Novel [NiFe]- and [FeFe]-hydrogenase gene transcripts indicative of active facultative aerobes and obligate anaerobes in earthworm gut contents

Abstract

The concomitant occurrence of molecular hydrogen (H(2)) and organic acids along the alimentary canal of the earthworm is indicative of ongoing fermentation during gut passage. Fermentative H(2) production is catalyzed by [FeFe]-hydrogenases and group 4 [NiFe]-hydrogenases in obligate anaerobes (e.g., Clostridiales) and facultative aerobes (e.g., Enterobacteriaceae), respectively, functional groups that might respond differently to contrasting redox conditions. Thus, the objectives of this study were to assess the redox potentials of the alimentary canal of Lumbricus terrestris and analyze the hydrogenase transcript diversities of H(2) producers in glucose-supplemented gut content microcosms. Although redox potentials in the core of the alimentary canal were variable on an individual worm basis, average redox potentials were similar. The lowest redox potentials occurred in the foregut and midgut regions, averaging 40 and 110 mV, respectively. Correlation plots between hydrogenase amino acid sequences and 16S rRNA gene sequences indicated that closely related hydrogenases belonged to closely related taxa, whereas distantly related hydrogenases did not necessarily belong to distantly related taxa. Of 178 [FeFe]-hydrogenase gene transcripts, 177 clustered in 12 Clostridiales-affiliated operational taxonomic units, the majority of which were indicative of heretofore unknown hydrogenases. Of 86 group 4 [NiFe]-hydrogenase gene transcripts, 79% and 21% were affiliated with organisms in the Enterobacteriaceae and Aeromonadaceae, respectively. The collective results (i) suggest that fermenters must cope with variable and moderately oxidative redox conditions along the alimentary canal, (ii) demonstrate that heretofore undetected hydrogenases are present in the earthworm gut, and (iii) corroborate previous findings implicating Clostridiaceae and Enterobacteriaceae as active fermentative taxa in earthworm gut content.

Fig. 1.

In situ redox potentials (E_h) of a radial microsensor profile of the crop/gizzard region of L. terrestris (A) and in situ redox potentials along the alimentary canal of L. terrestris (B). The worm radius equals 100% (i.e., 3 mm) in panel A. The pattern shown in panel A is representative of replicate analyses, and the means of 9 to 15 replicates per region are shown in panel B (error bars indicate positive standard deviations).

Fig. 2.

Phylogenetic tree of in silico-translated amino acid sequences derived from [FeFe]-hydrogenase gene transcripts (boldface) and closely related sequences. GenBank accession numbers are indicated in parentheses. Sequences correspond to residues 183 to 375 of the Desulfovibrio vulgaris hydrogenase (GenBank accession no. AAS96246). The consensus tree was drawn on the basis of neighbor-joining, maximum parsimony, and maximum likelihood trees. Solid and open circles, nodes congruent in all three analyses and two analyses, respectively. Branch lengths were based on the neighbor-joining analysis. Bootstrap values are means from maximum parsimony trees (100 resamplings) and neighbor-joining trees (1,000 resamplings) and are displayed only for nodes supported by all three analyses and values above 50%. Sequences were affiliated within 13 different OTUs (gray boxes) on the basis of an amino acid sequence threshold similarity of 80% (see Table S2 in the supplemental material). A total of 178 sequences were analyzed; representative sequences are shown for each OTU. The bar indicates a 0.1 change per amino acid.

Fig. 3.

Phylogenetic tree of in silico-translated amino acid sequences derived from [NiFe]-hydrogenase gene transcripts (boldface) and closely related sequences. GenBank accession numbers are indicated in parentheses. Sequences correspond to residues 341 to 710 of the E. coli hydrogenase 3 HycE protein (GenBank accession no. AAC75763). The consensus tree was drawn as described for Fig. 2. Bootstrap values are means from maximum parsimony trees (500 resamplings), maximum likelihood trees (10 resamplings), and neighbor-joining trees (1000 resamplings). A total of 106 sequences were analyzed; representative sequences are shown for each phylogenic position. The bar indicates a 0.1 change per amino acid.