Overexpression of the D-alanine racemase gene confers resistance to D- cycloserine in Mycobacterium smegmatis

Nancy E. Cáceres, N. Beth Harris, James F. Wellehan, Zhengyu Feng, Vivek Kapur, Raúl G. Barletta

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D-Cycloserine is an effective second-line drug against Mycobacterium avium and Mycobacterium tuberculosis. To analyze the genetic determinants of D-cycloserine resistance in mycobacteria, a library of a resistant Mycobacterium smegmatis mutant was constructed. A resistant clone harboring a recombinant plasmid with a 3.1-kb insert that contained the glutamate decarboxylase (gadA) and D-alanine racemase (alrA) genes was identified. Subcloning experiments demonstrated that alrA was necessary and sufficient to confer a D-cycloserine resistance phenotype. The D-alanine racemase activities of wild-type and recombinant M. smegmatis strains were inhibited by D-cycloserine in a concentration-dependent manner. The D-cycloserine resistance phenotype in the recombinant clone was due to the overexpression of the wild-type alrA gene in a multicopy vector. Analysis of a spontaneous resistant mutant also demonstrated overproduction of wild-type AlrA enzyme. Nucleotide sequence analysis of the overproducing mutant revealed a single transversion (G→T) at the alrA promoter, which resulted in elevated β- galactosidase reporter gene expression. Furthermore, transformants of Mycobacterium intracellulare and Mycobacterium bovis BCG carrying the M. smegmatis wild-type alrA gene in a multicopy vector were resistant to D- cycloserine, suggesting that AlrA overproduction is a potential mechanism of D-cycloserine resistance in clinical isolates of M. tuberculosis and other pathogenic mycobacteria. In conclusion, these results show that one of the mechanisms of D-cycloserine resistance in M. smegmatis involves the overexpression of the alta gene due to a promoter-up mutation.

Original languageEnglish (US)
Pages (from-to)5046-5055
Number of pages10
JournalJournal of bacteriology
Issue number16
StatePublished - Aug 1997

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology


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