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

Anna Blazejak,Axel Schippers

doi:10.3389/fmicb.2011.00253

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 108/g sediment close to the sediment surface to less than 105/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 104/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.

Highlights

Sulfate reduction plays a crucial role in the past and present global sulfur cycle, and may be regarded as one of the oldest metabolic pathways on Earth (Castresana and Moreira, 1999; Schen et al, 2001)
The abundance of sulfate-reducing prokaryotes (SRP) in marine sediments has been determined by a variety of methods including MPN-cultivation (Knoblauch et al, 1999), 16S rRNA slot-blot hybridization (Sahm et al, 1999), or FISH and CARD-FISH with 16S rRNA gene probes (Ravenschlag et al, 2000; Gittel et al, 2008)
The functional gene encoding for dissimilatory sulfite reductase of SRP shows a high similarity in different SRP (Wagner et al, 1998), a dsrA specific PCR primer set targeting both, Gram-positive and Gram-negative SRP species, was developed for competitive PCR quantification (Kondo et al, 2004)

Summary

Introduction

Sulfate reduction plays a crucial role in the past and present global sulfur cycle, and may be regarded as one of the oldest metabolic pathways on Earth (Castresana and Moreira, 1999; Schen et al, 2001). The functional gene encoding for dissimilatory sulfite reductase (dsrA) of SRP shows a high similarity in different SRP (Wagner et al, 1998), a dsrA specific PCR primer set targeting both, Gram-positive and Gram-negative SRP species, was developed for competitive PCR quantification (Kondo et al, 2004) These primers were used to design a quantitative, real-time PCR (Q-PCR) assay for dsrA for SRP quantification in subsurface marine sediments (Schippers and Neretin, 2006; Leloup et al, 2007, 2009; Nunoura et al, 2009; Webster et al, 2009; Schippers et al, 2010) and the Black Sea water column (Neretin et al, 2007)

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Results