Department of Civil and Environmental Engineering, Colorado State University, Fort Collins, CO, USA.
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA.
Appl Microbiol Biotechnol. 2019 May;103(10):4177-4192. doi: 10.1007/s00253-019-09762-7. Epub 2019 Apr 9.
Quantifying functional biomarker genes and their transcripts provides critical lines of evidence for contaminant biodegradation; however, accurate quantification depends on qPCR primers that contain no, or minimal, mismatches with the target gene. Developing accurate assays has been particularly challenging for genes encoding fumarate-adding enzymes (FAE) due to the high level of genetic diversity in this gene family. In this study, metagenomics applied to a field-derived, o-xylene-degrading methanogenic consortium revealed genes encoding FAE that would not be accurately quantifiable by any previously available PCR assays. Sequencing indicated that a gene similar to the napthylmethylsuccinate synthase gene (nmsA) was most abundant, although benzylsuccinate synthase genes (bssA) also were present along with genes encoding alkylsuccinate synthase (assA). Upregulation of the nmsA-like gene was observed during o-xylene degradation. Protein homology modeling indicated that mutations in the active site, relative to a BssA that acts on toluene, increase binding site volume and accessibility, potentially to accommodate the relatively larger o-xylene. The new nmsA-like gene was also detected at substantial concentrations at field sites with a history of xylene contamination.
量化功能生物标志物基因及其转录本为污染物生物降解提供了重要的证据；然而，准确的定量取决于 qPCR 引物，这些引物与目标基因没有或只有最小的不匹配。由于这个基因家族的遗传多样性很高，因此开发准确的检测方法对于编码延胡索酸添加酶（FAE）的基因来说一直具有特别大的挑战性。在这项研究中，应用于现场衍生的、以甲氧基为食的产甲烷共生物的宏基因组学揭示了编码 FAE 的基因，如果使用任何以前可用的 PCR 检测方法，这些基因都无法准确地定量。测序表明，一种类似于萘基甲基琥珀酸合酶基因（nmsA）的基因最为丰富，尽管苯甲基琥珀酸合酶基因（bssA）也存在，以及编码烷基琥珀酸合酶（assA）的基因。在邻二甲苯降解过程中观察到 nmsA 样基因的上调。蛋白质同源建模表明，与作用于甲苯的 BssA 相比，活性位点的突变会增加结合位点的体积和可及性，从而可能适应相对较大的邻二甲苯。在具有二甲苯污染历史的现场，也检测到大量的新型 nmsA 样基因。
