Civantos, C.; Paredes, C.; Murillo-Torres, M.; Botelho, J.; Sanchez-Romero, M. A.; Allsopp, L. P.; Bernal, P.

The Type VI secretion system (T6SS) is a bacterial nanoweapon that injects toxic effectors into prokaryotic and eukaryotic cells. It is widely found among gram-negative bacteria and provides a significant fitness advantage in interbacterial competition. Pseudomonas putida KT2440 possesses three T6SS clusters (K1-, K2- and K3-T6SS) that combat phytopathogens. This makes this strain a potent biocontrol agent that protects plants from pathogens and can be further enhanced by a better understanding of its T6SS regulation. Although the core components of T6SS are conserved, the elements controlling its regulation differ among bacterial species. T6SS activity is regulated by various factors acting at different levels, from transcription to post-translational modification, to ensure precise control of its activity. Here, we demonstrate the critical importance that the three Rsm proteins, RsmIEA, have in controlling the K1-T6SS structural components and related orphan elements at the post-transcriptional level in Pseudomonas putida. We identified multiple Rsm-binding sites responsible for directly repressing the translation of T6SS proteins (Hcp1 and Hcp5) and their associated effectors (Tke2 and Tke7). Derepression of K1-T6SS mRNA in the rsmIEA mutant led to enhanced translation and expression of the K1-T6SS components and effectors, and critically increased the number of cells in the population with assembled T6SS. This results in a greater capacity to secrete toxins and kill prey cells via the T6SS-dependent mechanism. Finally, we demonstrate the K1-T6SS ability to kill environmental pathogens, including Salmonella enterica and Erwinia amylovora.