Type VI secretion systems (T6SSs) are multi-protein secretory nano-machines that mediate inter-bacterial competition. Vibrio alginolyticus is an abundant gram-negative marine bacterium that efficiently kills other bacteria with its T6SS2. The V. alginolyticus T6SS2 gene cluster encodes a phosphatase, PppA, and a type II membrane-spanning Hanks-type threonine kinase, PpkA2, which have been implicated in the activation of T6S. Meanwhile, T6SS2 gene expression is under the control of quorum sensing. However, the role of PppA in T6SS2 activity is unclear. Here, our phosphoproteomic screen identified PppA as a novel PpkA2 substrate. Phosphorylation at threonine 253 (T253) of PppA is not conserved in other bacteria, suggesting that PppA may play a unique role in T6SS2 activation in V. alginolyticus. Interestingly, PppA phosphatase activity was modulated by the cognate kinase PpkA2, which implied that a homeostasis is required for optimal T6S activity. PppA and phosphorylation of PppA at T253 are important for T6S activity and T6SS2-mediated bacterial killing. Moreover, PppA and the phosphorylation of PppA are also essential for the expression of LuxR, the master regulator of quorum sensing, thus augmenting T6SS2 expression. Collectively, our data demonstrated that phosphorylation of PppA at T253 controls the activity of T6SS2, thereby enhancing the competitive fitness of V. alginolyticus.

VI型分泌系统（T6SSs）是介导细菌间竞争的多蛋白分泌型纳米机器。溶藻弧菌是一种革兰氏阴性海洋细菌，其T6SS2可以有效杀死其他细菌。该溶藻弧菌V. T6SS2基因簇编码磷酸酶，PPPA，和II型跨膜汉克斯型苏氨酸激酶，PpkA2，已经在T6S的活化有关。同时，T6SS2基因表达受到群体感应的控制。但是，尚不清楚PppA在T6SS2活性中的作用。在这里，我们的磷酸化蛋白质组学筛查确定PppA为新型PpkA2底物。PppA的苏氨酸253（T253）的磷酸化在其他细菌中并不保守，这表明PppA可能在TppSS2激活中起独特作用。溶藻弧菌。有趣的是，PppA磷酸酶的活性是由同源激酶PpkA2调节的，这意味着最佳的T6S活性需要动态平衡。在T253处PppA和PppA的磷酸化对于T6S活性和T6SS2介导的细菌杀伤很重要。此外，PppA和PppA的磷酸化对于LuxR的表达也很重要，LuxR是群体感应的主要调控因子，因此可以增强T6SS2的表达。总体而言，我们的数据表明，在T253处PppA的磷酸化控制了T6SS2的活性，从而增强了溶藻弧菌的竞争适应性。