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.

Mycobacterium Lrp/AsnC family transcriptional factor modulates the arginase pathway as both a sensor and a transcriptional repressor

L-Arginine is the precursor of nitric oxide(NO),a host immune effector against intracellular pathogens including Mycobacterium tuberculosis(M.tb).Pathogens including M.tb have evolved various strategies targeting arginine to block the production of NO for better survival and proliferation.However,L-arginine metabolism and regulation in Mycobacterium are poorly understood.Here,we report the identification of M.smegmatis MSMEG_1415(homolog of M.tb Rv2324)as an arginine-responsive transcriptional factor regulating the arginase pathway.In the absence of L-arginine,MSMEG_1415 acts as a repressor to inhibit the transcription of the roc(for arginine,ornithine catabolism)gene cluster,thereby switching off the arginase pathway.Treatment with L-arginine relieves the transcriptional inhibition of MSMEG_1415 on the roc gene cluster to activate the arginase pathway.Moreover,the L-arginine-MSMEG_1415 complex activates the transcription of the roc gene cluster by recognizing and binding a 15-bp palindrome motif,thereby preventing the excess accumulation of L-arginine in M.smegmatis.Physiologically,MSMEG_1415 confers mycobacteria resistance to starvation and fluoroquinolones exposure,suggestive of its important role in M.smegmatis persistence.The results uncover a unique regulatory mechanism of arginine metabolism in mycobacteria and identify M.tb Rv2324 as an attractive candidate target for the design of drugs against tuberculosis.