Presence of novel, potentially homoacetogenic bacteria in the rumen as determined by analysis of formyltetrahydrofolate synthetase sequences from ruminants

Abstract

Homoacetogens produce acetate from H(2) and CO(2) 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.

FIG. 1.

Schematic diagrams of amplicons of the partial formyltetrahydrofolate synthetase (FTHFS) gene and the encoded FTHFS peptide with selected motifs and functionally conserved residues. Different regions of the FTHFS gene were amplified using FTHFS PCR A, B, or C. The nucleotide and amino acid numbers and the designations of domains and structural and (putative) functional features are based on those of the FTHFS of M. thermoacetica (27, 41, 42, 44). aa, amino acids.

FIG. 2.

Phylogenetic tree based on 740 putative formyltetrahydrofolate (FTHFS) genes deposited in the GenBank database or obtained from clone libraries from rumen contents. Only sequences covering the full FTHFS PCR A length were included. The tree was generated by neighbor joining of PAM-corrected distances between deduced amino acid sequences. Bootstrap values more than 70% for both nucleotide and amino acid sequences (•) and for only amino acid sequences (○) are indicated at nodes. Clusters that contain known homoacetogenic, sulfate-reducing, and purinolytic bacteria and other cultured bacterial isolates are indicated by shading. The number of sequences (n) and the homoacetogen similarity (HS_c) score (expressed as a percentage) are indicated in parentheses as follows: (n, HS_c score). More details on which sequences obtained from different animals fall into which clusters are given in Table S2 in the supplemental material.

FIG. 3.

Similarity of FTHFS sequences to a hidden Markov model constructed with the FTFHS sequences of known homoacetogens (HoF-HMM). The significance of the matches of translated FTHFS sequences is expressed as an HMMER bit score; the higher the score, the better the match with HoF-HMM. The sequences are sequences from verified homoacetogens (▵), sequences from other cultured bacteria that do not grow as homoacetogens (□), sequences amplified from rumen contents in this study (• and ○), and sequences amplified from a range of other environments (×) (see Table S1 in the supplemental material). Sequences amplified from rumen contents in this study that fell into clusters with a homoacetogen similarity (HS_c) score of ≥90% (•) tended to have higher HMMER bit scores than sequences with an HS_c score of <90% (○).