Diversity of dissimilatory bisulfite reductase genes of bacteria associated with the deep-sea hydrothermal vent polychaete annelid Alvinella pompejana

Abstract

A unique community of bacteria colonizes the dorsal integument of the polychaete annelid Alvinella pompejana, which inhabits the high-temperature environments of active deep-sea hydrothermal vents along the East Pacific Rise. The composition of this bacterial community was characterized in previous studies by using a 16S rRNA gene clone library and in situ hybridization with oligonucleotide probes. In the present study, a pair of PCR primers (P94-F and P93-R) were used to amplify a segment of the dissimilatory bisulfite reductase gene from DNA isolated from the community of bacteria associated with A. pompejana. The goal was to assess the presence and diversity of bacteria with the capacity to use sulfate as a terminal electron acceptor. A clone library of bisulfite reductase gene PCR products was constructed and characterized by restriction fragment and sequence analysis. Eleven clone families were identified. Two of the 11 clone families, SR1 and SR6, contained 82% of the clones. DNA sequence analysis of a clone from each family indicated that they are dissimilatory bisulfite reductase genes most similar to the dissimilatory bisulfite reductase genes of Desulfovibrio vulgaris, Desulfovibrio gigas, Desulfobacterium autotrophicum, and Desulfobacter latus. Similarities to the dissimilatory bisulfite reductases of Thermodesulfovibrio yellowstonii, the sulfide oxidizer Chromatium vinosum, the sulfur reducer Pyrobaculum islandicum, and the archaeal sulfate reducer Archaeoglobus fulgidus were lower. Phylogenetic analysis separated the clone families into groups that probably represent two genera of previously uncharacterized sulfate-reducing bacteria. The presence of dissimilatory bisulfite reductase genes is consistent with recent temperature and chemical measurements that documented a lack of dissolved oxygen in dwelling tubes of the worm. The diversity of dissimilatory bisulfite reductase genes in the bacterial community on the back of the worm suggests a prominent role for anaerobic sulfate-reducing bacteria in the ecology of A. pompejana.

FIG. 1

Agarose gel of PCR amplification products generated by using primers (P94-F and P95-R) for dissimilatory bisulfite reductase genes. Template DNA was isolated from the epibiotic microbial community on the dorsal surface of A. pompejana (lanes 1 to 11) and from the pRKS72 plasmid that contains the dsvA and -B genes (dissimilatory bisulfite reductase of D. vulgaris) (22). A. pompejana specimens were collected from 13°N (lanes 1 to 5) and 9°N (lanes 6 to 11) of the East Pacific Rise. The microbial communities located on the anterior (ant [lanes 1 and 2 and 6 to 8]) and posterior (post [lanes 3 to 5 and 9 to 11]) of A. pompejana specimens were assayed separately. The dsv amplification product is 1.4 kb (22). The image was prepared with Adobe Photoshop.

FIG. 2

Agarose gel of the MboI restriction patterns of the 11 clone families identified in the clone library of dissimilatory bisulfite reductase gene fragments amplified from the A. pompejana epibiotic bacterial community. The image was prepared with Adobe Photoshop.

FIG. 3

Alignment of conceptual translations of the 11 clone families with the homologous region of the gene coding for the alpha subunit of the dissimilatory bisulfite reductases of D. vulgaris, D. gigas, Desulfobacterium autotrophicum, A. fulgidus, C. vinosum, and P. islandicum. Approximately 390 nucleotides from the 5′ end of each clone is represented. The alignment was made by using CLUSTAL. The boxes indicate identity greater than or equal to 65%, and dashes indicate gaps in the alignment.

FIG. 4

Dendrogram showing the relationships among the 11 clone families of dissimilatory bisulfite reductase genes and the alpha subunits of the dissimilatory bisulfite reductase genes of D. vulgarus, D. gigas, D. autotrophicum, A. fulgidus, C. vinosum, and P. islandicum. The analysis was made by using the neighbor-joining algorithm and 107 positions of aligned conceptual translations. The consensus of 100 bootstrap resamplings is shown with bootstrap values adjacent to the nodes.