Presence of Novel,Potentially Homoacetogenic Bacteria in the Rumen as Determined by Analysis of Formyltetrahydrofolate Synthetase Sequences from Ruminants

Homoacetogens produce acetate from H₂ and CO₂ via the Wood-Ljungdahl pathway. Some homoacetogens have been isolated from the rumen, but these organisms are expected to be only part of the full diversity present. To survey the presence of rumen homoacetogens, we analyzed sequences of formyltetrahydrofolate synthetase (FTHFS), a key enzyme of the Wood-Ljungdahl pathway. A total of 275 partial sequences of genes encoding FTHFS were PCR amplified from rumen contents of a cow, two sheep, and a deer. Phylogenetic trees were constructed using these FTHFS gene sequences and the translated amino acid sequences, together with other sequences from public databases and from novel nonhomoacetogenic bacteria isolated from the rumen. Over 90% of the FTHFS sequences fell into 34 clusters defined with good bootstrap support. Few rumen-derived FTHFS sequences clustered with sequences of known homoacetogens. Conserved residues were identified in the deduced FTHFS amino acid sequences from known homoacetogens, and their presence in the other sequences was used to determine a “homoacetogen similarity” (HS) score. A homoacetogen FTHFS profile hidden Markov model (HoF-HMM) was used to assess the homology of rumen and homoacetogen FTHFS sequences. Many clusters had low HS scores and HoF-HMM matches, raising doubts about whether the sequences originated from homoacetogens. In keeping with these findings, FTHFS sequences from nonhomoacetogenic bacterial isolates grouped in these clusters with low scores. However, sequences that formed 10 clusters containing no known isolates but representing 15% of our FTHFS sequences from rumen samples had high HS scores and HoF-HMM matches and so could represent novel homoacetogens.Feed ingested by ruminant animals is fermented in the rumen by a complex community of microbes. This community produces, among other products, the volatile fatty acids acetate, propionate, and butyrate, which are absorbed across the rumen wall and satisfy a large part of the animals'' carbon and energy requirements. Hydrogen gas (H₂) is also formed and is the major precursor of the methane (CH₄) formed in ruminant animals. This ruminant-derived CH₄ is a contributor to global greenhouse gas emissions (46) and also represents an energy loss for the animals (34). Proposed ruminant greenhouse gas mitigation strategies include using feeds that produce less CH₄ and more volatile fatty acids (31). Alternative strategies include interventions that slow or halt methanogenesis by vaccination, using natural inhibitors found in plants, and supplementing feed with fats and oils or small-molecule inhibitors (31, 32). In the absence of methanogenesis, accumulation of H₂ could lead to a decrease in the rate of feed fermentation (31, 53) and hence a decrease in animal productivity. Other microbes that use H₂ without producing methane could be valuable in conjunction with intervention strategies that inhibit methanogens. This possibility has sparked interest in possible inoculation of ruminants with alternative H₂ users.Bacteria that use the Wood-Ljungdahl pathway to produce acetate from CO₂ are metabolically (6) and phylogenetically (48) diverse and are designated “homoacetogens.” Homoacetogens grow with H₂ or other suitable electron donors, such as formate or sugars, plus CO₂ as a terminal electron acceptor, heterotrophically with organic substrates such as sugars and methoxylated compounds, or mixotrophically with, e.g., H₂ and organic substrates. Homoacetogens have been reported to occur in a normally functioning rumen, but they are unlikely to compete with methanogens for H₂ (24, 25, 34). However, homoacetogens could play an important role in the disposal of H₂ if methanogens are not established in or are eliminated from the rumen (11, 17). At present, it is not clear whether resident rumen homoacetogens could fulfill the H₂ disposal role or whether homoacetogens would have to be added to the rumen to take over this role from the methanogens.Cultivation-based enumeration techniques have shown that the sizes of rumen acetogen populations range from undetectable to 1.2 × 10⁹ per g of rumen contents and that the prevalence of these acetogens depends on diet, animal age, and time of sampling (5, 7, 23, 24). Several homoacetogens, including Acetitomaculum ruminis (15), Eubacterium limosum (14, 17), Blautia schinkii, and Blautia producta (11), have been isolated from ruminants. Homoacetogens have also been isolated from the kangaroo forestomach, whose function is analogous to that of the rumen, which suggests that homoacetogenesis may play a role in hydrogen removal in the low-methane-emission forestomach (37).Because homoacetogens occur in different lineages of bacteria (48), traditional 16S rRNA gene-based surveys provide little information on their prevalence. The formyltetrahydrofolate synthetase (FTHFS) gene (fhs) has been used as a functional marker for homoacetogens, as the enzyme that it encodes catalyzes a key step in the reductive acetogenesis pathway (26). The structure of the enzyme of the homoacetogen Moorella thermoacetica has been reported, and putative functional features have been identified (27, 41, 42). FTHFS sequences from true homoacetogens differ from their homologs in sulfate-reducing bacteria and in other bacteria that degrade purines and amino acids via the glycine synthase-glycine reductase pathway (12, 21, 22, 26). At present, only a limited number of FTHFS sequences have been deposited in databases, and the vast majority of them are partial sequences retrieved from complex microbial communities. FTHFS sequences have been surveyed in sludge (39, 43, 54), termites (40, 44), salt marsh plant roots (21), horse manure (22), cow manure, freshwater sediment, rice field soil, and sewage (54), but so far only one study has investigated bovine ruminal FTHFS sequences (30). The rumen FTHFS sequences had low levels of similarity to the FTHFS sequences of known homoacetogens and could be sequences of novel homoacetogens. To our knowledge, no bacteria with these unique FTHFS sequences have been identified.The aims of this study were to assess the diversity of FTHFS gene sequences retrieved from rumen samples and to screen novel rumen isolates for the presence of FTHFS genes and test their ability to grow as homoacetogens. We used alignments of FTHFS sequences to define a homoacetogen similarity score based on the presence of diagnostic amino acids and developed a hidden Markov model to assess the likelihood that FTHFS sequences of unknown origin are sequences from true homoacetogens that are able to use H₂ or alternative electron donors for reductive acetogenesis.