This chapter will deal mainly with the characteristics of bacteria from the rumen that have been successfully cultivated in the laboratory. For some ecosystems, particularly those dominated by slow-growing or specialized microorganisms, it has become clear that only a very small fraction (often <1%) of the total microbial diversity has been recovered by cultural methods (Amann et al., 1995) and that descriptions of the ecosystem based on the available isolated strains can be highly misleading. These discrepancies are apparent both from comparison of direct microscopic and culturable counts, and from direct analyses of ribosomal RNA sequence diversity. In the rumen, organisms surviving in significant numbers must have growth rates sufficient to counteract the constant dilution due to turnover of rumen contents, and there are indications that the discrepancies may be less extreme. Leedle et al (1982) found that the culturable count fluctuated with time after feeding between 14% and 74% of the direct microscopic count in the rumens of animals fed on two different diets. Since the viability of several rumen species is known to change upon starvation, the lower figure could partly reflect changes in the viability of known, culturable species. Thus it is to be hoped that the major obstacles to cultivation of the most numerous rumen bacteria have been overcome by the development of sufficiently rigorous anaerobic methods and of suitable isolation media. It remains likely, however, that some functionally important groups (e.g. obligate syntrophs) may not have been recovered; Mclnerney et al (1981) used co-culture with Desulfovibrio in the presence of sulphate to isolate a fatty acid-oxidizing bacterium similar to Syntrophomonas wolfei from bovine rumen contents.

The rumen bacteria

The inhabitants of the rumen microbial eco-system, a complex consortium of different microbial groups living in symbiotic relationship with the host, act synergistically for the bioconversion of lignocellulosic feeds intovolatile fatty acids which serve as a source of energy for the animals. The constraints, imposed by the host and the feed consumed by the animal, under which these microbes have to function, have been discussed. The eco-system is specialized and buffered in a narrow range of pH, which helps the animal to maintain a very well stabilized eco-system which is not disturbed by the incoming microbial contaminants into the fermentation sac (rumen) through feed and water intake. The microbial ecosystem is well studied for the rumen of domesticated animals like cattle, sheep and goat, but it is poorly studied in buffalo and wild ruminants. The necessity to use molecular biology techniques for identification and characterization of rumen microbes has been emphasized in this review.

Rumen microbial ecosystem

[This corrects the article on p. 517 in vol. 41.].

The biology of methanogenic bacteria.

The way in which the rumen has evolved as their first digestive organ potentially affords ruminants an efficiency of protein nutrition that is not available to non-ruminant herbivores. Protein is synthesized in the gut in the form of rumen microorganisms. The necessary energy is derived from plant polysaccharides such as cellulose, and the nitrogen is derived from ammonia and amino acids in the rumen. The energy and nitrogen sources can therefore be substrates of little value to most non-ruminants. Even more important, however, is the direct availability of that microbial protein for digestion and absorption by the host animal. Herbivores which employ hind-gut fermentation can only achieve the same efficiency of microbial protein utilization by coprophagy. In contrast, microbial protein is generally the ruminant’s principal source of amino acids.

Metabolism of nitrogen-containing compounds

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 peptide-directed 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.

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The genome sequence of the rumen methanogen Methanobrevibacter ruminantium reveals new possibilities for controlling ruminant methane emissions.

Obligately anaerobic, filterable microorganisms have been isolated from the rumens of cattle and sheep. On the basis of typical colonial appearance, lack of cell wall, filterability through a 450-nm membrane filter, absence of reversion to a bacterial form, and inhibition of growth by homologous antiserum, these organisms fit the description for organisms in the order Mycoplasmatales. They exhibit unique cultural, biochemical, and serological properties, and we have thus placed them in a new genus, Anaeroplasma. A. abactoclasticum is the type species of the new genus. Strain 6-1, a sterol-requiring strain, has been designated as the type strain of A. abactoclasticum and has been deposited in the American Type Culture Collection as ATCC 27879; a non-sterol-requiring strain, number 161, has been deposited as ATCC 27880. The relationship of Anaeroplasma abactoclasticum to Acholesplasma bactoclasticum, an anaerobic mycoplasma possessing extracellular proteolytic and bactoclastic enzymes, is discussed.

Anaeroplasma abactoclasticum gen.nov., sp.nov.: an Obligately Anaerobic Mycoplasma from the Rumen

Microorganisms in ruminal ingesta and pure cultures of anaerobic ruminal bacteria of different physiological and morphological groups incorporated (14)C from labeled 2-methylbutyrate during growth. The radioactivity was incorporated mainly into lipid and protein. Isoleucine was the only labeled amino acid found in acid hydrolysates of protein from either pure or mixed cultures. Radioactivity in isoleucine synthesized from 2-methylbutyrate-1-(14)C was entirely in carbon-2. Thus, the carboxylation of 2-methylbutyrate is a pathway for synthesis of isoleucine different from that operative in many aerobic and facultative microorganisms. The specific activity of isoleucine from 2-methylbutyrate by Bacteroides rumminicola 23 increased with higher concentrations of 2-methylbutyrate (2.6 to 44 x 10(-5)m) in the growth medium. At the highest concentration, the specific activity of isoleucine synthesized was 40% of the specific activity of the 2-methylbutyrate in the growth medium. The use of enzymatic casein hydrolysate, oxytocin, or vasopressin rather than ammonia as nitrogen source for growth of strain 23 depressed the incorporation of 2-methylbutyrate into isoleucine. Synthesis of isoleucine from 2-methylbutyrate appears to be an important reaction in the rumen.

Isoleucine Biosynthesis from 2-Methylbutyric Acid by Anaerobic Bacteria from the Rumen

Evidence for reductive carboxylation of succinate to synthesize alpha-ketoglutarate was sought in anaerobic heterotrophs from the rumen and from other anaerobic habitats. Cultures were grown in media containing unlabeled energy substrates plus [14C]succinate, and synthesis of cellular glutamate with a much higher specific activity than that of cellular asparate was taken as evidence for alpha-ketoglutarate synthase activity. Our results indicate alpha-ketoglutarate synthase functions in Selenomonas ruminantium, Veillonella alcalescens, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides uniformis, Bacteroides distasonis, and Bacteroides multiacidus. Evidence for this carboxylation was not found in strains representative of 10 other species.

Synthesis of alpha-ketoglutarate by reductive carboxylation of succinate in Veillonella, Selenomonas, and Bacteriodes species.

An anaerobic bacteriolytic mycoplasma was isolated by Robinson and Hungate and designated Acholeplasma bactoclasticum sp.n. We found that this strain and four similar isolates from the ovine rumen have a requirement for cholesterol and thus do not belong in the genus Acholeplasma. The morphological, cultural, and biochemical properties of these organisms are similar to those of a group of non-bacteriolytic anaerobic mycoplasmas that do not fit well in genera previously established for mycoplasmas and thus have been placed in a newly described genus, Anaeroplasma. We propose that the organisms isolated by Robinson and Hungate be transferred to the new genus as Anaeroplasma bactoclasticum (Robinson and Hungate) comb. nov., of which ATCC 27112 is the type strain.

Transfer of Acholeplasma bactoclasticum Robinson and Hungate to the Genus Anaeroplasma (Anaeroplasma bactoclasticum [Robinson and Hungate] comb.nov.): Emended Description of the Species

Microbes in ruminal contents incorporated (14)C into cells when they were incubated in vitro in the presence of [(14)C]carboxyl-labeled indole-3-acetic acid (IAA). Most of the cellular (14)C was found to be in tryptophan from the protein fractions of the cells. Pure cultures of several important ruminal species did not incorporate labeled IAA, but all four strains of Ruminococcus albus tested utilized IAA for tryptophan synthesis. R. albus did not incorporate (14)C into tryptophan during growth in medium containing either labeled serine or labeled shikimic acid. The mechanism of tryptophan biosynthesis from IAA is not known but appears to be different from any described biosynthetic pathway. We propose that a reductive carboxylation, perhaps involving a low-potential electron donor such as ferredoxin, is involved.

Isadore M. Robinson

Papers

Anaeroplasma abactoclasticum gen.nov., sp.nov.: an Obligately Anaerobic Mycoplasma from the Rumen

Isoleucine Biosynthesis from 2-Methylbutyric Acid by Anaerobic Bacteria from the Rumen

Synthesis of alpha-ketoglutarate by reductive carboxylation of succinate in Veillonella, Selenomonas, and Bacteriodes species.

Transfer of Acholeplasma bactoclasticum Robinson and Hungate to the Genus Anaeroplasma (Anaeroplasma bactoclasticum [Robinson and Hungate] comb.nov.): Emended Description of the Species

Tryptophan Biosynthesis from Indole-3-Acetic Acid by Anaerobic Bacteria from the Rumen