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首页> 外文期刊>Applied Microbiology >Enhanced Gene Detection Assays for Fumarate-Adding Enzymes Allow Uncovering of Anaerobic Hydrocarbon Degraders in Terrestrial and Marine Systems
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Enhanced Gene Detection Assays for Fumarate-Adding Enzymes Allow Uncovering of Anaerobic Hydrocarbon Degraders in Terrestrial and Marine Systems

机译：富马酸盐添加酶的增强的基因检测分析方法可以发现陆生和海洋系统中厌氧性烃降解物

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The detection of anaerobic hydrocarbon degrader populations via catabolic gene markers is important for the understanding of processes at contaminated sites. Fumarate-adding enzymes (FAEs; i.e., benzylsuccinate and alkylsuccinate synthases) have already been established as specific functional marker genes for anaerobic hydrocarbon degraders. Several recent studies based on pure cultures and laboratory enrichments have shown the existence of new and deeply branching FAE gene lineages, such as clostridial benzylsuccinate synthases and homologues, as well as naphthylmethylsuccinate synthases. However, established FAE gene detection assays were not designed to target these novel lineages, and consequently, their detectability in different environments remains obscure. Here, we present a new suite of parallel primer sets for detecting the comprehensive range of FAE markers known to date, including clostridial benzylsuccinate, naphthylmethylsuccinate, and alkylsuccinate synthases. It was not possible to develop one single assay spanning the complete diversity of FAE genes alone. The enhanced assays were tested with a range of hydrocarbon-degrading pure cultures, enrichments, and environmental samples of marine and terrestrial origin. They revealed the presence of several, partially unexpected FAE gene lineages not detected in these environments before: distinct deltaproteobacterial and also clostridial bssA homologues as well as environmental nmsA homologues. These findings were backed up by dual-digest terminal restriction fragment length polymorphism diagnostics to identify FAE gene populations independently of sequencing. This allows rapid insights into intrinsic degrader populations and degradation potentials established in aromatic and aliphatic hydrocarbon-impacted environmental systems.
机译：通过分解代谢基因标记检测厌氧烃降解物种群对于了解受污染地点的过程非常重要。已经建立了富马酸酯加成酶（FAE;即琥珀酸苄基酯和琥珀酸烷基酯合酶）作为厌氧烃降解物的特定功能标记基因。基于纯培养物和实验室扩增的一些最新研究表明，存在新的和深度分支的FAE基因谱系，例如梭菌苄基琥珀酸合酶和同系物，以及萘基甲基琥珀酸合酶。但是，已建立的FAE基因检测方法并不是针对这些新型谱系的，因此，它们在不同环境中的可检测性仍然不清楚。在这里，我们提出了一套新的平行引物组，用于检测迄今为止已知的FAE标记物的全面范围，包括梭菌苄基琥珀酸酯，萘基甲基琥珀酸酯和烷基琥珀酸酯合酶。不可能开发出一种跨越单独的FAE基因完全多样性的单一测定方法。使用一系列可降解碳氢化合物的纯培养物，浓缩物以及海洋和陆地来源的环境样品测试了增强的检测方法。他们揭示了以前在这些环境中未检测到的几种部分出乎意料的FAE基因谱系：独特的delteproteobacterial细菌和梭菌bssA同源物以及环境nmsA同源物。这些发现得到了双消化末端限制性片段长度多态性诊断技术的支持，以独立于测序鉴定FAE基因群体。这可以快速洞察固有的降解物种群以及在芳香族和脂族烃影响的环境系统中建立的降解潜力。

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