Overexpression of the D-Alanine Racemase Gene Confers Resistance to D-Cycloserine in Mycobacterium smegmatis

Authors

• Nancy E. Caceres, University of Nebraska - Lincoln
• N. Beth Harris, University of Nebraska - Lincoln
• James F. Wellehan, University of Minnesota, St. Paul, Minnesota
• Zhengyu Feng, University of Nebraska - Lincoln
• Vivek Kapur, University of Minnesota, St. Paul, MinnesotaFollow
• Raul G. Barletta, University of Nebraska - LincolnFollow

Document Type

Article

Date of this Version

8-1-1997

Comments

Published in JOURNAL OF BACTERIOLOGY, Aug. 1997, p. 5046–5055. Copyright © 1997, American Society for Microbiology. Used by permission.

Abstract

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 (G3T) at the alrA promoter, which resulted in elevated b-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 alrA gene due to a promoter-up mutation.