Real-Time PCR Quantification and Diversity Analysis of the Functional Genes aprA and dsrA of Sulfate-Reducing Prokaryotes in Marine Sediments of the Peru Continental Margin and the Black Sea

Abstract

Sulfate-reducing prokaryotes (SRP) are ubiquitous and quantitatively important members in many ecosystems, especially in marine sediments. However their abundance and diversity in subsurface marine sediments is poorly understood. In this study, the abundance and diversity of the functional genes for the enzymes adenosine 5'-phosphosulfate reductase (aprA) and dissimilatory sulfite reductase (dsrA) of SRP in marine sediments of the Peru continental margin and the Black Sea were analyzed, including samples from the deep biosphere (ODP site 1227). For aprA quantification a Q-PCR assay was designed and evaluated. Depth profiles of the aprA and dsrA copy numbers were almost equal for all sites. Gene copy numbers decreased concomitantly with depth from around 10(8)/g sediment close to the sediment surface to less than 10(5)/g sediment at 5 mbsf. The 16S rRNA gene copy numbers of total bacteria were much higher than those of the functional genes at all sediment depths and used to calculate the proportion of SRP to the total Bacteria. The aprA and dsrA copy numbers comprised in average 0.5-1% of the 16S rRNA gene copy numbers of total bacteria in the sediments up to a depth of ca. 40 mbsf. In the zone without detectable sulfate in the pore water from about 40-121 mbsf (Peru margin ODP site 1227), only dsrA (but not aprA) was detected with copy numbers of less than 10(4)/g sediment, comprising ca. 14% of the 16S rRNA gene copy numbers of total bacteria. In this zone, sulfate might be provided for SRP by anaerobic sulfide oxidation. Clone libraries of aprA showed that all isolated sequences originate from SRP showing a close relationship to aprA of characterized species or form a new cluster with only distant relation to aprA of isolated SRP. For dsrA a high diversity was detected, even up to 121 m sediment depth in the deep biosphere.

Keywords: ODP; aprA; deep biosphere; dsrA; real-time PCR; subsurface; sulfate-reducing prokaryotes.

Figure 1

Depth profiles of DNA copy numbers of the functional genes aprA and dsrA as marker for sulfate-reducing prokaryotes (SRP) and the 16S rRNA gene of total Bacteria at three sediment sites, surface (site 2MC, 0–0.35  mbsf) and deep (site 1227, 0–121.4  mbsf) sediments on the Peru margin, and in the Black Sea (site 20, 0–5.8  mbsf), and depth profile of pore water sulfate concentrations at site 1227 (0–135  mbsf, D’Hondt et al., 2004) on the Peru margin. ■, Bacteria; , dsrA; , aprA.

Figure 2

Gene aprA phylogeny based on deduced amino acid sequences of the aprA gene coding for the alpha subunit of the adenosine 5′ -phosphosulfate reductase. Sequences from this study are highlighted in light gray (Black Sea sediments) and dark gray (Peru margin sediments). Scale bar = 0.10 estimated substitutions per site. SOB, sulfide-oxidizing bacteria; SRB, sulfate-reducing bacteria; SRA, sulfate-reducing archaea.

Figure 3

Gene dsrA phylogeny based on deduced amino acid sequences of the dsrA gene coding for the alpha subunit of dissimilatory (bi)sulfite reductase. Sequences from this study are highlighted in light gray (Black Sea sediments) and dark gray (Peru margin sediments). Scale bar = 0.10 estimated substitutions per site.