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

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.

SUBMITTER: Ravishankar S

PROVIDER: S-EPMC4721953 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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

Ravishankar Sudha S Ambady Anisha A Swetha Rayapadi G RG Anbarasu Anand A Ramaiah Sudha S Sambandamurthy Vasan K VK

PloS one 20160121 1

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 cogn ...[more]

PMID: 26794499

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Project description:UnlabelledSuccinate:quinone oxidoreductase (Sdh) is a membrane-bound complex that couples the oxidation of succinate to fumarate in the cytoplasm to the reduction of quinone to quinol in the membrane. Mycobacterial species harbor genes for two putative sdh operons, but the individual roles of these two operons are unknown. In this communication, we show that Mycobacterium smegmatis mc(2)155 expresses two succinate dehydrogenases designated Sdh1 and Sdh2. Sdh1 is encoded by a five-gene operon (MSMEG_0416-MSMEG_0420), and Sdh2 is encoded by a four-gene operon (MSMEG_1672-MSMEG_1669). These two operons are differentially expressed in response to carbon limitation, hypoxia, and fumarate, as monitored by sdh promoter-lacZ fusions. While deletion of the sdh1 operon did not yield any growth phenotypes on succinate or other nonfermentable carbon sources, the sdh2 operon could be deleted only in a merodiploid background, demonstrating that Sdh2 is essential for growth. Sdh activity and succinate-dependent proton pumping were detected in cells grown aerobically, as well as under hypoxia. Fumarate reductase activity was absent under these conditions, indicating that neither Sdh1 nor Sdh2 could catalyze the reverse reaction. Sdh activity was inhibited by the Sdh inhibitor 3-nitroproprionate (3NP), and treatment with 3NP dissipated the membrane potential of wild-type or ?sdh1 mutant cells under hypoxia but not that of cells grown aerobically. These data imply that Sdh2 is the generator of the membrane potential under hypoxia, an essential role for the cell.ImportanceComplex II or succinate dehydrogenase (Sdh) is a major respiratory enzyme that couples the oxidation of succinate to fumarate in the cytoplasm to the reduction of quinone to quinol in the membrane. Mycobacterial species harbor genes for two putative sdh operons, sdh1 and sdh2, but the individual roles of these two operons are unknown. In this communication, we show that sdh1 and sdh2 are differentially expressed in response to energy limitation, oxygen tension, and alternative electron acceptor availability, suggesting distinct functional cellular roles. Sdh2 was essential for growth and generation of the membrane potential in hypoxic cells. Given the essentiality of succinate dehydrogenase and oxidative phosphorylation in the growth cycle of Mycobacterium tuberculosis, the potential exists to develop new antituberculosis agents against the mycobacterial succinate dehydrogenase. This enzyme has been proposed as a potential target for the development of new chemotherapeutic agents against intracellular parasites and mitochondrion-associated disease.

Dataset Information

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

Publications

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

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