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PMID:9260945
| Citation |
Cáceres, NE, Harris, NB, Wellehan, JF, Feng, Z, Kapur, V and Barletta, RG (1997) Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis. J. Bacteriol. 179:5046-55 |
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| 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. |
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| Keywords |
Alanine Racemase/biosynthesis; Alanine Racemase/genetics; Alanine Racemase/metabolism; Amino Acid Sequence; Antibiotics, Antitubercular/pharmacology; Cloning, Molecular; Cycloserine/pharmacology; Drug Resistance, Microbial/genetics; Gene Expression Regulation, Bacterial; Genomic Library; Molecular Sequence Data; Mutation; Mycobacterium/drug effects; Mycobacterium/enzymology; Mycobacterium/genetics; Mycobacterium avium Complex/genetics; Mycobacterium bovis/genetics; Open Reading Frames; Promoter Regions, Genetic; Sequence Alignment |
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Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0008784: alanine racemase activity |
ECO:0000314: |
F |
Figure 5: Depicts alanine racemase activity among different strains. Strain GPM14 shows marked overproduction of d-alanine racemase as a result of a single base change from T -> G in the alrA gene promoter as described in the "Results" section. |
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