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. 2002 Nov;46(11):3348-55.
doi: 10.1128/AAC.46.11.3348-3355.2002.

Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals, and antibiotics

Mamta Rawat  1 , Gerald L Newton, Mary Ko, Gladys J Martinez, Robert C Fahey, Yossef Av-Gay
Affiliations

Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals, and antibiotics

Mamta Rawat et al. Antimicrob Agents Chemother. 2002 Nov.

Abstract

Mycothiol (MSH; 1D-myo-inosityl 2-[N-acetyl-L-cysteinyl]amido-2-deoxy-alpha-D-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc(2)155 deficient in MSH production were produced by chemical mutagenesis as well as by transposon mutagenesis. One chemical mutant (mutant I64) and two transposon mutants (mutants Tn1 and Tn2) stably deficient in MSH production were isolated by screening for reduced levels of MSH content. The MSH contents of transposon mutants Tn1 and Tn2 were found to be less than 0.1% that of the parent strain, and the MSH content of I64 was found to be 1 to 5% that of the parent strain. All three strains accumulated 1D-myo-inosityl 2-deoxy-alpha-D-glucopyranoside to levels 20- to 25-fold the level found in the parent strain. The cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase (MshC) activities of the three mutant strains were < or =2% that of the parent strain. Phenotypic analysis revealed that these MSH-deficient mutants possess increased susceptibilities to free radicals and alkylating agents and to a wide range of antibiotics including erythromycin, azithromycin, vancomycin, penicillin G, rifamycin, and rifampin. Conversely, the mutants possess at least 200-fold higher levels of resistance to isoniazid than the wild type. We mapped the mutation in the chemical mutant by sequencing the mshC gene and showed that a single amino acid substitution (L205P) is responsible for reduced MSH production and its associated phenotype. Our results demonstrate that there is a direct correlation between MSH depletion and enhanced sensitivity to toxins and antibiotics.

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Figures

FIG. 1.
FIG. 1.
(A) Structure of MSH; (B) proposed MSH biosynthesis pathway. Ac, acetyl; CoA, coenzyme A.
FIG. 2.
FIG. 2.
Southern blot analysis of the three M. smegmatis::Tn611 clones. The DNA of M. smegmatis mc2155 [the wild type (WT)] was included as a control and, as expected, showed no hybridization signal. Genomic DNAs were digested with PstI and probed for hybridization with IS6100 as described by Perez et al. (19).
FIG. 3.
FIG. 3.
Tests of sensitivities of MSH-deficient mutants versus wild-type strain mc2155 to hydrogen peroxide. Closed circle, mc2155; open squares, mutant I64; open circle, mutant 49; upward closed triangles, mutant Tn1; downward open triangles, mutant Tn2.
FIG. 4.
FIG. 4.
Tests of sensitivity MSH-deficient mutants versus wild-type strain mc2155 to antibiotics. Antibiotics were applied to disks with concentrations of 0, 2, 7.8, 32, 125, and 250 μg (clockwise from the top).
FIG. 5.
FIG. 5.
Multiple-sequence alignment of partial MshC protein. The region containing the point mutation in mutant I64 (marked with boldface and underlined) was aligned to the MshC proteins of various actinomycetes. Completely identical amino acids are marked by asterisks, and dashes mark identity values of more than 60%.
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