title: Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis.
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 (G-->T) at the alrA promoter, which resulted in elevated beta-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.
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln 68583-0905, USA.
authors.forename: N E
authors.initials: NE
authors.lastname: Cáceres
authors.forename: N B
authors.initials: NB
authors.lastname: Harris
authors.forename: J F
authors.initials: JF
authors.lastname: Wellehan
authors.forename: Z
authors.initials: Z
authors.lastname: Feng
authors.forename: V
authors.initials: V
authors.lastname: Kapur
authors.forename: R G
authors.initials: RG
authors.lastname: Barletta
mesh_terms: D000411:Alanine Racemase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D003001:Cloning, Molecular; D003523:Cycloserine / Q000494:pharmacology*; D004352:Drug Resistance, Microbial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015698:Genomic Library; D008969:Molecular Sequence Data; D009154:Mutation; D009161:Mycobacterium / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D015269:Mycobacterium avium Complex / Q000235:genetics; D009163:Mycobacterium bovis / Q000235:genetics; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment
chemical_list: D000904:Antibiotics, Antitubercular; D003523:Cycloserine; D000411:Alanine Racemase