Genetic antagonism and hypermutability in Mycobacterium smegmatis

Abstract

Multidrug-resistant strains of Mycobacterium tuberculosis are a serious and continuing human health problem. Such strains may contain as many as four or five different mutations, and M. tuberculosis strains that are resistant to both streptomycin and rifampin contain mutations in the rpsL and rpoB genes, respectively. Coexisting mutations of this kind in Escherichia coli have been shown to interact negatively (S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 72:2084-2087, 1975; S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 74:1157-1161, 1977). We investigated this possibility in Mycobacterium smegmatis by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics. Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of M. smegmatis under selection either for one or for both antibiotics. Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains. The second antibiotic resistance mutation occurred at a lower frequency in both cases. Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process. Determinations of rates of mutation to antibiotic resistance in M. smegmatis showed that the frequencies were enhanced up to 10(4)-fold during stationary phase. If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.

FIG. 1

PCR strategy used to analyze the nucleotide sequence of hisD. The 1,338-nucleotide hisD coding sequence is represented by the rectangle, and the primers used to amplify and/or sequence the gene are indicated by arrows. The codon numbering is based on M. smegmatis hisD sequence data published by Hinshelwood and Stoker (13).

FIG. 2

EASPCR to identify the appearance of rpsL mutations in liquid culture before plating on selective media. (A) Outline of the procedure. (B) EASPCR of samples taken at different times. M, PCR marker; lane 1, negative control; lane 2, wild type; lane 3, Str^r mutant; lane 4, Rif^r mutant (3-day culture); lane 5, Rif^r mutant (8-day culture); lanes 6 to 9, same as lanes 2 to 5 but without MboII digestion before PCR II.

FIG. 3

Flow chart describing mutant isolation. Rif, rifampin; Str, streptomycin; ∗, selective medium contains the indicated drug.

FIG. 4

Growth phase-dependent hypermutability in M. smegmatis, shown as frequencies of the appearance of resistant mutants.