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Disruption of the adhC gene of Mycobacterium smegmatis mc(2)155, by standard gene replacement methods, revealed that there are two copies of this gene within a large duplication of the M. smegmatis mc(2)155 genome. M. smegmatis AdhC(+/-) and M. smegmatis AdhC(-/-) mutants were obtained when one or two adhC copies, respectively, were disrupted by homologous recombination. Southern blot analysis of DraI restriction digests of the DNA from these mutants and from wild-type M. smegmatis mc(2)155, resolved by PFGE, showed that the duplication size may be at least approximately 250 kb. The single and double knockout mutants were characterized and compared with the M. smegmatis wild-type. A growth disadvantage and a different morphology were associated with the loss of expression of one or both of the adhC copies, but both mutants were still acid-fast. Findings in this study indicate that the process of chromosomal duplication in M. smegmatis is ongoing and remains a potent source of genome dynamics. Hence, the M. smegmatis mc(2)155 genome might be larger than previously thought.

Disruption of adhC reveals a large duplication in the Mycobacterium smegmatis mc2155 genome