Phylogenetic diversity and community structure of sulfate-reducing bacteria in a salt marsh sediment

Phylogenetic diversity and community structure of sulfate-reducing bacteria (SRB) in a salt marsh sediment and rhizosphere of Spartina alterniflora were investigated. Uncultivated phylotypes were studied by selectively amplifying Desulfobacteriaceae 16S rRNA gene fragments from DNA extracted from salt marsh rhizosphere samples. An in vitro transcription technique was developed to synthesize reference RNAs containing sequences presumably identical to corresponding regions of the uncultivated organisms\u27 16S rRNAs. These reference RNAs were used in subsequent quantitative probing experiments. Oligonucleotide probes were designed to specifically target novel phylotypes and were tested for optimal hybridization wash conditions and target specificity. The newly designed probes were then applied together with eubacterial probes to determine the relative abundances of the novel phylotypes in the salt marsh sediment and rhizosphere. Lastly, 16S rRNA sequences of ten SRB isolates were analyzed and compared to sequences of other cultivated SRB and novel phylotypes retrieved directly from environmental samples. Two novel phylotypes were retrieved from rhizosphere samples, with A01 sharing 89.1% sequence identity with Desulfococcus multivorans and 4D19 sharing 96.3% sequence identity with Desulfosarcina variabilis. Additionally, six sequences were found that were extremely closely related to D. multivorans. Synthetic reference RNAs were successfully used in the optimization and application of probes A01-183 and 4D19-189, which specifically targeted A01 and 4D19, respectively. Mean relative abundances of A01-183 and 4D19-189 targets were 7.5% and 3.4%, respectively, suggesting that the target organisms of A01-183 and, to a lesser extent, 4D19-189 played a dominant role in the salt marsh sediment and rhizosphere. Phylogenetic analysis of SRB isolates placed all isolates within the Gram-negative mesophilic SRB group. Two isolates were members of the Desulfovibrionaceae family, with one a member of the genus Desulfovibrio and the other possibly representing a novel genus. The remaining eight isolates were members of the Desulfobacteriaceae family and were comprised of novel species within the genera Desulfobulbus, Desulfobacter, Desulfobacterium, and Desulfoarculus, as well as a novel genus most closely related to Desulfobotulus sapovorans. None of the SRB isolates appeared to be related to the phylotypes A01 or 4D19 at the species or genus level