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.Tuberculosis (TB) remains a major threat to public health. It is an important infectious disease, causing high morbidity and mortality worldwide, and the situation is made even worse by the emergence of drug-resistant strains of Mycobacterium tuberculosis (4). For example, multidrug-resistant (MDR) TB (resistant at least to rifampin and isoniazid) takes longer to treat (up to 2 years) with second-line drugs, which are expensive and have side effects. Even worse is extensively drugresistant TB, which is resistant to first-and second-line drugs (MDR plus resistance to any fluoroquinolone and at least one of three injectable second-line drugs: capreomycin, kanamycin, or amikacin). Thus, options for treatment are becoming seriously limited, returning TB control to the preantibiotic era (3,20). Drug resistance in M. tuberculosis is not caused by a universal mechanism for all drugs but can be caused by mutations of various chromosomal genes, as identified for MDR occurrence due to the sequential accumulation of mutations in different genes that provide resistance to individual drugs. The mutations connected to resistance can appear in targets of current drugs (e.g., inhA and kasA for isoniazid, rpoB for rifampin, and embCAB for ethambutol) or enzymes required for the intracellular activation o...

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