Development and use of molecular tool for rapid enumeration of sulphate-reducing bacteria and for assessment of their activity

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We developed a new PCR primer set and a new quantitative method using PCR are useful tool for the detection and the enumeration of sulpahte-reducing bacteria (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 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 and water samples from anaerobic aquatic environments. High densities of SRB were detected by the competitive PCR assuming that all SRB have a single copy of dsr. 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.We also developed a competitive RT-PCR method for the quantification of dsr mRNA in Desulfovibrio desulfuricans. The amount of dsr mRNA was determined at different growth conditions to find the relationship between the dsr mRNA content per cell and the rate of metabolism (cell specific sulphate reduction rate, csSRR). The maximum dsr mRNA content in SRB cell correlated with csSRR, indicating that dsr mRNA may be of value as molecular proxy assessing cellular sulphate reduction rates in nature.