated goat to prepare a ciliate fraction. Ciliate mixtures were incubated under mixtures of antibiotics for 48 h to eliminate extracellular bacteria. Individual cells of rumen ciliates were selected under microscopic observation after fixation with ethanol. Bacterial and archaeal 16S rRNA gene sequences were retrieved from each cell of three genera of ciliate. Two archaeal sequences related to Methanobrevibacter smithii were distributed to nearly all ciliate cells tested. These two methanogenic archaea were likely to be endosymbiotic methanogens commonly carried by the rumen ciliate, although some other sequences similar to the other genera were detected. A range of proteobacteria was retrieved from cells of P. multivesiculatum. Some sequences showed similarities to the previously known endosymbiotic proteobacteria. However, there were no proteobacteria that were carried by all the ciliate cells tested.     

Methyl coenzyme M reductase (<Emphasis Type="Italic">mcrA</Emphasis>) gene based phylogenetic analysis of methanogens population in Murrah buffaloes (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

The aim of the present study was to decipher the diversity of methanogens in rumen of Murrah buffaloes so that effective strategies can be made in order to mitigate methane emission from these methanogens. In the present study diversity of rumen methanogens in Murrah buffaloes (Bubalus bubalis) from North India was evaluated by using mcr-A gene library obtained from the pooled PCR product from four animals and by using MEGA4 software. A total of 104 clones were examined, revealing 26 different mcr-A gene sequences or phylotypes. Of the 26 phylotypes, 16 (64 of 104 clones) were less than 97% similar to any of the cultured strain of methanogens. Seven clone sequences were clustered with Methanomicrobium mobile and three clone sequences were clustered with Methanobrevibacter gottschalkii during the phylogenetic analysis. Uncultured group of methanogens comes out to be the major component of the methanogens community structure in Murrah buffaloes. Methanomicrobium phylotype comes out to be major phylotype among cultured methanogens followed by Methanobrevibacter phylotype. These results help in making effective strategies to check the growth of dominant communities in the rumen of this animal which in turn help in the reduction of methane emission in the environment and ultimately helps us in fighting with the problem of global warming.     

Abundance and genetic diversity of microbial polygalacturonase and pectate lyase in the sheep rumen ecosystem

Background

Efficient degradation of pectin in the rumen is necessary for plant-based feed utilization. The objective of this study was to characterize the diversity, abundance, and functions of pectinases from microorganisms in the sheep rumen.

Methodology/Principal Findings

A total of 103 unique fragments of polygalacturonase (PF00295) and pectate lyase (PF00544 and PF09492) genes were retrieved from microbial DNA in the rumen of a Small Tail Han sheep, and 66% of the sequences of these fragments had low identities (<65%) with known sequences. Phylogenetic tree building separated the PF00295, PF00544, and PF09492 sequences into five, three, and three clades, respectively. Cellulolytic and noncellulolytic Butyrivibrio, Prevotella, and Fibrobacter species were the major sources of the pectinases. The two most abundant pectate lyase genes were cloned, and their protein products, expressed in Escherichia coli, were characterized. Both enzymes probably act extracellularly as their nucleotide sequences contained signal sequences, and they had optimal activities at the ruminal physiological temperature and complementary pH-dependent activity profiles.

Conclusion/Significance

This study reveals the specificity, diversity, and abundance of pectinases in the rumen ecosystem and provides two additional ruminal pectinases for potential industrial use under physiological conditions.     

Phylogenetic analysis of 16S rRNA gene sequences reveals rumen bacterial diversity in Yaks (<Emphasis Type="Italic">Bos grunniens</Emphasis>)

Six matured male Yaks (Bos grunniens) with a mean live weight of 450 ± 23 kg (mean ± SD), were housed indoors in metabolism cages and fed pelleted lucerne (Medicago sativum). After an adjustment period of 24 days of feeding the diet, samples of rumen content were obtained for analysis of the bacteria in the liquor. The diversity of rumen bacteria was investigated by constructing a 16S rRNA gene clone library using the general bacterial primers F27 and R1492. A total of 130 clones, comprising nearly full length sequences (approx. 1.5 kb) were sequenced and submitted to BLAST and phylogenetic analysis. Using the criterion that similarity of 97% or greater with the sequences of cultivated bacteria, 16 clones were identified as Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Ruminococcus flavefaciens, Succiniclasticum ruminis, Selenomonas ruminantium and Prevotella ruminicola, respectively. A further 10 clones shared similarity ranging from 90 to 97% with cultivated bacteria but the similarity in sequences for the remaining 104 clones were less than 90% of those of cultivated bacteria. Using a phylogenetic analysis it was found that the majority of the clones identified (63.8%) were located in the Low G + C Subdivision, with most of the remainder (35.4% of clones) located in the Cytophaga-Flexibacter-Bacteroides phylum and one clone (0.8%) was identified as a Proteobacteria. It was apparent that Yaks have a large and diverse range of bacteria in the rumen content which differ from those of cattle and other ruminants.     

Systematics and evolution of ruminal species of the genus Prevotella

Bacterial species of the genusPrevotella represent a numerically dominant microbial population in the rumen of cattle. They belong to the phylogenetic divisionCytophaga-Flexibacter-Bacteroides (CFB) which is a large group of ecologically diverse bacteria with only a few shared traits. The phylogenetic descent from a common ancestor seems to be unquestionable, however, as judged from the small subunit ribosomal RNA analysis. Only 4 ruminalPrevotella species have been described to date, even though the sequence analysis of directly retrieved 16S rRNA genes indicates a large genetic diversity within this group of rumen bacteria. The closest relatives of ruminalPrevotella spp. are not surprisingly other species of the genusPrevotella, typically inhabiting the gastrointestinal tract, oral cavity and genital areas of other animals and man. The previous phylogenetic analysis showed that species of the genusPrevotella can be split into two groups or superclusters, the “ruminal” and the “non-ruminal prevotellas”. One of 4 currently described ruminalPrevotella spp.,i.e. P. albensis, has been placed outside the supercluster containing ruminalPrevotella spp. and within the supercluster containing the non-ruminalPrevotella spp. However, the number of available small subunit rRNA sequences from this species represents only a fraction of all known ruminalPrevotella sequences.