Acid-Induced Type VI Secretion System Is Regulated by ExoR-ChvG/ChvI Signaling Cascade in Agrobacterium tumefaciens

The type VI secretion system (T6SS) is a widespread, versatile protein secretion system in pathogenic Proteobacteria. Several T6SSs are tightly regulated by various regulatory systems at multiple levels. However, the signals and/or regulatory mechanisms of many T6SSs remain unexplored. Here, we report on an acid-induced regulatory mechanism activating T6SS in Agrobacterium tumefaciens, a plant pathogenic bacterium causing crown gall disease in a wide range of plants. We monitored the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) from A. tumefaciens and found that acidity is a T6SS-inducible signal. Expression analysis of the T6SS gene cluster comprising the imp and hcp operons revealed that imp expression and Hcp secretion are barely detected in A. tumefaciens grown in neutral minimal medium but are highly induced with acidic medium. Loss- and gain-of-function analysis revealed that the A. tumefaciens T6SS is positively regulated by a chvG/chvI two-component system and negatively regulated by exoR. Further epistasis analysis revealed that exoR functions upstream of the chvG sensor kinase in regulating T6SS. ChvG protein levels are greatly increased in the exoR deletion mutant and the periplasmic form of overexpressed ExoR is rapidly degraded under acidic conditions. Importantly, ExoR represses ChvG by direct physical interaction, but disruption of the physical interaction allows ChvG to activate T6SS. The phospho-mimic but not wild-type ChvI response regulator can bind to the T6SS promoter region in vitro and activate T6SS with growth in neutral minimal medium. We present the first evidence of T6SS activation by an ExoR-ChvG/ChvI cascade and propose that acidity triggers ExoR degradation, thereby derepressing ChvG/ChvI to activate T6SS in A. tumefaciens.VI型分泌系统（Type VI secretion system, T6SS）是致病变形菌门（Proteobacteria）中广泛分布、功能多样的蛋白质分泌系统。部分T6SS受到多层级、多类别的调控系统的严格调控，但目前仍有大量T6SS的信号通路与调控机制尚未得到阐明。本研究报道了根癌农杆菌（Agrobacterium tumefaciens）中一种由酸性环境诱导的T6SS激活调控机制——该菌株是一种可引发多种植物冠瘿瘤病的植物病原菌。本研究通过监测根癌农杆菌的T6SS标志性蛋白溶血素共调节蛋白（hemolysin-coregulated protein, Hcp）的分泌情况，发现酸性环境是可诱导T6SS激活的信号。对包含imp操纵子与hcp操纵子的T6SS基因簇进行表达分析后发现，在中性基础培养基中培养的根癌农杆菌几乎无法检测到imp基因的表达与Hcp蛋白的分泌，但在酸性培养基中二者均被显著诱导上调。功能缺失与功能获得性实验分析表明，根癌农杆菌的T6SS受到ChvG/ChvI双组分系统（two-component system）的正向调控，同时受到ExoR的负向调控。进一步的上位性分析（epistasis analysis）显示，在调控T6SS的通路中，ExoR作用于ChvG感应激酶（sensor kinase）的上游。蛋白水平检测发现，exoR缺失突变体中的ChvG蛋白含量显著升高，且过表达的ExoR的周质（periplasmic）形式在酸性条件下会被快速降解。重要的是，ExoR可通过直接物理相互作用抑制ChvG，而二者物理结合的阻断则会使ChvG得以激活T6SS。体外实验显示，磷酸化模拟型而非野生型的ChvI应答调控因子（response regulator）可结合T6SS启动子区域（promoter region），并能在中性基础培养基中培养时激活T6SS。本研究首次揭示了由ExoR-ChvG/ChvI级联通路介导的T6SS激活机制，并提出酸性环境会触发ExoR降解，从而解除ChvG/ChvI的抑制以激活根癌农杆菌的T6SS。