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 BTHI2691 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. Physiological and biophysical studies of Tke5 reveal is a pore-forming toxin that kills resilient phytopathogens via ion-selective membrane depolarisation. Neutralised by Tki5, Tke5 offers a targeted biocontrol mechanism to replace chemical pesticides.
Velázquez et al. (2026) studied this question.