Phosphorylation of PppA at threonine 253 controls T6SS2 expression and bacterial killing capacity in the marine pathogen Vibrio alginolyticus

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.