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

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

Methyl-Thiazoles: A Novel Mode of Inhibition with the Potential to Develop Novel Inhibitors Targeting InhA in Mycobacterium tuberculosis

InhA is a well validated Mycobacterium tuberculosis (Mtb) target as evidenced by the clinical success of isoniazid. Translating enzyme inhibition to bacterial cidality by targeting the fatty acid substrate site of InhA remains a daunting challenge. The recent disclosure of a methyl-thiazole series demonstrates that bacterial cidality can be achieved with potent enzyme inhibition and appropriate physicochemical properties. In this study, we report the molecular mode of action of a lead methyl-thiazole, along with analogues with improved CYP inhibition profile. We have identified a novel mechanism of InhA inhibition characterized by a hitherto unreported “Y158-out” inhibitor-bound conformation of the protein that accommodates a neutrally charged “warhead”. An additional novel hydrophilic interaction with protein residue M98 allows the incorporation of favorable physicochemical properties for cellular activity. Notably, the methyl-thiazole prefers the NADH-bound form of the enzyme with a <i>K</i>_d of ∼13.7 nM, as against the NAD⁺-bound form of the enzyme