Analysis of inducer dependence for growth.

<p>Identified recombinant colonies of S6094/KD-I, S3796/KD-I and ScysS/KD-I were grown with 100 μM IPTG until they reached mid-log phase, washed and the culture suspension in plain 7H9 broth was diluted and plated on plates with and without IPTG. The plates were incubated at 37°C for 48 hours and photographed. In all the cases, plate on left is without IPTG and plate on right is with IPTG. (<b>A</b>) S6094/KD-I; (<b>B</b>) S3796/KD-I and (<b>C</b>) ScysS/KD-I.</p

Inducer dependency for growth of <i>M</i>. <i>smegmatis</i> LeuRS, LysRS and CysRS conditional expression strains.

<p>The conditional expression strains SleuS/KD-I, SCysS/KD-I, S6094/KD-I and S3796/KD-I were grown in the presence of 100 μM IPTG till they reached mid-log phase, the cells were harvested, washed to remove traces of inducer and resuspended in fresh 7H9 broth to be used as inoculum. Several dilutions of these cultures were plated on 7H11 plates with and without IPTG. Plates were incubated at 37°C for 96 hours and the colonies were counted both at the end of 48 hours and 96 hours of incubation.</p

Transmembrane segment prediction analysis.

<p>TMHMM plots for <i>S</i>. <i>aureus</i> MprF (<b>A</b>), <i>P</i>. <i>aeruginosa</i> PA0290 (<b>B</b>), <i>M</i>. <i>tuberculosis</i> LysX (<b>C</b>) and MSMEG_3796 (<b>D</b>).</p

Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of <i>Mycobacterium smegmatis</i>

<div><p>Discovery of mupirocin, an antibiotic that targets isoleucyl-tRNA synthetase, established aminoacyl-tRNA synthetase as an attractive target for the discovery of novel antibacterial agents. Despite a high degree of similarity between the bacterial and human aminoacyl-tRNA synthetases, the selectivity observed with mupirocin triggered the possibility of targeting other aminoacyl-tRNA synthetases as potential drug targets. These enzymes catalyse the condensation of a specific amino acid to its cognate tRNA in an energy-dependent reaction. Therefore, each organism is expected to encode at least twenty aminoacyl-tRNA synthetases, one for each amino acid. However, a bioinformatics search for genes encoding aminoacyl-tRNA synthetases from <i>Mycobacterium smegmatis</i> returned multiple genes for glutamyl (GluRS), cysteinyl (CysRS), prolyl (ProRS) and lysyl (LysRS) tRNA synthetases. The pathogenic mycobacteria, namely, <i>Mycobacterium tuberculosis</i> and <i>Mycobacterium leprae</i>, were also found to possess two genes each for CysRS and LysRS. A similar search indicated the presence of additional genes for LysRS in gram negative bacteria as well. Herein, we describe sequence and structural analysis of the additional aminoacyl-tRNA synthetase genes found in <i>M</i>. <i>smegmatis</i>. Characterization of conditional expression strains of Cysteinyl and Lysyl-tRNA synthetases generated in <i>M</i>. <i>smegmatis</i> revealed that the canonical aminoacyl-tRNA synthetase are essential, while the additional ones are not essential for the growth of <i>M</i>. <i>smegmatis</i>.</p></div

A snapshot of structural alignment of MSMEG_5671 and <i>E</i>. <i>coli</i> YbaK.

<p>Structure of <i>E</i>. <i>coli</i> YbaK (gold) and the modeled structure of MSMEG_5671 (cyan) were superimposed using DALI server.</p

Inducer dependency for the growth of <i>M</i>. <i>tuberculosis</i> and <i>M</i>. <i>smegmatis</i> LeuRS conditional expression strains.

<p>Several confirmed recombinant colonies were grown in the presence of inducer till they reached mid-log phase, cells were washed, resuspended in fresh 7H9 broth and the cultures were either spotted or various dilutions plated for enumerating colony forming units (CFUs). Plates were incubated at 37°C for 28 days for <i>M</i>. <i>tuberculosis</i> strains and 48 hours for <i>M</i>. <i>smegmatis</i> strains, respectively. (<b>A</b>). Recombinant colonies (1 and 2) of TleuS/KD-P analysed for growth in the absence (left) and the presence (right) of P_<b>1</b>; N, -1 and -2 are the undiluted, 10⁻¹ and 10⁻² dilutions. (<b>B</b>). Culture dilutions of SleuS/KD-P and SleuS/KD-I strains were plated with and without P_<b>1</b> and IPTG, respectively. Bars in the graph represent CFU/ml calculated from the colony numbers that appeared on plates under each of the growth condition specified.</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

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

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