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

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 Rhsfamily 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 Cterminus 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.

Small protein Cgl2215 enhances phenolic tolerance by promoting MytA activity in Corynebacterium glutamicum

Corynebacterium glutamicum is a promising chassis microorganism for the bioconversion of lignocellulosic biomass owing to its good tolerance and degradation of the inhibitors generated in lignocellulosic pretreatments.Among the identified proteins encoded by genes within the C.glutamicum genome,nearly 400 are still functionally unknown.Based on previous transcriptome analysis,we found that the hypothetical protein gene cgl2215 was highly upregu-lated in response to phenol,ferulic acid,and vanillin stress.The cgl2215 deletion mutant was shown to be more sensitive than the parental strain to phenolic compounds as well as other environmental factors such as heat,ethanol,and oxidative stresses.Cgl2215 interacts with C.glutamicum mycoloyltransferase A(MytA)and enhances its in vitro esterase activity.Sensitivity assays of theΔmytA andΔcgl2215ΔmytA mutants in response to phenolic stress estab-lished that the role of Cgl2215 in phenolic tolerance was mediated by MytA.Furthermore,transmission electron microscopy(TEM)results showed that cgl2215 and mytA deletion both led to defects in the cell envelope structure of C.glutamicum,especially in the outer layer(OL)and electron-transparent layer(ETL).Collectively,these results indicate that Cgl2215 can enhance MytA activity and affect the cell envelope structure by directly interacting with MytA,thus playing an important role in resisting phenolic and other environmental stresses.