Beller Harry R, Kane Staci R, Legler Tina C, Alvarez Pedro J J
Lawrence Livermore National Laboratory, Livermore, California 94551, USA.
Environ Sci Technol. 2002 Sep 15;36(18):3977-84. doi: 10.1021/es025556w.
We have developed a real-time polymerase chain reaction (PCR) method that can quantify hydrocarbon-degrading bacteria in sediment samples based on a catabolic gene associated with the first step of anaerobic toluene and xylene degradation. The target gene, bssA, codes for the alpha-subunit of benzylsuccinate synthase. The primer-probe set for real-time PCR was based on consensus regions of bssA from four denitrifying bacterial strains; bssA sequences for two of these strains were determined during this study. The method proved to be sensitive (detection limit ca. 5 gene copies) and had a linear range of >7 orders of magnitude. We used the method to investigate how gasohol releases from leaking underground storage tanks could affect indigenous toluene-degrading bacteria. Microcosms inoculated with aquifer sediments from four different sites were incubated anaerobically with BTEX (benzene, toluene, ethylbenzene, and xylenes) and nitrate in the presence and absence of ethanol. Overall, population trends were consistent with observed toluene degradation activity: the microcosms with the most rapid toluene degradation also had the largest numbers of bssA copies. In the microcosms with the most rapid toluene degradation, numbers of bssA copies increased 100-to 1000-fold over the first 4 days of incubation, during which time most of the toluene had been consumed. These results were supported by slot blot analyses with unamplified DNA and by cloning and sequencing of putative bssA amplicons, which confirmed the real-time PCR method's specificity for bssA. Use of a companion real-time PCR method for estimating total eubacterial populations (based on 16S rDNA) indicated that, in some cases, ethanol disproportionately supported the growth of bacteria that did not contain bssA. The real-time PCR method for bssA could be a powerful tool for monitored natural attenuation of BTEX in fuel-contaminated groundwater. To our knowledge, this is the first reported molecular method that targets anaerobic, hydrocarbon-degrading bacteria based on a catabolic gene.
我们开发了一种实时聚合酶链反应（PCR）方法，该方法可基于与厌氧甲苯和二甲苯降解第一步相关的分解代谢基因，对沉积物样本中的烃降解细菌进行定量。目标基因bssA编码苄基琥珀酸合酶的α亚基。实时PCR的引物-探针组基于四种反硝化细菌菌株的bssA保守区域；本研究期间确定了其中两种菌株的bssA序列。该方法被证明具有灵敏性（检测限约为5个基因拷贝），线性范围大于7个数量级。我们使用该方法研究了地下储油罐泄漏的汽油醇如何影响本地甲苯降解细菌。用来自四个不同地点的含水层沉积物接种的微观模型，在有和没有乙醇的情况下，与BTEX（苯、甲苯、乙苯和二甲苯）和硝酸盐一起进行厌氧培养。总体而言，种群趋势与观察到的甲苯降解活性一致：甲苯降解最快的微观模型中bssA拷贝数也最多。在甲苯降解最快的微观模型中，bssA拷贝数在培养的前4天增加了100至1000倍，在此期间大部分甲苯已被消耗。未扩增DNA的狭缝印迹分析以及推定的bssA扩增子的克隆和测序支持了这些结果，证实了实时PCR方法对bssA的特异性。使用一种配套的实时PCR方法估计总真细菌种群（基于16S rDNA）表明，在某些情况下，乙醇对不含bssA的细菌生长的支持不成比例。针对bssA的实时PCR方法可能是监测燃料污染地下水中BTEX自然衰减的有力工具。据我们所知，这是首次报道的基于分解代谢基因靶向厌氧烃降解细菌的分子方法。
