群体感应(QS)系统控制响应于细胞密度的细菌群行为。在弧菌中,LuxR和AphA是响应于高或低细胞密度而控制基因表达的两个主QS调节因子(MQSR)。参与这两种MQSR和QS途径调节的其他调节因子仍有待确定。这里,我们进行了基于细菌单杂交(B1H)的转录因子(TFs)筛选,以鉴定可以直接调节由鱼病原体溶藻弧菌编码的285个TFs文库中luxR和aphA表达的TFs。在这些TFs中,总共有7个TFs被鉴定为与luxR和aphA的启动子结合,新的LysR型转录调节因子(LTTR)VqsA可以激活LuxR并抑制AphA转录。同时,LuxR和AphA对vqsA表达产生反馈抑制和激活,分别,表明VqsA协调QS,也受QS调节。此外,VqsA通过直接结合其自身的启动子区来抑制其自身的表达。luxR和aphA启动子区的VqsA结合位点以及LuxR的结合位点,Apha,通过电泳迁移率变化测定(EMSAs)和DNaseI足迹分析发现vqsA基因中的VqsA。最后,VqsA被证实在QS调节的表型中发挥重要作用,即,VI型分泌系统2(T6SS2)依赖性细菌间竞争,生物膜的形成,外毒素生产,和溶藻弧菌的体内毒力,我们的数据表明,VqsA是溶藻弧菌中重要的QS调节因子。IMPORTANCE群体感应(QS)系统的调节机制研究将有助于了解细菌的发病机制和确定有效的QS干扰(QSI)靶标。这里,我们系统地筛选了调节主QS调节因子(MQSRs)LuxR和AphA表达的转录因子(TFs),和一个新的LysR型转录调节因子,VqsA,已确定。我们的数据阐明了介导LuxR之间相互作用的机制,Apha,和VqsA以及这些调节器对QS的表达和输出的影响。由于vqsA破坏而导致的毒力基因表达受损表明,VqsA在QS调节和发病机理中起着重要作用,并且可能是第三个MQSR参与弧菌感应环境信号以协调QS反应。这项研究将有助于制定干预QS和有效控制困扰水产养殖业的病原体的策略。
The quorum sensing (QS) system controls bacterial group behaviors in response to cell density. In vibrios, LuxR and AphA are two master QS regulators (MQSRs) controlling gene expression in response to high or low cell density. Other regulators involved in the regulation of these two MQSRs and QS pathways remain to be determined. Here, we performed bacterial one-hybrid (B1H)-assay-based screens of transcriptional factors (TFs) to identify TFs that can directly regulate the expression of luxR and aphA from a library of 285 TFs encoded by the fish pathogen Vibrio alginolyticus A total of 7 TFs were identified to bind to the promoters of both luxR and aphA Among these TFs, the novel LysR-type transcriptional regulator (LTTR) VqsA could activate LuxR and repress AphA transcription. Meanwhile, LuxR and AphA exerted feedback inhibition and activation of vqsA expression, respectively, indicating that VqsA coordinates QS and is also regulated by QS. In addition, VqsA inhibited its own expression by directly binding to its own promoter region. The VqsA-binding sites in the promoter regions of luxR and aphA as well as the binding sites of LuxR, AphA, and VqsA in the vqsA gene were uncovered by electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analysis. Finally, VqsA was verified to play essential roles in QS-regulated phenotypes, i.e., type VI secretion system 2 (T6SS2)-dependent interbacterial competition, biofilm formation, exotoxin production, and in vivo virulence of V. alginolyticus Collectively, our data showed that VqsA is an important QS regulator in V. alginolyticusIMPORTANCE Investigation of the mechanism of regulation of quorum sensing (QS) systems will facilitate an understanding of bacterial pathogenesis and the identification of effective QS interference (QSI) targets. Here, we systematically screened transcriptional factors (TFs) that modulate the expression of the master QS regulators (MQSRs) LuxR and AphA, and a novel LysR-type transcriptional regulator, VqsA, was identified. Our data illuminated the mechanisms mediating the interaction among LuxR, AphA, and VqsA as well as the effects of these regulators on the expression and output of QS. The impaired expression of virulence genes as a result of vqsA disruption demonstrated that VqsA is an important player in QS regulation and pathogenesis and may be the third
MQSR involved in sensing environmental signals by vibrios to coordinate QS responses. This study will facilitate the development of strategies to interfere with QS and effectively control this pathogen that plagues the aquaculture industry.