Functional gene analysis suggests different acetogen populations in the bovine rumen and tammar wallaby forestomach

Abstract

Reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonacetogens were identified by low "homoacetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae acetogens but without close sequences from cultured isolates. These sequences probably originated from novel acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems.

FIG. 1.

CLUSTAL W alignment of partial ACS amino acid sequences. Position 1 in this alignment corresponds to amino acid 495 in M. thermoacetica (34) and the start of domain 3 of ACS (10). Primers ACS_f and ACS_r have been marked, and residues important in coordinating the active site of ACS are indicated by an asterisk.

FIG. 2.

Maximum-likelihood trees of 16S rRNA gene and FTHFS amino acid sequences for comparison of tree topology. Bootstrap values of ≥75% are shown at nodes as closed circles for both tree construction methods or open circles for maximum-likelihood only. In the interest of space, Carboxydothermus has been abbreviated “Carbox.” Marked classifications are indicated by capital letters as follows: A, Ruminococcaceae; B, Blautia group; C, Lachnospiraceae; D, Clostridiaceae; E, Eubacteriaceae; F, Veillonellaceae; G, Peptococcaceae; H, Thermoanaerobacteraceae; I, Syntrophomonadaceae; J, Desulfobacteraceae; K, Desulfobulbaceae; L, Syntrophobacteraceae; M, Desulfohalobiaceae; N, Nitrospiraceae; O, unclassified Planctomycetales; P, methanogenic archaea. The scale bar represents 10% sequence divergence.

FIG. 3.

Maximum-likelihood trees of 16S rRNA gene and ACS amino acid sequences for comparison of tree topology. Bootstrap values of ≥75% are shown at nodes as closed circles for both tree construction methods or open circles for maximum likelihood only. In the interest of space Carboxydothermus has been abbreviated “Carbox.” Marked classifications are indicated by capital letters as follows: A, Ruminococcaceae; B, Blautia group; C, Lachnospiraceae; D, Clostridiaceae; E, Eubacteriaceae; F, Veillonellaceae; G, Peptococcaceae; H, Thermoanaerobacteraceae; I, Syntrophomonadaceae; J, Desulfobacteraceae; K, Desulfobulbaceae; L, Syntrophobacteraceae; M, Desulfohalobiaceae; N, Nitrospiraceae; O, unclassified Planctomycetales; P, methanogenic archaea. The scale bar represents 10% sequence divergence.

FIG. 4.

Phylogenetic analysis of deduced ACS amino acid sequences from the bovine rumen (bovine) and the tammar wallaby forestomach (macropod). GenBank accession numbers of reference sequences are shown after the species names. Bootstrap values of ≥75% are shown at nodes as closed circles for both tree construction methods or open circles for maximum likelihood only. The scale bar represents 10% sequence divergence.

FIG. 5.

Phylogenetic analysis of deduced FTHFS amino acid sequences from the bovine rumen (bovine) and the tammar wallaby forestomach (macropod). GenBank accession numbers of reference sequences are shown after the species names. Bootstrap values of ≥75% are shown at nodes as closed circles for both tree construction methods or open circles for maximum likelihood only. HS scores are included in brackets for OTUs recovered in the present study. The scale bar represents 10% sequence divergence.