A Study of Mutability in Streptomycin-Dependent Strains of Escherichia Coli

Abstract

Mutations from streptomycin-dependence to non-dependence were studied in different streptomycin-dependent (Sd) strains of E. coli B/r, with respect to a comparison of mutation rates, mutagen stability, and patterns of delayed appearance of induced mutants. With MnCl2 as the mutagen, Sd strains were grown in aerated cultures of nutrient broth containing streptomycin for 24-36 hrs. at 37[degree], then centri-fuged, washed in 0.3 [image] NaCl, resuspended in 0.04% MnCl2, and incubated for 1 hr. at 37[degree]. An untreated control sample was re-suspended in broth without MnCl2. Dilutions of control and treated suspensions were plated on nutrient agar containing streptomycin (50 [mu]g./ml.) and incubated for 48 hrs., then further dilutions were plated on plain nutrient agar and incubated for 5 days at 37[degree]. Colonies growing on nutrient agar without streptomycin represented mutations from dependence to non-dependence. A preliminary survey of 70 strains with respect to spontaneous and induced reversions showed that none was mutagen stable and all differed with respect to reversion frequency. However, this difference was in part influenced by the no. of residual cell divisions on the plain nutrient agar, since in several strains the cells had failed to divide, thereby preventing mutation. This supports previous evidence that most of the reversions detected as colonies on plain nutrient agar occur during residual cell divisions after plating. Survival of cells after treatment with MnCl2 varied from 10 to 80% in different expts. In order to detn. differences among 15 Sd strains with respect to mutation rates, each strain was treated with MnCl2 and 4 plates were spread for each dilution of the control and treated samples. Records were made of the no. of bacteria plated and also of the no. of residual divisions, from which was calculated a spontaneous mutation rate, and corrections were made for spontaneous mutations. A high degree of variability was observed among mutation rates detd. for each strain at different times, but significant differences also existed among strains. To detn. the pattern of delayed appearance of induced mutants, 12 strains were selected which passed through a min.-no. of residual divisions on plain nutrient agar. By plating treated and control samples on a series of agar plates containing increasing concns. of streptomycin (not exceeding 1 [mu]g./ml.), which increased the no. of residual divisions, the reversion frequency could be detd. after each successive division. A large increase in no. of colonies occurred when a streptomycin concn. range of 0.4-1 [mu]g./ml. was used, but the colonies varied in size, and the smaller colonies represented "semidependent" mutants. When the complete reversion frequencies had been corrected for the "semidependent" types, most of the strains used had a majority of the latter type. Based on complete reversions, 3 strains gave similar patterns of delayed appearance of induced mutants, 100% expression being reached at the 5th or 6th division and 50% at 3-4 1/2 divisions. However, the reversions produced by one strain were all streptomycin sensitive, by a 2d strain all resistant, and by a 3d 63% resistant and 37% sensitive.