Physical interaction of MsTAG (Ms5082) with MsParA and its effect on mycobacterial growth in response to DNA damage induction.

<p>(A) Bacterial two-hybrid assays for the interaction of MsTAG with MsParA performed as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038276#s2" target="_blank">Materials and Methods</a>’. (B) Co-IP assays. Exponentially growing cells of recombinant <i>M. smegmatis</i> containing MsTAG-expression plasmid were harvested, resuspended and lysed. Co-IP assays were performed as described under ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038276#s2" target="_blank">Materials and Methods</a>’. Right panel shows a negative control using an unrelated anti-Ms3759 anti-serum. (C) MMS sensitivity assays for the MsTAG-overexpressing <i>M. smegmatis</i> strains. Growth of the recombinant mycobacterial strains were examined in the presence or absence of 0.012% MMS. Aliquots were taken at the indicated times and the CFU was measured. Each analysis was performed in triplicate. Representative growth curves are shown. The recombinant mycobacterial strains are indicated above the panels. (D) Scanning electron microscopy assay of cell morphology. The recombinant mycobacterial strains were grown in the presence of 0.012% MMS and SEM observation was carried out as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038276#s2" target="_blank">Materials and Methods</a>’. Representative images are shown. The images were taken at 8000× magnification. Bars, 2 µm.</p

The <i>M. tuberculosis</i> MtTAG interacts with MsParA and affects the growth of <i>M. smegmatis</i>.

<p>(<b>A</b>) Effects of MtTAG on the ATPase activity of MsParA. (<b>B</b>) Effects of MtTAG and its mutant variants on the growth of <i>M. smegmatis</i>. Growth patterns of <i>M. smegmatis</i> strains overexpressing MtTAG and its mutant variant (E48A) in the presence of MMS were determined as described under ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038276#s2" target="_blank">Materials and Methods</a>’. (<b>C</b>) Scanning electron microscopy assay of cell morphology. The cells were grown in 7H9 media supplemented with 0.012% MMS and SEM observation was carried out as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038276#s2" target="_blank">Materials and Methods</a>’. Representative images taken at 8000× magnification are shown.</p

Effects of MsTAG and its co-expression with MsParA on mycobacterial growth and morphology.

<p>(<b>A</b>) A portion of an alignment of 3-methyladenine DNA glycosylase is shown with conserved catalytic residues Glu (E) indicated by an arrow. (<b>B</b>) Comparative growths of <i>E. coli</i> overexpressing the <i>Tag</i> gene b3459 and <i>M. smegmatis</i> strain overexpressing MsTAG on 7H10 agar plates with or without 0.012% MMS at 37°C. (<b>C</b>) Co-IP assays for the interaction between the MsTAG-E46A mutant and MsParA. (<b>D</b>) MMS sensitivity assays. Growth of <i>M. smegmatis</i> strains overexpressing MsTAG or its mutant variant (E46A) and those co-expressing MsTAG and MsParA in 7H9 medium with and without 0.012% MMS were compared. Aliquots were taken at the indicated times and the OD600 was measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038276#s2" target="_blank">Materials and Methods</a>’. Each analysis was performed in triplicate. Representative growth curves are shown. (<b>E</b>) Scanning electron microscopy assay of cell morphology. The experiment was carried out as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038276#s2" target="_blank">Materials and Methods</a>’. The recombinant mycobacterial strains were grown in 7H9 medium supplemented with 0.012% MMS. Representative images are shown. The images were taken at 8000× magnification. Bars, 2 µm.</p

MsParA affects the growth and morphology of <i>M. smegmatis</i>.

<p>The wild-type and mutant strains were grown on the surface of solid agar medium and in the liquid 7H9 medium. (<b>A</b>) Strains were grown on 7H10 agar plates supplemented with 30 µg/ml Kanamycin (Kan) at 37°C for 48 hours. (<b>B</b>) Monitoring of growth on 7H9 medium of the <i>M. smegmatis</i> wild-type (Ms/pMV361), MsParA deletion strain (Msm-MsParA::hyg/pMV361) and MsParA complementation strain (Msm-MsParA::hyg/pMV361MsParA) by OD600 analysis as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038276#s2" target="_blank">Materials and Methods</a>”. (<b>C</b>) Scanning electron microscopy assay of cell morphology. The experiment was carried out as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038276#s2" target="_blank">Materials and Methods</a>”. Representative images are shown. The images were taken at 15,000× magnification. Bars, 1 µm.</p

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

<p>(<b>A</b>) 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>B</b>) qRT-PCR assays conducted to analyze changes in gene expression of <i>katG</i> in <i>M. smegmatis</i> over-expressing OxyS after H₂O₂ (2 mM) treatment. The experiment was carried out as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030186#s4" target="_blank">Materials and Methods</a>” 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. (<b>C</b>) EMSA assays for the interactions of the mutant proteins with <i>katGp</i>. 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. (<b>D</b>) 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.</p

List of identified OxyS orthologous proteins from different mycobacteria species.

<p>List of identified OxyS orthologous proteins from different mycobacteria species.</p

OxyS is a LysR-type regulator in <i>M. tuberculosis</i> and is involved in regulation of <i>katG</i>.

<p>(<b>A</b>) Conserved domain analysis of <i>M. tuberculosis</i> 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>B</b>) The modeled structure of OxyS was obtained using the automated comparative protein modeling web server SWISS-MODEL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030186#pone.0030186-Arnold1" target="_blank">[34]</a> and CbnR protein (a LysR family transcriptional regulator in Ralstonia eutropha NH9) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030186#pone.0030186-Muraoka1" target="_blank">[35]</a> as a template (PDB ID: 1iz1). (<b>C</b>) Detection of OxyS protein by western blotting in the recombinant mycobacterial strains. An inducible system for conditional gene over-expression in mycobacteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030186#pone.0030186-Stover1" target="_blank">[36]</a> was used to over-express OxyS in <i>M. smegmatis</i>. Lane 1, purified His-OxyS; Lane 2, cell lysate from Msm/pMV261; Lane 3, cell lysate from Msm/pMV261-OxyS. (<b>D</b>) qRT-PCR assays conducted to analyze changes in gene expression in <i>M. smegmatis</i>. The experiment was carried out as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030186#s4" target="_blank">Materials and Methods</a>” section. The 16S rRNA gene, <i>rrs</i>, 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.</p

Construction of the MsParA (Ms6939) knockout strain of <i>M. smegmatis</i> and Southern blot assays.

<p>(<b>A</b>) Schematic representation of the recombination strategy for the removal of MsParA from the genome of <i>M. smegmatis</i>. (<b>B</b>) A map of the recombinant vector pMindMsParA containing upstream and downstream sequences of MsParA, and the gene that confers resistance against hygromycin. (<b>C</b>) Schematic representation of the size of a <i>BstE</i> II-digested DNA fragment from the genomic DNA of Msm/WT strain (upper panel) and MsParA knockout strain (lower panel). The probe is indicated with a black bar. (<b>D</b>) Southern blot assays. A 300 bp probe corresponding to the sequences of the MsParA upstream genomic fragment of <i>M. smegmatis</i> was obtained by PCR and labeled with digoxigenin dUTP (Boehringer Mannheim, Inc., Germany). The probe was used to detect the size change of the <i>BstE</i> II-digested genomic fragment of <i>M. smegmatis</i> before and after recombination.</p

OxyS interacts with the regulatory region of <i>katG</i> in <i>M. tuberculosis</i>.

<p>(<b>A</b>) The regulatory sequence of the <i>katG</i> gene was cloned into the upstream of <i>HIS3-aadA</i> reporter genes of the bacterial one-hybrid reporter vector pBXcmT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030186#pone.0030186-Guo1" target="_blank">[19]</a>. (<b>B</b>) The interaction between OxyS and the promter region of <i>katG</i> was measured by bacterial one-hybrid analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030186#pone.0030186-Guo1" target="_blank">[19]</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030186#pone.0030186-Guo1" target="_blank">[19]</a>. <i>Rv3911p</i> (the promoter of the <i>Rv3911</i> gene) was also used as a negative control. (<b>C</b>) <i>in vivo</i> ChIP assays for the interaction of OxyS with the <i>katG</i> promoter in <i>M. tuberculosis</i>. DNA recovered from the immunoprecipitates was amplified with primers specific for either <i>katGp</i> or a negative control promoter <i>Rv3911p</i>. ‘+’ 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. (<b>D</b>) EMSA assays for the binding of OxyS to the <i>katG</i> 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.</p

Effects of H₂O₂ on the growth of recombinant mycobacterial strains and the interaction of OxyS with <i>katG</i> promoter in <i>M. smegmatis</i>.

<p>(<b>A</b>) Effects of H₂O₂ on the growth of recombinant mycobacterial strains. The antimicrobial activity of H₂O₂ against <i>M. smegmatis</i> was determined using a modified bacterial growth time course assay described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030186#s4" target="_blank">Materials and Methods</a>”. <i>M. smegmatis</i> 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>B</b>) ChIP assays for the effect of H₂O₂ on the interaction of OxyS and its mutant proteins with <i>katG</i> promoter in <i>M. smegmatis</i>. The experiments were carried out as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030186#s4" target="_blank">Materials and Methods</a>”. Recombinant <i>M. smegmatis</i> 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 <i>MsmkatGp</i>. ‘+’ 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.</p