Growth dependence of <i>inhA</i>/KD/DO on hemin.

<p>Wild type <i>M</i>. <i>smegmatis</i> (WT), <i>inhA</i>/KD/DO and <i>inhA</i>/KD/DO/<i>hemH</i> were grown till mid log phase, washed and dilutions plated on two sets of 7H11 plates, one set supplemented with 50 μg/ml hygromycin, 40 μg/ml hemin and either 0, 10 or 50 μM IPTG, another set supplemented with 50 μg/ml hygromycin and either 0, 10 or 50 μM IPTG. Solid bars (no hemin (0H), shaded bars (40 μg/ml hemin (40H)). This data is representative of 2 independent experiments.</p

Minimum IPTG requirement of the <i>rpoB</i> conditional expression strains.

<p>Cultures of wild-type <i>M</i>. <i>smegmatis</i>, <i>rpoB</i>/KD/SO and <i>rpoB</i>/KD/DO were plated on 7H11 plates supplemented with 50 μg/ml hygromycin and different concentrations of IPTG. Wild-type <i>M</i>. <i>smegmatis</i> mc²155 served as control. The numbers above the agar plates indicate the μM IPTG concentration supplemented in the respective plates.</p

Growth dependence of <i>inhA</i>/KD/DO on hemin.

<p>Wild type <i>M</i>. <i>smegmatis</i> (WT), <i>inhA</i>/KD/DO and <i>inhA</i>/KD/DO/<i>hemH</i> were grown till mid log phase, washed and dilutions plated on two sets of 7H11 plates, one set supplemented with 50 μg/ml hygromycin, 40 μg/ml hemin and either 0, 10 or 50 μM IPTG, another set supplemented with 50 μg/ml hygromycin and either 0, 10 or 50 μM IPTG. Solid bars (no hemin (0H), shaded bars (40 μg/ml hemin (40H)). This data is representative of 2 independent experiments.</p

IPTG inducible conditional expression vectors with promoter-operator sequences.

<p><b>(A)</b> Vector maps of conditional expression vectors with single lac operator (left) and double lac operator (right); <b>(B)</b> Promoter-operator sequences present in the two conditional expression vectors.</p

Filamentation assay.

<p><i>M</i>. <i>smegmatis</i> and <i>ftsZ</i>/KD/DO cultures grown at 37°C with different concentrations of IPTG were sampled at 48 hours of incubation. A smear of these cells on the microscope slides were stained with Carbol Fuschin followed by light microscopy.</p

List of plasmids and strains used in this study.

<p>*References</p><p>List of plasmids and strains used in this study.</p

Assessment of regulation of β-galactosidase expression from the conditional expression vectors.

<p><i>lacZ</i>/KD/SO (top panel) and <i>lacZ</i>/KD/DO (bottom panel) were grown at 37°C in the absence and the presence of different concentrations of IPTG and 40 μg/ml of X-gal. Wild-type <i>M</i>. <i>smegmatis</i> mc²155 (WT) was used as control which received 1000 μM IPTG and 40 μg/ml X-gal. The numbers indicate the μM IPTG concentrations used in the respective tubes.</p

Growth kinetics of <i>rpoB</i>, <i>inhA</i> and <i>ftsZ</i> KD strains.

<p>KD cultures grown with minimum IPTG required were harvested, washed and used for preparing inoculum of cultures for growth kinetic studies. A master culture of each KD strain with ~10⁶ CFU/ml was prepared, split into many aliquots where each aliquot received a different IPTG concentration. They were grown at 37°C. The effect on the growth was monitored by measuring the survivors periodically by plating them on 7H11 plates supplemented with IPTG. <i>rpoB</i> (<b>A</b>), <i>inhA</i> (<b>B</b>) and <i>ftsZ</i> (<b>C</b>). The graphs are representative results obtained from 3 independent experiments.</p