McbG, a LysR Family Transcriptional Regulator, Activates the mcbBCDEF Gene Cluster Involved in the Upstream Pathway of Carbaryl Degradation in Pseudomonas sp. Strain XWY-1
McbG, a LysR Family Transcriptional Regulator, Activates the mcbBCDEF Gene Cluster Involved in the Upstream Pathway of Carbaryl Degradation in Pseudomonas sp. Strain XWY-1
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McbG是一个LysR家族转录调控因子，可激活参与假单胞菌西维因上游降解途径的mcbBCDEF基因簇。XWY-1菌株
DOI：
10.1128/aem.02970-20
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发表时间：
2021-02
影响因子：
4.4
通讯作者：
Zhijian Ke;Yidong Zhou;Wankui Jiang;Mingliang Zhang;Hui Wang;Yijun Ren;J. Qiu;Minggen Cheng;Q. Hong
中科院分区：
文献类型：
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作者：
Zhijian Ke;Yidong Zhou;Wankui Jiang;Mingliang Zhang;Hui Wang;Yijun Ren;J. Qiu;Minggen Cheng;Q. Hong
假单胞菌属菌株XWY-1是一株降解西维因的菌株，它利用西维因作为唯一的碳和能源进行生长。参与西维因降解的功能基因已有报道。摘要尽管在假单胞菌中已经报道了参与西维因降解的酶编码基因。菌株XWY-1，目前尚未鉴定出调节子。在负责西维因降解上游途径(从西维因到水杨酸盐)的mcbABCDEF簇中，mcbBCDEF基因是结构性表达的，而mcbBCDEF是由mcbBCDEF诱导的，1-萘酚是西维因被MCBA降解的产物。在本研究中，我们鉴定了McbG，mcbBCDEF簇的转录激活因子。McbG是一种由315个氨基酸组成的蛋白质，其相对分子质量为35.7 kDa。它属于LysR家族的转录调控因子，与来自Sphingobium氯苯胆碱ATCC 39723的五氯酚降解转录激活蛋白PcpR有28.48%的同源性。基因中断和互补研究表明，在菌株XWY-1中，mcbG对于mcbBCDEF簇的转录是必不可少的，以响应1-萘酚。凝胶迁移率改变分析(EMSA)和DNase I足迹分析结果表明，McbG与mcbBCDEF启动子区域的25bp基序结合。基序中的回文序列TATCGATA对于McbG结合是必不可少的。结合部位位于-10盒和转录起始部位之间。此外，McbG还可以抑制自身的转录。EMSA结果表明，mcbG启动子区域的25bp基序在McbG与mcbG启动子结合过程中起着重要作用。本研究揭示了菌株XWY-1降解西维因上游途径的调控机制。McbG的发现增加了LysR转录调控家族中调控模型的多样性。重要假单胞菌属。菌株XWY-1是一株降解西维因的菌株，它利用西维因作为唯一的碳和能源进行生长。参与西维因降解的功能基因已有报道。然而，监管机制尚未得到调查。以往的研究表明，在菌株XWY-1中，负责西维因水解制1-萘酚的MCBA基因是结构性表达的。在本研究中，我们发现了一个LysR类转录调控因子McbG，它激活mcbBCDEF基因簇，负责将1-萘酚降解为水杨酸，并抑制其自身的转录。McbG在mcbBCDEF启动子区域的DNA结合部位含有回文序列，影响McbG与DNA的结合。这些发现加深了我们对西维因微生物降解机理的理解。
Pseudomonas sp. strain XWY-1 is a carbaryl-degrading strain that utilizes carbaryl as the sole carbon and energy source for growth. The functional genes involved in the degradation of carbaryl have already been reported. ABSTRACT Although enzyme-encoding genes involved in the degradation of carbaryl have been reported in Pseudomonas sp. strain XWY-1, no regulator has been identified yet. In the mcbABCDEF cluster responsible for the upstream pathway of carbaryl degradation (from carbaryl to salicylate), the mcbA gene is constitutively expressed, while mcbBCDEF is induced by 1-naphthol, the hydrolysis product of carbaryl by McbA. In this study, we identified McbG, a transcriptional activator of the mcbBCDEF cluster. McbG is a 315-amino-acid protein with a molecular mass of 35.7 kDa. It belongs to the LysR family of transcriptional regulators and shows 28.48% identity to the pentachlorophenol (PCP) degradation transcriptional activation protein PcpR from Sphingobium chlorophenolicum ATCC 39723. Gene disruption and complementation studies reveal that mcbG is essential for transcription of the mcbBCDEF cluster in response to 1-naphthol in strain XWY-1. The results of the electrophoretic mobility shift assay (EMSA) and DNase I footprinting show that McbG binds to the 25-bp motif in the mcbBCDEF promoter area. The palindromic sequence TATCGATA within the motif is essential for McbG binding. The binding site is located between the –10 box and the transcription start site. In addition, McbG can repress its own transcription. The EMSA results show that a 25-bp motif in the mcbG promoter area plays an important role in McbG binding to the promoter of mcbG. This study reveals the regulatory mechanism for the upstream pathway of carbaryl degradation in strain XWY-1. The identification of McbG increases the variety of regulatory models within the LysR family of transcriptional regulators. IMPORTANCE Pseudomonas sp. strain XWY-1 is a carbaryl-degrading strain that utilizes carbaryl as the sole carbon and energy source for growth. The functional genes involved in the degradation of carbaryl have already been reported. However, the regulatory mechanism has not been investigated yet. Previous studies demonstrated that the mcbA gene, responsible for hydrolysis of carbaryl to 1-naphthol, is constitutively expressed in strain XWY-1. In this study, we identified a LysR-type transcriptional regulator, McbG, which activates the mcbBCDEF gene cluster responsible for the degradation of 1-naphthol to salicylate and represses its own transcription. The DNA binding site of McbG in the mcbBCDEF promoter area contains a palindromic sequence, which affects the binding of McbG to DNA. These findings enhance our understanding of the mechanism of microbial degradation of carbaryl.