Delivery of an Rhs‐family nuclease effector reveals direct penetration of the gram‐positive cell envelope by a type VI secretion system in <i>Acidovorax citrulli</i>

Abstract

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, <i>Acidovorax citrulli</i> (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 <i>rhsB</i> significantly impairs the ability of killing <i>Bacillus subtilis</i> while ectopic expression of immunity proteins RimB1 or RimB2 confers protection. We demonstrate that the AC T6SS not only can effectively outcompete <i>Escherichia coli</i> and <i>B. subtilis in planta</i> 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.