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

我们开发了一套新的PCR引物和一种新的PCR定量方法，为自然环境中硫酸盐还原菌（SRB）的检测和计数提供了有用的工具。设计了SRB异化亚硫酸盐还原酶基因（dsr）的PCR引物。使用单组dsr特异性引物进行PCR扩增，从27株SRB菌株（包括革兰氏阴性和阳性种）中获得了预期大小的PCR产物。利用该引物对60个沉积物DNA克隆进行了测序，结果表明所有克隆均与SRB的预测dsr密切相关。这些结果表明，利用新设计的引物对自然样品中SRB的选择性检测是有用的。该引物组利用比dsr靶区短约20%的竞争者，通过dsr选择性竞争PCR估计细胞数量。该方法适用于厌氧水生环境中的沉积物和水样。假设所有SRB都有一个dsr拷贝，通过竞争性PCR检测到高密度的SRB。我们的研究结果表明，新开发的针对dsr的竞争性PCR技术是一种快速、可重复地原位估计SRB数量的强大工具，优于使用培养依赖技术。我们还开发了一种竞争性的RT-PCR方法来定量脱硫弧菌中的dsr mRNA。测定不同生长条件下dsr mRNA的表达量，寻找细胞dsr mRNA含量与代谢速率（细胞特异性硫酸盐还原率，csSRR）之间的关系。SRB细胞中dsr mRNA的最大含量与csSRR相关，表明dsr mRNA可能作为自然界中评估细胞硫酸盐还原速率的分子代理。

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.