Two LysR Family Transcriptional Regulators, McbH and McbN, Activate the Operons Responsible for the Midstream and Downstream Pathways, Respectively, of Carbaryl Degradation in Pseudomonas sp. Strain XWY-1
Two LysR Family Transcriptional Regulators, McbH and McbN, Activate the Operons Responsible for the Midstream and Downstream Pathways, Respectively, of Carbaryl Degradation in Pseudomonas sp. Strain XWY-1
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两个LysR家族转录调控因子McbH和McbN分别激活负责假单胞菌西维因降解中游和下游途径的操纵子。XWY-1菌株
DOI：
10.1128/aem.02060-21
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发表时间：
2021-12
影响因子：
4.4
通讯作者：
Zhijian Ke;Qian Zhu;Siyuan Gao;Mingliang Zhang;Mingli Jiang;Yijun Ren;Yali Liu;Yidong Zhou;J. Qiu;Q. Hong
中科院分区：
文献类型：
--
作者：
Zhijian Ke;Qian Zhu;Siyuan Gao;Mingliang Zhang;Mingli Jiang;Yijun Ren;Yali Liu;Yidong Zhou;J. Qiu;Q. Hong
在假单胞菌菌株XWY-1中参与西维因完全降解途径的酶编码基因包括mcbABCDEF、mcbIJKLM和mcbOPQ。以前的研究表明，mcbA基因，负责水解西维因1-萘酚，是组成型表达和mcbBCDEF的转录调节McbG。以前，我们鉴定了一种LysR家族转录调节因子McbG，它激活了假单胞菌菌株XWY-1中西维因降解的上游途径（从西维因到水杨酸酯）中涉及的mcbBCDEF基因簇（Z.克，Y. Zhou，W. Jiang，M. Zhang等人，Appl Environ Microbiol 87：e02970-20，2021，https：//doi.org/10.1128/AEM.02970-20）。在这项研究中，我们确定了McbH和McbN，分别激活mcbIJKLM簇（负责从水杨酸到龙胆酸的中游途径）和mcbOPQ簇（负责从龙胆酸到丙酮酸和富马酸的下游途径）。它们都属于转录调节因子LysR家族。基因破坏和互补研究表明，McbH是必不可少的响应水杨酸和McbN是必不可少的响应龙胆酸的mcbOPQ簇的转录的mcbIJKLM簇。电泳迁移率变动分析（EMSA）和DNA酶I足迹法结果表明，McbH与mcbIJKLM启动子区52 bp的基序结合，McbN与mcbOPQ启动子区58 bp的基序结合。McbH与mcbIJKLM启动子结合的关键序列是一个13 bp的基序，符合LysR家族的典型特征。然而，不同于LysR家族调节子结合位点序列的典型特征的12-bp基序被鉴定为McbN结合mcbOPQ启动子的关键序列。本研究揭示了西维因降解的中游和下游途径的调节机制，并进一步加深了我们对LysR转录调节因子家族的了解（及其大小）。重要信息假单胞菌菌株XWY-1中西维因完全降解途径中涉及的酶编码基因包括mcbABCDEF、mcbIJKLM和mcbOPQ。以前的研究表明，mcbA基因，负责水解西维因1-萘酚，是组成型表达和mcbBCDEF的转录调节McbG。然而，mcbIJKLM和mcbOPQ的转录调控机制尚未研究。在这项研究中，我们确定了两个LysR型转录调节因子，McbH和McbN，分别激活mcbIJKLM簇（负责水杨酸盐降解为龙胆酸盐）和mcbOPQ簇（负责龙胆酸盐降解为丙酮酸盐和富马酸盐）。13 bp基序是McbH与mcbIJKLM启动子结合的关键，12 bp基序不同于LysR型转录调节子（LTTR）结合序列的典型特征，影响McbN与启动子的结合。这些发现有助于扩大对西维因微生物降解调控机制的理解。
The enzyme-encoding genes involved in the complete degradation pathway of carbaryl in Pseudomonas sp. strain XWY-1 include mcbABCDEF, mcbIJKLM, and mcbOPQ. Previous studies demonstrated that the mcbA gene, responsible for hydrolysis of carbaryl to 1-naphthol, is constitutively expressed and that the transcription of mcbBCDEF was regulated by McbG. ABSTRACT Previously, a LysR family transcriptional regulator, McbG, that activates the mcbBCDEF gene cluster involved in the upstream pathway (from carbaryl to salicylate) of carbaryl degradation in Pseudomonas sp. strain XWY-1 was identified by us (Z. Ke, Y. Zhou, W. Jiang, M. Zhang, et al., Appl Environ Microbiol 87:e02970-20, 2021, https://doi.org/10.1128/AEM.02970-20). In this study, we identified McbH and McbN, which activate the mcbIJKLM cluster (responsible for the midstream pathway, from salicylate to gentisate) and the mcbOPQ cluster (responsible for the downstream pathway, from gentisate to pyruvate and fumarate), respectively. They both belong to the LysR family of transcriptional regulators. Gene disruption and complementation study reveal that McbH is essential for transcription of the mcbIJKLM cluster in response to salicylate and McbN is indispensable for the transcription of the mcbOPQ cluster in response to gentisate. The results of electrophoretic mobility shift assay (EMSA) and DNase I footprinting showed that McbH binds to the 52-bp motif in the mcbIJKLM promoter area and McbN binds to the 58-bp motif in the mcbOPQ promoter area. The key sequence of McbH binding to the mcbIJKLM promoter is a 13-bp motif that conforms to the typical characteristics of the LysR family. However, the 12-bp motif that is different from the typical characteristics of the LysR family regulator binding site sequence is identified as the key sequence for McbN to bind to the mcbOPQ promoter. This study revealed the regulatory mechanisms for the midstream and downstream pathways of carbaryl degradation in strain XWY-1 and further our knowledge of (and the size of) the LysR transcription regulator family. IMPORTANCE The enzyme-encoding genes involved in the complete degradation pathway of carbaryl in Pseudomonas sp. strain XWY-1 include mcbABCDEF, mcbIJKLM, and mcbOPQ. Previous studies demonstrated that the mcbA gene, responsible for hydrolysis of carbaryl to 1-naphthol, is constitutively expressed and that the transcription of mcbBCDEF was regulated by McbG. However, the transcription regulation mechanisms of mcbIJKLM and mcbOPQ have not been investigated yet. In this study, we identified two LysR-type transcriptional regulators, McbH and McbN, which activate the mcbIJKLM cluster (responsible for the degradation of salicylate to gentisate) and the mcbOPQ cluster (responsible for the degradation of gentisate to pyruvate and fumarate), respectively. The 13-bp motif is critical for McbH to bind to the promoter of mcbIJKLM, and 12-bp motif different from the typical characteristics of the LysR-type transcriptional regulator (LTTR) binding sequence affects the binding of McbN to the promoter. These findings help to expand the understanding of the regulatory mechanism of microbial degradation of carbaryl.