Pseudomonas aeruginosa is a major nosocomial pathogen in which the type Ⅵ secretion system (T6SS) contributes to interbacterial competition and virulence. While most strains encode three T6SSs, additional T6SS clusters have been identified in clinical isolates through comparative genomics, but their functions and effector biology remain undefined. Here, we identify TseMt as a major antibacterial effector associated with an H4‐T6SS in a clinical P. aeruginosa isolate LYSZa7. TseMt is a periplasmically active toxin whose activity is neutralized by a cognate immunity protein, TsiMt. Biochemical assays show that TseMt binds membranes andate and reveal its role in mediating bacterial com forms ion‐conducting pores, establishing it as a pore‐forming effector. A 3.0 Å cryo‐electron microscopy structure reveals a distinct three‐domain architecture comprising an N‐terminal MIX‐like domain, a central α‐helical scaffold, and a C‐terminal toxin domain. Genetic analysis and structural modeling indicate that TseMt is delivered through a dedicated PAAR−VgrG−chaperone pathway. Together, these findings define the structural basis, functional mechanism, and delivery pathway of the H4‐T6SS effector TseMt from a clinical P. aeruginosa isolate and reveal its role in mediating bacterial competition.

The type Ⅵ secretion system (T6SS) is a double-tubular nanomachine widely found in gram-negative bacteria. Its spear-like Hcp tube is capable of penetrating a neighboring cell for cytosol-to-cytosol protein delivery. However, gram-positive bacteria have been considered impenetrable to such T6SS action. Here we report that the T6SS of a plant pathogen, Acidovorax citrulli (AC), could deliver an Rhs-family nuclease effector RhsB to kill not only gram-negative but also gram-positive bacteria. Using bioinformatic, biochemical, and genetic assays, we systematically identified T6SS-secreted effectors and determined that RhsB is a crucial antibacterial effector. RhsB contains an N-terminal PAAR domain, a middle Rhs domain, and an unknown C-terminal domain. RhsB is subject to self-cleavage at both its N- and C-terminal domains and its secretion requires the upstream-encoded chaperone EagT2 and VgrG3. The toxic C-terminus of RhsB exhibits DNase activities and such toxicity is neutralized by either of the two downstream immunity proteins, RimB1 and RimB2. Deletion of rhsB significantly impairs the ability of killing Bacillus subtilis while ectopic expression of immunity proteins RimB1 or RimB2 confers protection. We demonstrate that the AC T6SS not only can effectively outcompete Escherichia coli and B. subtilis in planta but also is highly potent in killing other bacterial and fungal species. Collectively, these findings highlight the greatly expanded capabilities of T6SS in modulating microbiome compositions in complex environments.