The mycobacterial LysR-type regulator OxyS responds to oxidative stress and negatively regulates expression of the catalase-peroxidase gene

Abstract

Protection against oxidative stress is one of the primary defense mechanisms contributing to the survival of Mycobacterium tuberculosis in the host. In this study, we provide evidence that OxyS, a LysR-type transcriptional regulator functions as an oxidative stress response regulator in mycobacteria. Overexpression of OxyS lowers expression of the catalase-peroxidase (KatG) gene in M. smegmatis. OxyS binds directly with the katG promoter region and a conserved, GC-rich T-N(11)-A motif for OxyS binding was successfully characterized in the core binding site. Interestingly, the DNA-binding activity of OxyS was inhibited by H(2)O(2), but not by dithiothreitol. Cys25, which is situated at the DNA-binding domain of OxyS, was found to have a regulatory role for the DNA-binding ability of OxyS in response to oxidative stress. In contrast, the other three cysteine residues in OxyS do not appear to have this function. Furthermore, the mycobacterial strain over-expressing OxyS had a higher sensitivity to H(2)O(2). Thus, OxyS responds to oxidative stress through a unique cysteine residue situated in its DNA-binding domain and negatively regulates expression of the katG gene. These findings uncover a specific regulatory mechanism for mycobacterial adaptation to oxidative stress.

Figure 1. OxyS is a LysR-type regulator in M. tuberculosis and is involved in regulation of katG.

(A) Conserved domain analysis of M. tuberculosis OxyS. The conserved domains in the N-terminus and C-terminus of OxyS were found by searching the CDD database on the NCBI website. (B) The modeled structure of OxyS was obtained using the automated comparative protein modeling web server SWISS-MODEL and CbnR protein (a LysR family transcriptional regulator in Ralstonia eutropha NH9) as a template (PDB ID: 1iz1). (C) Detection of OxyS protein by western blotting in the recombinant mycobacterial strains. An inducible system for conditional gene over-expression in mycobacteria was used to over-express OxyS in M. smegmatis. Lane 1, purified His-OxyS; Lane 2, cell lysate from Msm/pMV261; Lane 3, cell lysate from Msm/pMV261-OxyS. (D) qRT-PCR assays conducted to analyze changes in gene expression in M. smegmatis. The experiment was carried out as described in the “Materials and Methods” section. The 16S rRNA gene, rrs, was used as an internal control for normalization. Target genes were amplified using specific primers. Expression levels of all genes were normalized to the levels of 16S rRNA gene transcripts, and fold-changes in expression of each gene were calculated. Representative data are shown.

Figure 2. OxyS interacts with the regulatory region of katG in M. tuberculosis.

(A) The regulatory sequence of the katG gene was cloned into the upstream of HIS3-aadA reporter genes of the bacterial one-hybrid reporter vector pBXcmT . (B) The interaction between OxyS and the promter region of katG was measured by bacterial one-hybrid analysis . Left panel: bacterial one-hybrid plates. Right panel: an outline of the plates in the left panel. Each unit represents the corresponding co-transformant in the plates. CK+: co-transformant containing pBX-Rv2031p and pTRG-Rv3133c as a positive control. CK−: co-transformant containing pBX-Rv2031p and pTRG-Rv3133c-deltaC as a negative control . Rv3911p (the promoter of the Rv3911 gene) was also used as a negative control. (C) in vivo ChIP assays for the interaction of OxyS with the katG promoter in M. tuberculosis. DNA recovered from the immunoprecipitates was amplified with primers specific for either katGp or a negative control promoter Rv3911p. ‘+’ refers to the immunoprecipitate obtained with OxyS antibodies, whereas ‘−’ refers to the control in which ChIP was carried out without any primary antibodies. ‘Input’ refers to total genomic DNA prior to IP reaction and was used as a positive control in PCR. (D) EMSA assays for the binding of OxyS to the katG promoter. The EMSA reactions (10 µl) for measuring mobility shift contained FITC-labeled DNA and increasing amount of OxyS (100 nM, 200 nM, 300 nM, 400 nM, 500 nM and 600 nM). The protein/DNA complexes are indicated by arrows on the right of the panels.

Figure 3. Identification of the binding sites for OxyS in the promoter region of katG.

(A) DNase I footprinting assays were employed to assess the binding sequence of OxyS. The experiments were carried out as described under the “Materials and Methods” section. The ladders are shown in the right panel and the nucleotide sequences obtained are listed. The protected regions are underlined. (B) Summary of OxyS footprinting analysis in the M. tuberculosis katG promoter region. The DNA sequence where OxyS was found to bind corresponds with the katG promoter region from −180 to −1. OxyS footprint regions (underlined) and the position of the katG translation start site are indicated. The T-N₁₁-A motif conserved in the DNA binding sites of LysR regulators is indicated on the sequence. Two OxyS binding boxes are indicated. (C) A blast assay for the conserved sequence motif recognition by OxyS (OxyS box1) among different mycobacterial species. Sequence alignment was carried and visualized locally using the BioEdit software. The conserved T-N₁₁-A DNA-binding site in LysR regulators is shown at the bottom. M. tu, Mycobacterium tuberculosis H37Rv; M. bo, Mycobacterium bovis AF2122/97; M. in, Mycobacterium intracellulare ATCC 13950; M. av, Mycobacterium avium subsp. paratuberculosis K-10; M. sm, Mycobacterium smegmatis str. mc²155; M. ka, Mycobacterium kansasii ATCC 12478; M. ma, Mycobacterium marinum M; M. ul, Mycobacterium ulcerans Agy99. (D) DNA-binding assays for OxyS on OxyS box1 and OxyS box1mut substrates. The EMSA reactions (10 µl) for measuring mobility shift contained FITC-labeled DNA substrate and increasing amount of OxyS (0 nM, 100 nM, 200 nM and 300 nM). The conserved “TG” and “GA” in OxyS box1 were replaced by “CC” in OxyS box1mut substrate. The protein/DNA complexes are indicated by arrows on the right of the panels.

Figure 4. Effect of H₂O₂ on the DNA binding ability of OxyS and its mutant variants.

(A) Effects of H₂O₂ and DTT on the DNA binding activity of OxyS were measured by EMSA. The concentration of OxyS in lanes 1 to 10 was 400 nM. The increasing concentration of H₂O₂ or DTT is indicated at the top of the panels. (B) qRT-PCR assays conducted to analyze changes in gene expression of katG in M. smegmatis over-expressing OxyS after H₂O₂ (2 mM) treatment. The experiment was carried out as described in the “Materials and Methods” section. Expression levels of all genes were normalized to the levels of 16S rRNA gene transcripts, and the fold-changes in expression were calculated. Representative data are shown. (C) EMSA assays for the interactions of the mutant proteins with katGp. The protein/DNA complexes are indicated by arrows on the right of the panels. The increasing concentrations of proteins are indicated at the top of the panels. (D) Effects of H₂O₂ on the DNA binding activity of the mutant proteins. The concentration of proteins was 400 nM. The concentration of H₂O₂ is indicated at the top of the panels. The protein/DNA complexes are indicated by arrows on the right of the panels.

Figure 5. Effects of H₂O₂ on the growth of recombinant mycobacterial strains and the interaction of OxyS with katG promoter in M. smegmatis.

(A) Effects of H₂O₂ on the growth of recombinant mycobacterial strains. The antimicrobial activity of H₂O₂ against M. smegmatis was determined using a modified bacterial growth time course assay described under “Materials and Methods”. M. smegmatis strains were grown in LB at 37°C overnight. This culture was then diluted (1∶100) in 5 ml of fresh LB broth containing the indicated concentration of H₂O₂, and the culture was again incubated at 37°C with shaking at 220 rpm for three days. Aliquots were taken at the indicated times, and the number of CFUs per ml (cfu/ml) was determined. Each analysis was performed in triplicate. Symbols are the average of three replicates, and error bars indicate the SDs (Standard Deviation) of three replicate samples. The recombinant mycobacterial strains are indicated. (B) ChIP assays for the effect of H₂O₂ on the interaction of OxyS and its mutant proteins with katG promoter in M. smegmatis. The experiments were carried out as described under “Materials and Methods”. Recombinant M. smegmatis strain over-expressing OxyS or its mutant proteins was treated with H₂O₂ (lane 2, 0 mM; lane 3, 2 mM) before cross-link. DNA recovered from the immunoprecipitates was amplified with primers specific for MsmkatGp. ‘+’ refers to the immunoprecipitate obtained with OxyS antibodies, whereas ‘−’ refers to the control in which ChIP was carried out without any primary antibodies. ‘Input’ refers to total genomic DNA prior to IP reaction and was used as a positive control in PCR.