A Quorum Sensing-Regulated Type VI Secretion System Containing Multiple Nonredundant VgrG Proteins Is Required for Interbacterial Competition in Chromobacterium violaceum.

The environmental pathogenic bacterium Chromobacterium violaceum kills Gram-positive bacteria by delivering violacein packed into outer membrane vesicles, but nothing is known about its contact-dependent competition mechanisms. In this work, we demonstrate that C. violaceum utilizes a type VI secretion system (T6SS) containing multiple VgrG proteins primarily for interbacterial competition. The single T6SS of C. violaceum contains six vgrG genes, which are located in the main T6SS cluster and four vgrG islands. Using T6SS core component-null mutant strains, Western blotting, fluorescence microscopy, and competition assays, we showed that the C. violaceum T6SS is active and required for competition against Gram-negative bacteria such as Pseudomonas aeruginosa but dispensable for C. violaceum infection in mice. Characterization of single and multiple vgrG mutants revealed that, despite having high sequence similarity, the six VgrGs show little functional redundancy, with VgrG3 showing a major role in T6SS function. Our coimmunoprecipitation data support a model of VgrG3 interacting directly with the other VgrGs. Moreover, we determined that the promoter activities of T6SS genes increased at high cell density, but the produced Hcp protein was not secreted under such condition. This T6SS growth phase-dependent regulation was dependent on CviR but not on CviI, the components of a C. violaceum quorum sensing (QS) system. Indeed, a ΔcviR but not a ΔcviI mutant was completely defective in Hcp secretion, T6SS activity, and interbacterial competition. Overall, our data reveal that C. violaceum relies on a QS-regulated T6SS to outcompete other bacteria and expand our knowledge about the redundancy of multiple VgrGs. IMPORTANCE The type VI secretion system (T6SS) is a contractile nanomachine used by many Gram-negative bacteria to inject toxic effectors into adjacent cells. The delivered effectors are bound to the components of a puncturing apparatus containing the protein VgrG. The T6SS has been implicated in pathogenesis and, more commonly, in competition among bacteria. Chromobacterium violaceum is an environmental bacterium that causes deadly infections in humans. In this work, we characterized the single T6SS of C. violaceum ATCC 12472, including its six VgrG proteins, regarding its function and regulation. This previously undescribed C. violaceum T6SS is active, regulated by QS, and required for interbacterial competition instead of acute infection in mice. Among the VgrGs, VgrG3, encoded outside the main T6SS cluster, showed a major contribution to T6SS function. These results shed light on a key contact-dependent killing mechanism used by C. violaceum to antagonize other bacteria.

The MarR family regulator OsbR controls oxidative stress response, anaerobic nitrate respiration, and biofilm formation in Chromobacterium violaceum.

BACKGROUND: Chromobacterium violaceum is an environmental opportunistic pathogen that causes rare but deadly infections in humans. The transcriptional regulators that C. violaceum uses to sense and respond to environmental cues remain largely unknown. RESULTS: Here, we described a novel transcriptional regulator in C. violaceum belonging to the MarR family that we named OsbR (oxidative stress response and biofilm formation regulator). Transcriptome profiling by DNA microarray using strains with deletion or overexpression of osbR showed that OsbR exerts a global regulatory role in C. violaceum, regulating genes involved in oxidative stress response, nitrate reduction, biofilm formation, and several metabolic pathways. EMSA assays showed that OsbR binds to the promoter regions of several OsbR-regulated genes, and the in vitro DNA binding activity was inhibited by oxidants. We demonstrated that the overexpression of osbR caused activation of ohrA even in the presence of the repressor OhrR, which resulted in improved growth under organic hydroperoxide treatment, as seem by growth curve assays. We showed that the proper regulation of the nar genes by OsbR ensures optimal growth of C. violaceum under anaerobic conditions by tuning the reduction of nitrate to nitrite. Finally, the osbR overexpressing strain showed a reduction in biofilm formation, and this phenotype correlated with the OsbR-mediated repression of two gene clusters encoding putative adhesins. CONCLUSIONS: Together, our data indicated that OsbR is a MarR-type regulator that controls the expression of a large number of genes in C. violaceum, thereby contributing to oxidative stress defense (ohrA/ohrR), anaerobic respiration (narK1K2 and narGHJI), and biofilm formation (putative RTX adhesins).