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Evaluation of NAD+-dependent DNA ligase of mycobacteria as a potential target for antibiotics

Abstract number: 1733_1164

Rychta E., Korycka-Machala M., Brzostek A., Sayer H., Rumijowska-Galewicz A., Bowater R., Dziadek J.

Objectives: The aim of this study was verification of the essentiality of NAD+-dependent DNA ligase (LigA) in mycobacteria and its evaluation as a putative target for new antituberculosis drugs.

Methods: A homologous recombination system was used to assess essentiality of the mycobacterial gene for NAD+-dependent DNA ligase (ligA). Conditional mutants carrying a single ligA gene controlled with chemically inducible promoters were constructed. The protein level was measured with western blot using antibodies raised against LigA of Mycobacterium tuberculosis.

Results: Since NAD+-dependent DNA ligase was postulated as a useful target for new antibiotics, we evaluated if ligA is essential for the viability of mycobacteria. Using a homologous recombination system we found that wild type ligA gene cannot be deleted from the chromosome of Mycobacterium smegmatis. We were able to delete the native ligA in M. smegmatis by integrating in to the attB site of chromosomal DNA an extra copy of M. smegmatis or M. tuberculosis ligA, with expression controlled by chemically inducible promoters. The resultant conditional mutants allow studies of the effect of LigA depletion on growth of mycobacteria. Interestingly, the LigA protein from M. smegmatis can be substituted with the NAD+-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The investigation of LigA conditional mutants revealed that mycobacterial cells are not affected by large changes to the levels of LigA. The strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria.

Conclusion: NAD+-dependent DNA ligase is essential for mycobacterial viability, but only low levels of protein are required. Thus, further investigations are required to determine if NAD+-dependent DNA ligase could be useful as an antibiotic target in mycobacteria.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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