title: Targeted mutagenesis of the Mycobacterium smegmatis mca gene, encoding a mycothiol-dependent detoxification protein.
abstract: Mycothiol (MSH), a functional analogue of glutathione (GSH) that is found exclusively in actinomycetes, reacts with electrophiles and toxins to form MSH-toxin conjugates. Mycothiol S-conjugate amidase (Mca) then catalyzes the hydrolysis of an amide bond in the S conjugates, producing a mercapturic acid of the toxin, which is excreted from the bacterium, and glucosaminyl inositol, which is recycled back to MSH. In this study, we have generated and characterized an allelic exchange mutant of the mca gene of Mycobacterium smegmatis. The mca mutant accumulates the S conjugates of the thiol-specific alkylating agent monobromobimane and the antibiotic rifamycin S. Introduction of M. tuberculosis mca epichromosomally or introduction of M. smegmatis mca integratively resulted in complementation of Mca activity and reduced levels of S conjugates. The mutation in mca renders the mutant strain more susceptible to electrophilic toxins, such as N-ethylmalemide, iodoacetamide, and chlorodinitrobenzene, and to several oxidants, such as menadione and plumbagin. Additionally we have shown that the mca mutant is also more susceptible to the antituberculous antibiotic streptomycin. Mutants disrupted in genes belonging to MSH biosynthesis are also more susceptible to streptomycin, providing further evidence that Mca detoxifies streptomycin in the mycobacterial cell in an MSH-dependent manner.
authors.affiliation: Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, British Columbia V5Z 3J5, Canada.
authors.forename: Mamta
authors.initials: M
authors.lastname: Rawat
authors.forename: Mandeep
authors.initials: M
authors.lastname: Uppal
authors.forename: Gerald
authors.initials: G
authors.lastname: Newton
authors.forename: Micah
authors.initials: M
authors.lastname: Steffek
authors.forename: Robert C
authors.initials: RC
authors.lastname: Fahey
authors.forename: Yossef
authors.initials: Y
authors.lastname: Av-Gay
mesh_terms: D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D003545:Cysteine; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D004187:Disaccharides / Q000096:biosynthesis / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006020:Glycopeptides; D007294:Inositol; D007460:Iodoacetamide / Q000494:pharmacology; D016254:Mutagenesis, Insertional; D020102:Mycobacterium smegmatis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics; D009285:Naphthoquinones / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D011720:Pyrazoles; D012294:Rifamycins / Q000378:metabolism; D013307:Streptomycin / Q000494:pharmacology; D013438:Sulfhydryl Compounds; D024483:Vitamin K 3 / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001643:Bridged Bicyclo Compounds; D004137:Dinitrochlorobenzene; D004187:Disaccharides; D004791:Enzyme Inhibitors; D006020:Glycopeptides; D009285:Naphthoquinones; D016877:Oxidants; D011720:Pyrazoles; D012294:Rifamycins; D013438:Sulfhydryl Compounds; C089265:mycothiol; D007294:Inositol; D024483:Vitamin K 3; D000581:Amidohydrolases; C413523:mycothiol S-conjugate amidase; D003545:Cysteine; D005033:Ethylmaleimide; C032779:rifamycin S; C028381:monobromobimane; D013307:Streptomycin; C014758:plumbagin; D007460:Iodoacetamide