C.M.S. is funded by the program Juan de la Cierva Incorporación (IJC2018-036923-I) and Proyectos dirigidos por jóvenes investigadores de la Universidad
de Málaga (B1-2021_21). P.B. is supported by the
MCIN/AEI/10.13039/501100011033 Spanish agency
through the Ramon y Cajal RYC2019-026551-I
and the “Generation of knowledge” Ref: PID2021-
123000OB-I00 “NextGenerationEU/PRTR” grants., Peer reviewed

Pseudomonas putida is a plant-beneficial rhizobacterium that encodes multiple type-VI secretion systems (T6SS) to outcompete phytopathogens in the rhizosphere. Among its antibacterial effectors, Tke5 (a member of the BTH_I2691 protein family) is a potent pore-forming toxin that disrupts ion homeostasis without causing considerable membrane damage. Tke5 harbours an N-terminal MIX domain, which is required for T6SS-dependent secretion in other systems. Many MIX domain-containing effectors require T6SS adaptor proteins (Tap) for secretion, but their molecular mechanisms of adaptor-effector binding remain elusive. Here, we report the 2.8 Å cryo-EM structure of the Tap3-Tke5 complex of P. putida strain KT2440, providing structural and functional insights into how effector Tke5 is recruited by its cognate adaptor protein Tap3. Functional dissection shows that the α-helical region of Tke5 is sufficient to kill intoxicated bacteria, while its β-rich region likely contributes to target membrane specificity. These findings delineate a mechanism of BTH_I2691 proteins for Tap recruitment and toxin activity, contributing to our understanding of a widespread yet understudied toxin family., The authors would like to thank Diamond Light Source for sample screening and data collection access and support of the cryo-EM facilities at the UK’s national Electron Bio-imaging Centre (eBIC) [under proposals EM BI38262], funded by the Wellcome Trust, MRC and BBSRC. We thank Pablo Guerra from the IBMB-CSIC CryoEM Platform for assistance during microscope data acquisition (experiment AV-20240320043). The authors acknowledge funding from Project IU16-014045 (CRYO-TEM) by the Generalitat de Catalunya and by “ERDF A way of making Europe”, a European Union initiative. This study was supported by the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033/ FEDER, UE projects PID2021-123000OB-I00, PID2024-159235OB-I00, and CNS2022-135585 to PB, and projects PID2021-127816NB-I00 and PID2024-155225NB-I00 to DAJ) and the Basque Government (IT1745-22 to DAJ). MZZ and JAA acknowledge support from the Basque Government predoctoral program (PRE_2023_1_0100 and PRE_2021_1_0164, respectively)., Peer reviewed

Pseudomonas putida is a plant-beneficial rhizobacterium that encodes multiple type-VI secretion systems (T6SS) to outcompete phytopathogens in the rhizosphere. Among its antibacterial effectors, Tke5 (a member of the BTH_I2691 protein family) is a potent pore-forming toxin that disrupts ion homeostasis without causing considerable membrane damage. Tke5 harbours an N-terminal MIX domain, which is required for T6SS-dependent secretion in other systems. Many MIX domain-containing effectors require T6SS adaptor proteins (Tap) for secretion, but their molecular mechanisms of adaptor-effector binding remain elusive. Here, we report the 2.8 Å cryo-EM structure of the Tap3-Tke5 complex of P. putida strain KT2440, providing structural and functional insights into how effector Tke5 is recruited by its cognate adaptor protein Tap3. Functional dissection shows that the α-helical region of Tke5 is sufficient to kill intoxicated bacteria, while its β-rich region likely contributes to target membrane specificity. These findings delineate a mechanism of BTH_I2691 proteins for Tap recruitment and toxin activity, contributing to our understanding of a widespread yet under-studied toxin family., Ministerio de Ciencia e Innovación (MCIN): PID2021-127816NB-I00 PID2021-123000OB-I00 PID2024-155225NB-I00 PID2024-159235OB-I00 CNS2022-135585;
Eusko Jaurlaritza (Gobierno Vasco): IT1745-22;
Generalitat de Catalunya (Government of Catalonia): IU16-014045, Peer reviewed