摘要
CopRS/CopABCD是细菌用以维持铜内环境稳定的一个系统,虽然已在荧光假单胞菌(Pseudomonas fluorescens)中发现了CopRS/CopABCD系统的同源物,但其潜在的功能还未知。本实验在一个鱼类致病菌P. fluorescens(TSS)中鉴定到了一个基因簇,由copR、copS、copC和copD组成,但缺乏copAB。copR、copS、copC和copD基因的敲除实验发现copRSCD基因簇与TSS抗铜性相关,而且copRS操纵子和copCD操纵子在转录水平上受亚抑制水平的铜诱导。双元调控系统中的调控蛋白CopR不仅激活copCD表达,而且还激活copRS的表达。凝胶滞缓实验显示CopR能直接与copCD和copRS的启动子区域结合。干扰copR的正常表达不仅影响细菌的生长,而且还影响到细菌生物膜的形成、对鱼的侵染力和在组织中的存活力。本实验还筛选到一个CopR的突变体C104,该突变体因缺失N端的信号接受域而成为一个具有组成性活性的调控蛋白,C104在TSS中表达时导致菌株的毒力降低。本实验所发现的P. fluorescens CopR与细菌致病力之间的关系以前未见报道。
CopRS/CopABCD is one of the known systems that control copper homeostasis in bacteria. Though CopRS/CopABCD homologues are found to exist in Pseudomonas fluorescens, the potential role of this system in P. fluorescens has not been investigated. In this study a genetic cluster, consisting of copR, S, C, and D but lacking copAB, was identified in a pathogenic P. fluorescens strain (TSS) isolated from diseased fish. The copRSCD cluster was demonstrated to be required for full copper resistance by knockout of copR, copS, copC and copD. The copRS operon and copCD operon were regulated at the transcription level by Cu. Expression of copCD is regulated directly by the two-component response regulator CopR, which also regulates its own expression. From the exprement of EMSA, we found that CopR can interact directly with the promoter of copCD and copRS. Interruption of the regulated expression of copR affected bacterial growth, biofilm formation, and tissue dissemination and survival. A mutant CopR, which lacks the N-terminal signal receiver domain and is constitutively active, was found to have an attenuating effect on bacterial virulence when expressed in TSS. To our knowledge, this is the first report that suggests a link between CopR and bacterial pathogenicity in P. fluorescens.
引文
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