This Article Full Text Full Text (PDF) Other Versions of this Article: AAC.00254-07v1 51/8/2888    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Korycka-Machala, M. Articles by Dziadek, J. Search for Related Content PubMed PubMed Citation Articles by Korycka-Machala, M. Articles by Dziadek, J.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, August 2007, p. 2888-2897, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00254-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Evaluation of NAD⁺-Dependent DNA Ligase of Mycobacteria as a Potential Target for Antibiotics

Malgorzata Korycka-Machala,¹ Ewelina Rychta,² Anna Brzostek,¹ Heather R. Sayer,³ Anna Rumijowska-Galewicz,¹ Richard P. Bowater,³ and Jarosaw Dziadek^1*

Centre of Medical Biology, Polish Academy of Sciences, Lodz, Poland,¹ Department of Genetics of Microorganisms, University of Lodz, Lodz, Poland,² School of Biological Sciences, University of East Anglia, Norwich, United Kingdom³

Received 20 February 2007/ Returned for modification 20 April 2007/ Accepted 23 May 2007

Mycobacteria contain genes for several DNA ligases, including ligA, which encodes a NAD⁺-dependent enzyme that has been postulated to be a target for novel antibacterial compounds. Using a homologous recombination system, direct evidence is presented that wild-type ligA cannot be deleted from the chromosome of Mycobacterium smegmatis. Deletions of native ligA in M. smegmatis could be obtained only after the integration of an extra copy of M. smegmatis or Mycobacterium tuberculosis ligA into the attB site of the chromosome, with expression controlled by chemically inducible promoters. The four ATP-dependent DNA ligases encoded by the M. smegmatis chromosome were unable to replace the function of LigA. Interestingly, the LigA protein from M. smegmatis could be substituted with the NAD⁺-dependent DNA ligase of Escherichia coli or the ATP-dependent ligase of bacteriophage T4. The conditional mutant strains allowed the analysis of the effect of LigA depletion on the growth of M. smegmatis. The protein level of the conditional mutants was estimated by Western blot analysis using antibodies raised against LigA of M. tuberculosis. This revealed that a strong overproduction or depletion of LigA did not affect the growth or survival of mycobacteria under standard laboratory conditions. In conclusion, although NAD⁺-dependent DNA ligase is essential for mycobacterial viability, only low levels of protein are required for growth. These findings suggest that very efficient inhibition of enzyme activity would be required if NAD⁺-dependent DNA ligase is to be useful as an antibiotic target in mycobacteria. The strains developed here will provide useful tools for the evaluation of the efficacy of any appropriate compounds in mycobacteria.

Published ahead of print on 4 June 2007.

This article has been cited by other articles:

• Aniukwu, J., Glickman, M. S., Shuman, S. (2008). The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends. Genes Dev. 22: 512-527 [Abstract] [Full Text]