Hitherto unknown [Fe-Fe]-hydrogenase gene diversity in anaerobes and anoxic enrichments from a moderately acidic fen

Abstract

Newly designed primers for [Fe-Fe]-hydrogenases indicated that (i) fermenters, acetogens, and undefined species in a fen harbor hitherto unknown hydrogenases and (ii) Clostridium- and Thermosinus-related primary fermenters, as well as secondary fermenters related to sulfate or iron reducers might be responsible for hydrogen production in the fen. Comparative analysis of [Fe-Fe]-hydrogenase and 16S rRNA gene-based phylogenies indicated the presence of homologous multiple hydrogenases per organism and inconsistencies between 16S rRNA gene- and [Fe-Fe]-hydrogenase-based phylogenies, necessitating appropriate qualification of [Fe-Fe]-hydrogenase gene data for diversity analyses.

FIG. 1.

Distribution of conserved amino acid (aa) motifs (black rectangles) in bacterial [Fe-Fe]-hydrogenases. FS4B and FS4A are [4Fe4S]-cluster binding sites (13). P1, P2, and P3 are highly conserved motifs coordinating the H-cluster (30). Amino acid positions are given in parentheses and are according to amino acid residues of the D. vulgaris hydrogenase (GenBank accession no. AAS96246). Abbreviations: N, amino terminal; C, carboxy terminal. The fragments used for the [Fe-Fe]-hydrogenase trees in Fig. 3 and Fig. S1 in the supplemental material and in Fig. 2 and Fig. S2 in the supplemental material are shaded gray and hatched, respectively.

FIG. 2.

Phylogenetic tree of amplified [Fe-Fe]-hydrogenase genes (boldface) and closely related sequences (residues 183 to 375 of the D. vulgaris hydrogenase [see Fig. 1]). The origin of enrichment-derived sequences (enrich_clone) is indicated by the following capital letters: A, 10⁻⁴ dilution of fen soil; B, 10⁻⁷ dilution of preincubated, nonsupplemented fen soil slurry; C, 10⁻² dilution of preincubated, TSB (0.3 g liter⁻¹)-supplemented fen soil slurry (for details see materials and methods in the supplemental data). The consensus tree was drawn based on neighbor-joining, maximum-parsimony, and maximum-likelihood trees. Incongruencies of tree topologies between the three methods were indicated by multifurcations. Branch lengths and bootstrap values (500 resamplings) were based on the maximum-parsimony analysis. Bootstrap values are displayed for nodes supported by all three analyses. The bar indicates 0.1 estimated change per amino acid. See Fig. S2 in the supplemental material for a tree that includes all presently available pure-culture [Fe-Fe]-hydrogenases.

FIG. 3.

Comparison of [Fe-Fe]-hydrogenase- and 16S rRNA gene-based phylogenetic trees (residues 35 to 389 of the D. vulgaris hydrogenase) (see Fig. 1). Both trees are consensus trees calculated and displayed as described for Fig. 2. Consistent monophyletic groups between both trees are shaded in gray. Examples of hydrogenases and hosts with inconsistent phylogenies are in boldface. Bars indicates 0.1 estimated change per amino acid or nucleotide. See Fig. S1 in the supplemental material for trees that include all presently available pure-culture [Fe-Fe]-hydrogenases and corresponding 16S rRNA genes.