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

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

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 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

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

Homology of <i>M. tuberculosis</i> and <i>M. smegmatis</i> proteins present in the thymidylate synthesis pathway.

<p>1 =  % identical amino-acids;</p><p>2 =  classified on the basis of chemical properties (e.g. polar vs. non-polar) of the respective amino-acids side chains.</p

The <i>dut</i> gene is essential in <i>M. smegmatis</i>.

<p>M stands for the 1 kb DNA marker from Fermentas. (<b>A</b>) Identification of SCO strain by colony PCR. SCO strains were generated by homolog recombination of p2Nbk-<i>dut</i>h with chromosomal copy of <i>dut</i>. Chromosomal DNA from <i>M. smegmatis</i> mc ²-155 was used as a positive control yielding the 486 bp fragment (lane WT); the suicide vector integration due to single-crossover event yielded the 860 bp fragment. (lane SCO). (<b>B</b>) Colony PCR analysis of the generated double crossover (DCO) strains. For demonstration, only a subset of 19 samples are shown here. The identical numbers represent samples from the same cell line. The potential DCO cell lines were screened for both the WT copy (indicated as 2, 3) and for the disrupted deletion mutant <i>dut</i> gene (labeled as 2′ 3′). The lengths of the expected PCR product for the wild type (WT) <i>dut</i> gene and for the disrupted <i>dut</i> mutant were 0.7 and 1.1 kb, respectively. (<b>C</b>) Southern blot analysis of DCOs. The probe used to perform the hybridization corresponds to the 1.5 kb WT (lane 1) and the 3.3 kb disrupted <i>dut</i> deletion mutant (lane 2 and 3) restriction fragment, respectively.</p

The Δ-loop mutant dUTPase is unable to rescue the lethal phenotype despite its normal expression level.

<p>(<b>A</b>) Colony PCR analysis of the generated double crossover (DCO) strains. 88 strains were screened and no mutant cell line could be isolated. For demonstration, only a subset of the samples are shown here. M stands for the 1 kb DNA marker from Fermentas. The identical numbers represent samples from the same cell line. Every cell line was screened for both the WT copy (indicated as 1, 2, 3, 4) and for the disrupted mutant <i>dut</i> gene (labeled as 1′ 2′ 3′ 4′). The lengths of the expected PCR product for the wild type (WT) <i>dut</i> gene and for the disrupted <i>dut</i> mutant were 0.7 and 1.1 kb, respectively. (<b>B</b>) Western-blot analysis of FLAG-tagged WT and Δ-loop dUTPase expression in <i>M. smegmatis</i> transformed with the appropriate construct.</p