P-147 - Characterization of two chimeric type VI secretion system effectors in plant-associated Pseudomonas spp.

The Type VI secretion system (T6SS) is a bacterial nanoweapon that delivers diverse effectors mediating interbacterial competition and host interactions. Many T6SS effectors (T6Es) contain only a single catalytic domain that exerts specific antibacterial activity, but the significance of multi-catalytic domain effectors with functional versatility remains largely unexplored. In this study, we identified two homologous chimeric T6Es from ecologically distinct plant-associated Pseudomonas species: PK5712 from the plant growth-promoting rhizobacterium P. kribbensis XP1-6 and PSPTO5413L from the phytopathogenic bacterium P. syringae pv. tomato (Pst) DC3000. Structural and functional analyses using AlphaFold and Foldseek revealed three catalytic domains in these two T6Es, including autolysin-, lysostaphin-, and lysozyme-like domains, suggesting their targets on bacterial cell wall. Interspecies competition assays revealed that PSPTO5413L exhibits strong antibacterial activity in Pst DC3000, whereas PK5712 plays a limited role in XP1-6. Remarkably, the expression of PK5712 in Pst DC3000 could restore the reduced interbacterial competition ability of PSPTO5413L mutant against E. coli. Additionally, in silico predictions and mutagenesis analysis also identified the corresponding immunity proteins regulating their toxicity. This study highlights the role of chimeric T6Es and the power of in silico approaches in revealing molecular functions of T6Es.