Inactivation of mshB, a key gene in the mycothiol biosynthesis pathway in Mycobacterium smegmatis.
The mshB gene encoding N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. Disruption of mshB in Mycobacterium smegmatis resulted in decreased production of mycothiol (5-10 % of the parent strain mc(2)155) but did not abolish mycothiol synthesis completely. Complementation of the MshB(-) mutants with the mshB gene resulted in increased mycothiol production towards the exponential and stationary phases of the bacterial growth cycle. These results suggest that another enzyme is capable of mycothiol biosynthesis by providing N-acetylglucosaminylinositol deacetylation activity in the absence of MshB. One of the candidate enzymes capable of carrying out such reactions is the MshB orthologue mycothiol amide hydrolase, MCA. However, epichromosomal expression of mca in the MshB(-) mutants did not restore mycothiol levels to the level of the parent strain. Unlike other mutants, which have little or no detectable levels of mycothiol, the MshB(-) mutant did not exhibit increased resistance to isoniazid. However, the MshB(-) mutant was resistant to ethionamide. Phenotypic analysis of other mutants lacking mycothiol revealed that MshA(-) mutants also exhibit ethionamide resistance but that a MshC(-)mutant was sensitive to ethionamide, suggesting that mycothiol or its early intermediates influence ethionamide activation.
Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, British Columbia, Canada V5Z 3J5.
Mamta
M
Rawat
Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.
Svetozar
S
Kovacevic
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia.
Helen
H
Billman-Jacobe
Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, British Columbia, Canada V5Z 3J5.
Yossef
Y
Av-Gay
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D003545:Cysteine; D004187:Disaccharides / Q000096:biosynthesis*; D005000:Ethionamide / Q000494:pharmacology; D005816:Genetic Complementation Test; D006020:Glycopeptides; D007294:Inositol; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D018384:Oxidative Stress; D011720:Pyrazoles; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013438:Sulfhydryl Compounds
D001426:Bacterial Proteins; D004187:Disaccharides; D006020:Glycopeptides; D011720:Pyrazoles; D013438:Sulfhydryl Compounds; C089265:mycothiol; D007294:Inositol; D000581:Amidohydrolases; C417275:N-acetyl-1-D-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase; D003545:Cysteine; D005000:Ethionamide