Amos Benjamin K, Sung Youlboong, Fletcher Kelly E, Gentry Terry J, Wu Wei-Min, Criddle Craig S, Zhou Jizhong, Löffler Frank E
School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0512, USA.
Appl Environ Microbiol. 2007 Nov;73(21):6898-904. doi: 10.1128/AEM.01218-07. Epub 2007 Sep 7.
Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 x 10(6) and 1 x 10(4) 16S rRNA gene copies per mul of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per mul of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 x 10(7) +/- 0.1 x 10(7) per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.
洛弗利地杆菌菌株SZ可将六价铀（U(VI)）还原为四价铀（U(IV)），并且是金属还原地杆菌属中首个能够利用四氯乙烯（PCE）作为生长支持性电子受体的成员。使用针对16S rRNA基因的特异性引物对进行直接PCR和巢式PCR，可将菌株SZ与其他已知的氯乙烯脱氯细菌以及密切相关的地杆菌分离株区分开来，包括其亲缘关系最近的培养菌株硫还原地杆菌（G. thiogenes）。直接PCR和巢式PCR的检测限分别约为每微升模板DNA 1×10⁶和1×10⁴个16S rRNA基因拷贝。定量实时PCR（qPCR）方法将灵敏度提高到每微升模板DNA低至30个16S rRNA基因拷贝，但特异性较低。熔解曲线分析以及扩增曲线形状的比较可识别假阳性信号，并在单独分析时将菌株SZ与硫还原地杆菌区分开来。当具有高度序列相似性的目标（菌株SZ）DNA和非目标（硫还原地杆菌）DNA混合时，这些指标的可靠性较低，这表明qPCR方案的开发不仅应评估特异性，还应探索非目标DNA对定量准确性的影响。使用特异性工具在PCE脱氯菌群中检测到了类似菌株SZ的扩增子，包括生物强化菌群KB-1以及两个受氯乙烯污染的地下水样品。在田纳西州橡树岭铀和氯代溶剂污染的综合现场规模地下研究挑战（IFC）场地进行生物刺激后，在22个地下水样品中的13个中也检测到了类似菌株SZ的扩增子。类似菌株SZ的细胞数量从低于检测水平增加到每升地下水2.3×10⁷±0.1×10⁷个，这表明类似菌株SZ的生物体有助于污染物转化。洛弗利地杆菌菌株SZ特异性工具将有助于监测铀和/或氯代溶剂污染场地（如橡树岭IFC场地）的生物修复工作。
