<span style="font-size: 19.0pt;mso-bidi-font-size:14.0pt;font-family:"Times New Roman","serif"; color:black">Permeabilization and <i><span style="font-size:19.5pt; mso-bidi-font-size:14.5pt;font-family:"Times New Roman","serif";color:black">in situ </span></i><span style="font-size:19.0pt;mso-bidi-font-size:14.0pt; font-family:"Times New Roman","serif";color:black">adsorption studies during growth and coumarin production <span style="font-size:20.0pt;mso-bidi-font-size: 15.0pt;font-family:"Times New Roman","serif";color:black;mso-bidi-font-weight: bold">in<b> </b><span style="font-size:19.0pt;mso-bidi-font-size:14.0pt; font-family:"Times New Roman","serif";color:black">hairy root cultures of <i><span style="font-size:19.5pt;mso-bidi-font-size:14.5pt;font-family:"Times New Roman","serif"; color:black">Cichorium intybus </span></i><span style="font-size:19.0pt; mso-bidi-font-size:14.0pt;font-family:"Arial","sans-serif";color:black; mso-bidi-font-weight:bold">L. </span></span></span></span></span>

564-571<span style="font-size: 13.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif";="" color:black"="">Effect of addition of a penneabilizing agent dimethyl sulfoxide (DMSO) and a solid adsorbent, XAD -7, on growth and coumarin production in hairy root cultures of <span style="font-size:13.5pt;mso-bidi-font-size: 8.5pt;font-family:" times="" new="" roman","serif";color:black"="">C. intybus was studied. Continuous permeabilization of the hairy root cultures <span style="font-size: 13.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif";="" color:black"="">of  C. <span style="font-size:13.5pt;mso-bidi-font-size: 8.5pt;font-family:" times="" new="" roman","serif";color:black"="">intybus with DMSO has been shown to be an effective strategy for enhanced release of coumarins while preserving the root viability. DMSO at 0.2 % <span style="font-size:13.0pt;mso-bidi-font-size:8.0pt; font-family:" times="" new="" roman","serif";color:black"="">(v/v) level showed the maximum growth and coumarin production but was less as compared to control on day 28. Treatment of cells with increasing concentrations of DMSO (0.3 - 0.6 % <span style="font-size:13.0pt;mso-bidi-font-size:8.0pt; font-family:" times="" new="" roman","serif";color:black"="">v/v) to hairy root cultures of <span style="font-size:14.0pt;mso-bidi-font-size:9.0pt;font-family: " times="" new="" roman","serif";color:black"="">C. <span style="font-size: 13.5pt;mso-bidi-font-size:8.5pt;font-family:" times="" new="" roman","serif";="" color:black"="">intybus, <span style="font-size:13.0pt;mso-bidi-font-size: 8.0pt;font-family:" times="" new="" roman","serif";color:black"="">showed an inverse relationship with growth and coumarin production. Growth and production of coumarins increased with 1 <span style="font-size:13.0pt;mso-bidi-font-size: 8.0pt;font-family:" arial","sans-serif";color:black"="">% media filtrate (MF) of cultures of Phytopthora parasirica <span style="font-size:13.0pt; mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif";color:black"="">var. <span style="font-size:13.5pt;mso-bidi-font-size:8.5pt;font-family: " times="" new="" roman","serif";color:black"="">nicotiana treatment. It was observed that treatment with DMSO (0.2 % <span style="font-size:13.0pt;mso-bidi-font-size:8.0pt; font-family:" times="" new="" roman","serif";color:black"="">v/v) and 1 % <span style="font-size:13.0pt;mso-bidi-font-size:8.0pt; font-family:" times="" new="" roman","serif";color:black"="">MF of P. parasitica <span style="font-size:13.0pt;mso-bidi-font-size: 8.0pt;font-family:" times="" new="" roman","serif";color:black"="">showed the better growth and coumarin production with an increased release of coumarins as compared to the control and other treatments. It was observed that treatment of hairy root cultures with XAD -7 resulted in lesser growth and coumarin production as compared to control during the culture period. Addition of XAD -7 along with 1 <span style="font-size:13.0pt;mso-bidi-font-size:8.0pt; font-family:" arial","sans-serif";color:black"="">% <span style="font-size: 13.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif";="" color:black"="">MF of <span style="font-size:13.5pt;mso-bidi-font-size: 8.5pt;font-family:" times="" new="" roman","serif";color:black"="">P. parasitica showed enhanced growth , coumarin production and increased adsorption as compared to control and lone XAD-7 treatment. Combined addition of DMSO I <span style="font-size:13.0pt;mso-bidi-font-size: 8.0pt;font-family:" times="" new="" roman","serif";color:black"="">XAD-7 with fungal elicitor showed synergistic response in terms of biomass and coumarin production. Excretion of coumarins in both the cases was dependent on the presence of DMSO I <span style="font-size:13.0pt;mso-bidi-font-size: 8.0pt;font-family:" times="" new="" roman","serif";color:black"="">XAD-7. These result s showed that continuous permeabilization of hairy root cultures of C. <span style="font-size:13.5pt;mso-bidi-font-size: 8.5pt;font-family:" times="" new="" roman","serif";color:black"="">intybus by using DMSO at 0.2 <span style="font-size:13.0pt; mso-bidi-font-size:8.0pt;font-family:" arial","sans-serif";color:black"="">% (v/v) level coupled with 1 <span style="font-size:13.0pt; mso-bidi-font-size:8.0pt;font-family:" arial","sans-serif";color:black"="">% MF of <span style="font-size:13.5pt;mso-bidi-font-size: 8.5pt;font-family:" times="" new="" roman","serif";color:black"="">P. parasitica maintained viability of tissues and produced coumarins at higher level. </span

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

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

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

Scintillation Proximity Assay for Inhibitors of Escherichia coli MurG and, Optionally, MraY

MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[(3)H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis—penicillin G, moenomycin, and bacitracin—had no effect. Surprisingly, however, the β-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents