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首页> 外文期刊>Applied Microbiology >Characterization of Novel Polycyclic Aromatic Hydrocarbon Dioxygenases from the Bacterial Metagenomic DNA of a Contaminated Soil
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Characterization of Novel Polycyclic Aromatic Hydrocarbon Dioxygenases from the Bacterial Metagenomic DNA of a Contaminated Soil

机译：新型多环芳烃双加氧酶从细菌土壤基因组DNA的表征。

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Ring-hydroxylating dioxygenases (RHDs) play a crucial role in the biodegradation of a range of aromatic hydrocarbons found on polluted sites, including polycyclic aromatic hydrocarbons (PAHs). Current knowledge on RHDs comes essentially from studies on culturable bacterial strains, while compelling evidence indicates that pollutant removal is mostly achieved by uncultured species. In this study, a combination of DNA-SIP labeling and metagenomic sequence analysis was implemented to investigate the metabolic potential of main PAH degraders on a polluted site. Following in situ labeling using [~(13)C]phenanthrene, the labeled metagenomic DNA was isolated from soil and subjected to shotgun sequencing. Most annotated sequences were predicted to belong to Betaproteobacteria , especially Rhodocyclaceae and Burkholderiales , which is consistent with previous findings showing that main PAH degraders on this site were affiliated to these taxa. Based on metagenomic data, four RHD gene sets were amplified and cloned from soil DNA. For each set, PCR yielded multiple amplicons with sequences differing by up to 321 nucleotides (17%), reflecting the great genetic diversity prevailing in soil. RHDs were successfully overexpressed in Escherichia coli , but full activity required the coexpression of two electron carrier genes, also cloned from soil DNA. Remarkably, two RHDs exhibited much higher activity when associated with electron carriers from a sphingomonad. The four RHDs showed markedly different preferences for two- and three-ring PAHs but were poorly active on four-ring PAHs. Three RHDs preferentially hydroxylated phenanthrene on the C-1 and C-2 positions rather than on the C-3 and C-4 positions, suggesting that degradation occurred through an alternate pathway.
机译：环羟基化双加氧酶（RHD）在受污染部位发现的多种芳香烃，包括多环芳香烃（PAH）的生物降解中，起着至关重要的作用。当前关于RHD的知识主要来自对可培养细菌菌株的研究，而有力的证据表明，污染物的去除主要是由未培养的物种实现的。在这项研究中，结合了DNA-SIP标记和宏基因组序列分析，以研究污染部位主要PAH降解子的代谢潜力。使用[〜（13）C]菲原位标记后，从土壤中分离出标记的宏基因组DNA，并进行shot弹枪测序。预计大多数带注释的序列都属于Betaproteobacteria，尤其是红景天科和Burkholderiales，这与以前的发现一致，表明该站点上的主要PAH降解物与这些分类群相关。根据宏基因组学数据，从土壤DNA扩增并克隆了四个RHD基因集。对于每组，PCR产生多个扩增子，其序列相差高达321个核苷酸（17％），反映了土壤中普遍存在的巨大遗传多样性。 RHDs在大肠杆菌中成功地过表达，但是完整的活性需要两个电子载体基因的共表达，这两个基因也从土壤DNA克隆而来。值得注意的是，两个RHD与鞘氨醇单胞菌的电子载体结合时表现出更高的活性。四个RHD对二环和三环PAH表现出明显不同的偏好，但对四环PAH的活性较差。三个RHD优先在C-1和C-2位置而不是C-3和C-4位置使羟基羟基化，表明降解是通过替代途径发生的。

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