Role of IncP-1β Plasmids pWDL7::rfp and pNB8c in Chloroaniline Catabolism as Determined by Genomic and Functional Analyses
Role of IncP-1β Plasmids pWDL7::rfp and pNB8c in Chloroaniline Catabolism as Determined by Genomic and Functional Analyses
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DOI：
10.1128/aem.07480-11
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发表时间：
2012-02-01
影响因子：
4.4
通讯作者：
Top, E. M.
中科院分区：
文献类型：
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作者：
Krol, J. E.;Penrod, J. T.;Top, E. M.
宽宿主范围的分解代谢质粒在细菌降解人造化合物中起着重要作用。为了深入了解这些质粒在氯苯胺降解中的作用，我们确定了IncP-1氯苯胺降解质粒pWDL7::rfp及其近亲pNB8c的首个完整核苷酸序列，以及3-氯苯胺（3-CA）氧化基因的表达模式、功能和生物增强潜力。基于骨干蛋白的系统发育分析，这两个质粒都是IncP-1 β亚群中一个不同分支的成员。质粒几乎相同，但pWDL7::rfp携带一个重复的反向分解代谢转座子Tn6063，包含一个假定的3-CA氧化基因簇dcaQTA1A2BR， pNB8c只包含一个转座子拷贝。在两个质粒上均未检测到芳香环裂解途径的基因，表明只存在上部3-CA降解途径。高拷贝数载体表达的dcaA1A2B基因产物可在大肠杆菌中将3-CA转化为4-氯儿茶酚。质粒之间dca启动子区域的细微差异以及3-CA对pNB8c dca基因转录的诱导缺失可能解释了先前pNB8c不赋予3-CA转化的发现。用pWDL7::rfp对活性污泥进行生物强化，加速了3-CA的去除，但仅在存在额外碳源的情况下。成功的生物增强需要在本地细菌中与氯儿茶酚切割基因互补的上通路基因。因此，这些质粒的基因组序列有助于解释其分解代谢活动的分子基础。
Broad-host-range catabolic plasmids play an important role in bacterial degradation of man-made compounds. To gain insight into the role of these plasmids in chloroaniline degradation, we determined the first complete nucleotide sequences of an IncP-1 chloroaniline degradation plasmid, pWDL7::rfp and its close relative pNB8c, as well as the expression pattern, function, and bioaugmentation potential of the putative 3-chloroaniline (3-CA) oxidation genes. Based on phylogenetic analysis of backbone proteins, both plasmids are members of a distinct clade within the IncP-1 beta subgroup. The plasmids are almost identical, but whereas pWDL7::rfp carries a duplicate inverted catabolic transposon, Tn6063, containing a putative 3-CA oxidation gene cluster, dcaQTA1A2BR, pNB8c contains only a single copy of the transposon. No genes for an aromatic ring cleavage pathway were detected on either plasmid, suggesting that only the upper 3-CA degradation pathway was present. The dcaA1A2B gene products expressed from a high-copy-number vector were shown to convert 3-CA to 4-chlorocatechol in Escherichia coll. Slight differences in the dca promoter region between the plasmids and lack of induction of transcription of the pNB8c dca genes by 3-CA may explain previous findings that pNB8C does not confer 3-CA transformation. Bioaugmentation of activated sludge with pWDL7::rfp accelerated removal of 3-CA, but only in the presence of an additional carbon source. Successful bioaugmentation requires complementation of the upper pathway genes with chlorocatechol cleavage genes in indigenous bacteria. The genome sequences of these plasmids thus help explain the molecular basis of their catabolic activities.