Tke5 is a Pseudomonas putida toxin that kills plant pathogens by depolarising membranes.

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 with a significant fitness advantage, allowing this biocontrol agent to thrive in plant-related polymicrobial environments and prevent phytopathogen infections. Despite its agricultural significance, the toxin repertoire of P. putida, particularly those secreted via the K2- and K3-T6SSs, remains poorly understood. We present a comprehensive molecular study of Tke5, a potent toxin encoded within the K3-T6SS, which represents the initial biophysical and functional analysis of the BTH_I2691 family. Our data demonstrate that Tke5 is a pore-forming toxin that disrupts bacterial membranes through selective ion transport, inducing membrane depolarisation and cell death. Tke5 is neutralised by Tki5 in the inner membrane of Gram-negative bacteria. Unlike detergent-like pore-forming toxins, Tke5 preserves overall membrane integrity, avoiding large, non-specific disruptions. This mechanism offers a powerful approach to targeting resilient phytopathogens. This study reveals a previously undescribed mode of action within a widespread yet understudied toxin family. Our findings highlight the potential of P. putida as a biocontrol agent, offering alternatives to chemical pesticides by exploiting novel toxin mechanisms, crucial for developing effective strategies to combat plant pathogens.