P_Rv0560c induction by structural analogs of salicylate in <i>M. tuberculosis</i>.

<p>Promoter activity was measured in <i>M. tuberculosis</i> transformants grown under aerobic growth conditions. A. Promoter activity of P_Rv0560c after treatment with 0.4 mM of compound for 3 d. Results are the average and standard deviation of three independent transformants assayed in duplicate. Activity is given in Miller Units. B. Chemical structures of compounds of interest. A significant difference compared using Student's t-test to the untreated control is marked by an * for p<0.05) ** for p<0.01, *** for p<0.0001.</p

Identification of the promoter and regulatory elements.

<p>A. DNA sequence of the P_Rv0560c region. The predicted translation start site of Rv0560c according to TubercuList is marked with **. Protein sequences of Rv0561c and Rv0560c are shown. Potential −10 promoter elements (PM1, PM2, PM3) are underlined. The −35 and extended −10 element are in bold. A palindromic moitif is indicated by grey shading. B. Promoter activity following mutation of the promoter region. <i>M. tuberculosis</i> transformants were grown under aerobic growth conditions in the absence/presence of 0.4 mM salicylate. Results are the average and standard deviation of three independent transformants assayed in duplicate. Activity is given in Miller Units. A significant difference compared to the wild type is marked by an * (p<0.05).</p

P_Rv0560c induction kinetics after exposure to salicylate in <i>M. tuberculosis</i>.

<p>A and B. Promoter activity was measured in <i>M. tuberculosis</i> transformants grown under aerobic growth conditions exposed to 0.4 mM salicylate (A and B), or 0.2 mM or 0.4 mM salicylate (C) or with the use of LacZ tagged for degradation(D). Results are the average and standard deviation of three independent transformants assayed in duplicate. Activity is given in Miller Units. LacZ-ASV was tagged with AANDENYAASV; LacZ-LAA was tagged with AANDENYALAA.</p

P₂₇₈ promoter activity was not induced by stress treatments.

<p>A) P₂₇₈ promoter activity in standing liquid cultures. Treatments were: 50 µg/mL of chlorpromazine for 3 h, 10 µg/mL of menadione for 3 h, 10 µg/mL of valinomycin for 3h, 6 µg/mL of vancomycin for 90 min. B) Promoter activity in rolling cultures. Treatments were: 42°C for 1 h, 10 mM diamide for 1 h C) Promoter activity in response to diamide treatment in <i>M. tuberculosis</i> CDC1551. D) Promoter activity in response to diamide and vancomycin treatments in presence or absence of streptomycin selection. Stress treatments were 10 mM diamide for 1 h or 6 µg/mL of vancomycin for 90 min. The average and standard deviation of three independent transformants assayed in duplicate is given. ß-galactosidase activity is given in Miller Units - measured as nmol of O-nitrophenol produced over time (min) per mg of protein. Activity is given in Miller Units (MU)- measured as nmol of O-nitrophenol produced per min per mg of protein. The background activity from pSM128 (control vector) was 6±3 MU under the different conditions tested.</p

Identification of key residues in a regulatory region of the <i>clpP1P2</i> operon.

<p>A) Sequence of the region upstream of <i>clpP1P2</i>. The <i>tig</i> stop codon, −10 promoter element, and <i>clpP1</i> start codons are underlined. The 18 nucleotides that constitute the regulatory region are boxed in grey. The CGC region mutated is underlined in bold. B) Identification of a regulatory region. The CGC motif (underlined bold) was mutated to AAA. C) Mapping of a regulatory binding site. Single nucleotide substitutions in P₂₇₈ were made by SDM. Residues A or T were mutated to G or and residues C or G were mutated to A. Results are the average activity of three independent transformants assayed in duplicate ± standard deviation. Activity is given in Miller Units- measured as nmol of O-nitrophenol produced per min per mg of protein. A significant difference of activity compared to wild-type P₂₇₈ is marked by an *(p<0.05) using the student’s t-test (unpaired, two sided).</p

Protein turnover in strains over-expressing ClpP subunits.

<p>A to D) <i>M. tuberculosis</i> transformants were grown to late exponential phase in standing liquid cultures in presence of succinate +/− acetamide (0.1% w/v) and cell-free extracts were prepared and β-galactosidase activity measured. Empty bars: uninduced conditions (succinate); Grey striped bars: induced conditions (succinate+acetamide). A significant difference measured by the student’s t-test (unpaired, two sided) compared to the induced LacZ level in the WT strain is marked by an *(p<0.05). E) Three <i>M. tuberculosis</i> transformants carrying LacZ-ASV were grown to late exponential phase in standing liquid cultures in presence of acetamide (0.1% w/v) and cell-free extracts were prepared. Treatments were 10 mM diamide for 1 h or 6 µg/mL of vancomycin for 90 min. A significant difference from untreated WT is marked by an *(p<0.05). Results are the average activity of three independent transformants assayed in duplicate ± standard deviation. Activity is given in Miller Units- measured as nmol of O-nitrophenol produced per min per mg of protein. Strains are- WT: wild-type; P1: over-expressing ClpP1; P2: over-expressing ClpP2; P1P2: over-expressing ClpP1 and ClpP2.</p

Promoter activity during aerobic growth, hypoxia, and reaeration in <i>M. tuberculosis.</i>

<p>A) <i>M. tuberculosis</i> transformants harbouring P₂₇₈ were grown in aerobic culture. Results are the average activity of three transformants against average OD₅₈₀. A significant difference, measured by the student’s t-test (unpaired, two sided), compared to promoter activity at OD₅₈₀ = 0.15 is marked by an *(p<0.05). B) P₂₇₈ promoter activity in the Wayne model of hypoxia. <i>M. tuberculosis</i> liquid cultures were inoculated to a theoretical starting OD₅₈₀ of 0.004 in DTA medium. A significant difference compared to activity at day 0 is marked by an *(p<0.05) using the student’s t-test (unpaired, two sided). C) P₂₇₈ promoter activity after reaeration. Long term hypoxic cultures (12 weeks) were used to inoculate medium and grown in aerobic rolling cultures. Cell-free extracts were prepared once the cultures reached an OD₅₈₀ of 0.3. Results are the average activity of three independent transformants assayed in duplicate ± standard deviation. Activity is given in Miller Units- measured as nmol of O-nitrophenol produced per min per mg of protein.</p

<i>clpP1</i> and <i>clpP2</i> are co-transcribed as an operon.

<p>A) Chromosomal organisation of <i>clpP1</i> and <i>clpP2</i>. Regions amplified for RT-PCR are marked. B) Limiting dilution semi-quantitiative RT-PCR. RNA was extracted from <i>M. tuberculosis</i> grown to late exponential phase in liquid cultures and cDNA was synthesised from 1 µg of RNA. Serial 4- fold dilutions of cDNA were used as a template for PCR using primers specific for <i>clpP1</i> (P1), <i>clpP2</i> (P2), the <i>clpP1</i>-<i>clpP2</i> junction (P1P2) and <i>sigA</i>. C: no RT control; B: no template blank, M: markers.</p

Identification of the promoter of the <i>clpP1P2</i> operon.

<p>A) The regions upstream of <i>clpP1</i> or <i>clpP2</i> tested for promoter activity are marked. B) P₁₂₅ activity in <i>M. tuberculosis</i>. C) P<i>_clpP2</i> activity in <i>M. tuberculosis</i>. Promoter activity was measured in transformants grown to late exponential phase in standing liquid cultures. Results are the average activity ± standard deviation of three independent transformants assayed in duplicate. Activity is given in Miller Units (MU)- measured as nmol of O-nitrophenol produced per min per mg of protein. Control = pSM128 empty vector control.</p

Identification of the −10 promoter element.

<p>A) Sequence of the P₁₂₅ region upstream of <i>clpP1P2.</i> Putative −10 elements (10A and 10B) are boxed. The residues mutated are in bold. The predicted ClpP1 start codon and Tig stop codons are indicated. B) Identification of the −10 element. The following mutations were made - 10A: TAGTGT mutated to <b>C</b>AGTG<b>G</b>; 10B: TAGAAG mutated to <b>CG</b>GAAG. Results are the average activity of three independent transformants assayed in duplicate ± standard deviation. Activity is given in Miller Units (MU)- measured as nmol of O-nitrophenol produced per min per mg of protein. The background activity from pSM128 (control vector) was 4±2 MU. A significant difference, measured by the student’s t-test (unpaired, two sided), compared to the control vector (pSM128) is marked by an *(p<0.05).</p