TssW-PpkA-Fha axis controls the positioning and initiation of the type VI secretion system in Acidovorax citrulli.

Understanding how bacteria spatially regulate their molecular weaponry is key to unraveling their virulence and survival strategies in competitive ecosystems. The type VI secretion system (T6SS) is a highly potent contractile nanomachine that promotes bacterial survival by targeting competitors and hosts. While T6SS functionality has been extensively linked to the diversity of secreted effectors, the role of accessory components in spatially regulating its activity remains less understood. Here, we uncover that two membrane components, PpkA and TssW, are crucial modulators of T6SS efficacy in the plant pathogen Acidovorax citrulli, which possesses broad-spectrum antibacterial and antifungal activity. PpkA, an inner-membrane kinase, is essential for constitutive T6SS activity through its interactions with structural components and the assembly scaffold Fha. TssW, an outer-membrane anchor, determines T6SS positioning via interactions spanning the inner and outer membranes. Notably, PpkA promotes T6SS assembly by phosphorylating Fha and forming liquid-liquid phase-separated condensates that selectively recruit T6SS structural components at the membrane interface. These findings reveal a novel mechanism for accessory proteins in T6SS positioning and efficacy, highlighting the importance of spatial regulation in bacterial competition and virulence. IMPORTANCE: How cells determine where to assemble a macromolecular complex is a fundamental question in biology since the localization of these complexes is directly linked to functions. In bacteria, the type VI secretion system (T6SS) relies on effective positioning to target competitor and host cells in contact-dependent interactions. This study identifies a PpkA-TssW-Fha axis that orchestrates T6SS localization and activation through membrane anchoring and liquid-liquid phase separation at the inner membrane interface. These new insights can help us not only better understand how the T6SS functions but also better design T6SS-based solutions for therapeutic targeting of drug-resistant and T6SS-susceptible bacterial and fungal pathogens.