Lee Patrick K H, Macbeth Tamzen W, Sorenson Kent S, Deeb Rula A, Alvarez-Cohen Lisa
Department of Civil and Environmental Engineering, University of California, Berkeley, CA 94720-1710, USA.
Appl Environ Microbiol. 2008 May;74(9):2728-39. doi: 10.1128/AEM.02199-07. Epub 2008 Mar 7.
Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of "Dehalococcoides" spp. in the groundwater of a trichloroethene-dense nonaqueous-phase liquid site at Fort Lewis, WA, that was sequentially subjected to biostimulation and bioaugmentation. Dehalococcoides cells carrying the tceA, vcrA, and bvcA genes were indigenous to the site. The sum of the three identified RDase gene copy numbers closely correlated to 16S rRNA gene copy numbers throughout the biostimulation and bioaugmentation activity, suggesting that these RDase genes represented the major Dehalococcoides metabolic functions at this site. Biomarker quantification revealed an overall increase of more than 3 orders of magnitude in the total Dehalococcoides population through the 1-year monitoring period (spanning biostimulation and bioaugmentation), and measurement of the respective RDase gene concentrations indicated different growth dynamics among Dehalococcoides cells. The Dehalococcoides cells containing the tceA gene consistently lagged behind other Dehalococcoides cells in population numbers and made up less than 5% of the total Dehalococcoides population, whereas the vcrA- and bvcA-containing cells represented the dominant fractions. Quantification of transcripts in groundwater samples verified that the 16S rRNA gene and the bvcA and vcrA genes were consistently highly expressed in all samples examined, while the tceA transcripts were detected inconsistently, suggesting a less active physiological state of the cells with this gene. The production of vinyl chloride and ethene toward the end of treatment supported the physiological activity of the bvcA- and vcrA-carrying cells. A clone library of the expressed RDase genes in field samples produced with degenerate primers revealed the expression of two putative RDase genes that were not previously monitored with RT-qPCR. The level of abundance of one of the putative RDase genes (FtL-RDase-1638) identified in the cDNA clone library tracked closely in field samples with abundance of the bvcA gene, suggesting that the FtL-RDase-1638 gene was likely colocated in genomes containing the bvcA gene. Overall, results from this study demonstrate that quantification of biomarker dynamics at field sites can provide useful information about the in situ physiology of Dehalococcoides strains and their associated activity.
定量聚合酶链反应（qPCR）与逆转录（RT）相结合，以分析16S rRNA基因以及三个还原性脱卤酶（RDase）基因（tceA、vcrA和bvcA）的基因拷贝数和转录本，这些基因作为华盛顿州刘易斯堡一个三氯乙烯密集型非水相液体场地地下水中“脱卤球菌属”物种的生物标志物。携带tceA、vcrA和bvcA基因的脱卤球菌细胞是该场地原生的。在整个生物刺激和生物强化活动过程中，三个已鉴定的RDase基因拷贝数之和与16S rRNA基因拷贝数密切相关，这表明这些RDase基因代表了该场地脱卤球菌的主要代谢功能。生物标志物定量显示，在为期1年的监测期（涵盖生物刺激和生物强化）内，脱卤球菌总数总体增加了超过3个数量级，对各个RDase基因浓度的测量表明脱卤球菌细胞之间存在不同的生长动态。含有tceA基因的脱卤球菌细胞在数量上始终落后于其他脱卤球菌细胞，占脱卤球菌总数的比例不到5%，而含有vcrA和bvcA的细胞占主导部分。对地下水样品中转录本的定量分析证实，在所有检测样品中，16S rRNA基因以及bvcA和vcrA基因始终高度表达，而tceA转录本的检测结果不一致，这表明携带该基因的细胞生理状态不太活跃。处理末期氯乙烯和乙烯的产生支持了携带bvcA和vcrA的细胞的生理活性。用简并引物构建的现场样品中表达的RDase基因克隆文库显示，有两个假定的RDase基因表达，而之前用逆转录定量聚合酶链反应（RT-qPCR）未对其进行监测。在cDNA克隆文库中鉴定出的一个假定的RDase基因（FtL-RDase-1638）的丰度水平在现场样品中与bvcA基因的丰度密切相关，这表明FtL-RDase-1638基因可能位于含有bvcA基因的基因组中。总体而言，本研究结果表明，对现场生物标志物动态进行定量分析可以提供有关脱卤球菌菌株原位生理学及其相关活性的有用信息。
