Tke5 is a novel Pseudomonas putida toxin that depolarises membranes killing plant pathogens

The soil bacterium Pseudomonas putida injects toxic proteins into neighbouring competitors including resilient phytopathogens using the Type VI secretion system (T6SS). The secretion of toxins endows P. putida a significant fitness advantage, allowing this biocontrol agent to thrive in plant-related polymicrobial environments and prevent phytopathogen infections. However, despite the significance of P. putida in agriculture and biotechnology, the identity and mechanisms of action of most of its toxins remain largely unexplored. This is in part because only one of the three T6SSs identified in P. putida (K1-T6SS) is active under laboratory conditions, limiting the study of toxins secreted through the K2- and K3-T6SSs. We present the first comprehensive molecular characterisation of Tke5, a potent toxin encoded within the K3-T6SS, which represents the first functional analysis of the BTH_I2691 protein family within the cl49522 superfamily. Our findings reveal that Tke5 is a novel pore-forming toxin that disrupts bacterial membranes through selective ion transport rather than by forming large, non-specific membrane disruptions. This mechanism leads to membrane depolarisation and bacterial cell death while preserving overall membrane integrity, distinguishing it from conventional detergent-like pore-forming toxins. This discovery not only contributes to our fundamental understanding of a widespread yet understudied toxin family, but also introduces a powerful new mechanism for targeting even the most resilient phytopathogens, potentially revolutionising the biocontrol capabilities of P. putida . Our work highlights the importance of studying bacterial toxins in the context of biocontrol, offering alternatives to combat plant pathogens without relying on traditional chemical pesticides.