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

Abstract

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).

Keywords: Biofilm formation; Chromobacterium violaceum; MarR family regulator; Nitrate respiration; OsbR regulon; Oxidative stress; Transcriptome analysis.

Fig. 1

Construction and analyses of C. violaceum strains with deletion (∆osbR) or overexpression (WT(osbR)) of osbR. a The levels of OsbR in the indicated strains were analyzed by western blot using a polyclonal anti-OsbR antiserum. Equal protein loading was confirmed by Ponceau staining. Full-length blot and Ponceau-stained membrane are presented in Additional file 3, Fig. S3. b Growth curves of the indicated strains in LB medium. All strains showed a similar growth profile

Fig. 2

Microarray analyses revealed that ObsR regulates many genes involved in several biological processes. a Venn diagrams comparing the differentially expressed genes from the microarray analyses of this work (osbR strains) with published data (WT treated with the oxidant CHP). b, c Functional categorization by Gene Ontology (GO) of the differentially expressed genes from the comparison wild-type versus ∆osbR (b) and WT(osbR) versus ∆osbR(pJN105) (c)

Fig. 3

Validation by Northern blot of selected genes regulated by OsbR. RNA samples from the indicated C. violaceum strains (the same strains used in the microarray analyses) were transferred to a membrane and hybridized with specific radiolabeled probes for the selected genes (indicated by black arrows in the gene maps). The selected genes were repressed (narX, narJ, and CV_3659) or activated (CV_0568 and ohrA) by OsbR. Grey arrows indicate neighbor genes also differentially expressed according to microarray analyses. The rRNA were used as a loading control (Bottom gels). Full-length blot and RNA loading gel are presented in Additional file 3, Fig. S4

Fig. 4

OsbR directly activates and represses genes from its regulon. EMSA assay was performed with radiolabeled probes corresponding to the promoter regions of the indicated genes incubated with increasing concentrations of OsbR. The coding region of CV_0208 was used as a negative control. Competition assays were performed with an excess of specific (S) or unspecified (N) unlabeled fragments

Fig. 5

OsbR oxidation causes dimerization and inhibition of its DNA binding activity. a Formation of covalent OsbR dimers under oxidative stress in vitro. After 2 hours of exposure to oxidants or DTT, the His-OsbR protein was analyzed by non-reducing SDS-PAGE. NT refers to nontreated protein. Monomeric and dimeric forms are indicated by M and D, respectively. Asterisk indicates a third unknown multimeric form. b EMSA assay with oxidized OsbR. Prior to incubation with the DNA probes, His-OsbR was oxidized with 1 mM CHP for 1 hour (for the osbR probe, OsbR was oxidized with the indicated CHP concentrations)

Fig. 6

OsbR is required for fully ohrA expression and protection against oxidative stress. a Northern blot of ohrA using the indicated strains untreated (−) or treated (+) with 100 μM CHP for 10 min. The same film was exposed for 24 h (top) or 4 days (bottom). Ribosomal RNA loading is shown below. Full-length blot and RNA loading gel are presented in Additional file 3, Fig. S5. b, c Growth curves of different strains under oxidative stress. The indicated C. violaceum strains were grown in LB treated with either 120 μM TBHP (b) or 55 μM CHP (c)

Fig. 7

OsbR affects C. violaceum growth under anaerobic conditions. The indicated strains of C. violaceum were diluted to OD₆₀₀ = 0.1 in LB medium or LB plus 0.5% NaNO₃. The cultures were grown without agitation in an anaerobic jar and the OD₆₀₀ was measured after 48 h. Statistical analysis was performed by Two-way ANOVA and P value < 0.05 was considered significant. P value < 0.05 = *; P value < 0.01 = **; P value < 0.001 = ***; P value < 0.0001 = ****

Fig. 8

OsbR represses biofilm formation in C. violaceum. Strains were diluted to OD₆₀₀ = 0.01 in LB medium, and the biofilm formation was measured after growth in static condition for 24 h. Upper: Representative test tubes for each strain after biofilm staining with violet crystal. Bottom: Quantification of biofilm formation by measurement of OD₆₀₀. Statistical analysis was performed with One-way ANOVA with P value < 0.05 being considered significant. ****, P value < 0.0001