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. 2016 Jan 21;11(1):e0147188.
doi: 10.1371/journal.pone.0147188. eCollection 2016.

Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of Mycobacterium smegmatis

Sudha Ravishankar  1 , Anisha Ambady  1 , Rayapadi G Swetha  2 , Anand Anbarasu  2 , Sudha Ramaiah  2 , Vasan K Sambandamurthy  1
Affiliations

Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of Mycobacterium smegmatis

Sudha Ravishankar et al. PLoS One. .

Abstract

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 Mycobacterium smegmatis returned multiple genes for glutamyl (GluRS), cysteinyl (CysRS), prolyl (ProRS) and lysyl (LysRS) tRNA synthetases. The pathogenic mycobacteria, namely, Mycobacterium tuberculosis and Mycobacterium leprae, 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 M. smegmatis. Characterization of conditional expression strains of Cysteinyl and Lysyl-tRNA synthetases generated in M. smegmatis revealed that the canonical aminoacyl-tRNA synthetase are essential, while the additional ones are not essential for the growth of M. smegmatis.

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Conflict of interest statement

Competing Interests: Sudha Ravishankar, Anisha Ambady and Vasan K. Sambandamurthy are employed by AstraZeneca India Pvt Ltd. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Fig 1
Fig 1. A snapshot of structural alignment of MSMEG_5671 and E. coli YbaK.
Structure of E. coli YbaK (gold) and the modeled structure of MSMEG_5671 (cyan) were superimposed using DALI server.
Fig 2
Fig 2. Transmembrane segment prediction analysis.
TMHMM plots for S. aureus MprF (A), P. aeruginosa PA0290 (B), M. tuberculosis LysX (C) and MSMEG_3796 (D).
Fig 3
Fig 3. Inducer dependency for the growth of M. tuberculosis and M. smegmatis LeuRS conditional expression strains.
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 M. tuberculosis strains and 48 hours for M. smegmatis strains, respectively. (A). Recombinant colonies (1 and 2) of TleuS/KD-P analysed for growth in the absence (left) and the presence (right) of P1; N, -1 and -2 are the undiluted, 10−1 and 10−2 dilutions. (B). Culture dilutions of SleuS/KD-P and SleuS/KD-I strains were plated with and without P1 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.
Fig 4
Fig 4. Analysis of inducer dependence for growth.
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. (A) S6094/KD-I; (B) S3796/KD-I and (C) ScysS/KD-I.
Fig 5
Fig 5. Inducer dependency for growth of M. smegmatis LeuRS, LysRS and CysRS conditional expression strains.
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.
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Grants and funding

The authors received no specific funding for this work. AstraZeneca India Pvt Ltd. provided support in the form of salaries for authors SR, AA and VKS but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.