Affiliation:
1. Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln 68583-0905, USA.
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Reference55 articles.
1. Ausubel F. M. R. Brent R. E. Kingston D. D. Moore J. G. Seidman J. A. Smith and K. Struhl (ed.). 1989. Current protocols in molecular biology p. 1-8. Greene Publishing Associates and Wiley-Interscience New York N.Y.
2. Time-dependent inhibition of Bacillus stearothermophilus alanine racemase by (1-aminoethyl)phosphonate isomers by isomerization to noncovalent slowly dissociating enzyme-(1-aminoethyl)phosphonate complexes;Badet B.;Biochemistry,1986
3. An efficient and high-yielding method for isolation of RNA from mycobacteria;Bashyam M. D.;BioTechniques,1994
4. A study of the mycobacterial transcriptional apparatus: identification of novel features in promoter elements;Bashyam M. D.;J. Bacteriol.,1996
5. Analysis of the Escherichia coli genome. IV. DNA sequence of the region from 89.2 to 92.8 minutes;Blattner F. R.;Nucleic Acids Res.,1993
Cited by
134 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献