Detection and Enumeration of Sulphate-Reducing Bacteria in Estuarine Sediments by Competitive PCR

Abstract

The distribution of sulphate-reducing bacteria (SRB) in the sediments of the Colne River estuary, Essex, UK covering different saline concentrations of sediment porewater was investigated by the use of quantitative competitive PCR. Here, we show that a new PCR primer set and a new quantitative method using PCR are useful tools for the detection and the enumeration of SRB in natural environments. A PCR primer set selective for the dissimilatory sulphite reductase gene (dsr) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from all 27 SRB strains tested, including Gram-negative and positive species. Sixty clones derived from sediment DNA using the primers were sequenced and all were closely related with the predicted dsr of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsr selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsr. This procedure was applied to sediment samples from the River Colne estuary, Essex, UK together with simultaneous measurement of in situ rates of sulphate reduction. High densities of SRB ranging from 0.2 − 5.7 × 10⁸ cells ml^{− 1} wet sediment were estimated by the competitive PCR assuming that all SRB have a single copy of dsr. Using these estimates cell specific sulphate reduction rates of 10^{− 17} to 10^{− 15} mol of SO₄ ^{2 −} cell^{− 1} day^{− 1} were calculated, which is within the range of, or lower than, those previously reported for pure cultures of SRB. Our results show that the newly developed competitive PCR technique targeted to dsr is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.

Keywords:

• competitive PCR
• dissimilatory sulphite reductase gene
• estuarine sediments
• sulphate reduction rates

This study was supported in part by a Grant-in-Aid for Scientific Research (No. 12760130) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Fukui Prefectural Fund for the Promotion of Science to RK. We thank Tania Cresswell-Maynard, John W. Green and Junichi Takeuchi, University of Essex, for their field assistance.