Selective action of amphotericin B in the isolation of fermentation mutants of yeast.

Amphotericin B and other polyene antibiotics have been shown to be useful agents for the isolation of auxotrophic mutants of yeast by virtue of their ability to selectively eliminate large numbers of wild-type cells from mixed populations (A. G. Moat, I. J. Barnes, and E. H. McCurley, J. Bacteriol. 92:297, 1966; A. G. Moat, N. Peters, and A. M. Srb, J. Bacteriol. 77:673, 1957; R. Snow, Nature 211:206, 1966; 0. Stromnes and R. K. Mortimer, J. Bacteriol. 95:197, 1968). In the experiments described in this report, evidence is presented which indicates that amphotericin B (Fungizone) is also effective in the isolation of fermentative mutants of yeast. Saccharomyces cerevisiae 173-1/a, a haploid, ad', gal+ yeast was used as the parental strain. S. cerevisiae S-732-C, a haploid, try-, his-, gat-, adstrain was used as the gab control. Stock cultures were grown on broth containing 2% glucose, 1 % peptone and yeast extract, and 0.5% phosphate at pH 5.6. For solid medium, 2% agar was added. All incubations were carried out at 32 C. Eosin-methylene blue (EMB) media have been used for years to distinguish fermenters from nonfermenters in bacterial populations (J. E. HoltHarris and 0. Teague, J. Infect. Diseases 18:596, 1916). They have also been employed for the detection of the ability of yeast colonies to ferment galactose (S. Spiegelman et al., Proc. Natl. Acad. Sci. U.S. 36:591, 1950; B. Rotman and S. Spiegelman, J. Bacteriol. 69:492, 1953). However, the conditions employed in the earlier studies with yeast did not provide for adequate development of gab variants (A. James, J. Bacteriol. 67:237, 1953). A modified EMB medium containing galactose and glucose in a ratio of 20:1 was developed. On this medium, the known gal+ strain (S. cerevisiae 173-1/a) developed a deep purple colony; a known gab strain (S. cerevisiae S-732-C) grew as well as the wild type and exhibited a slight pink coloration after 48 hr. Since yeasts require a source of fermentable carbohydrate in order to take up most nutrients from the medium (R. Thorne, Wallerstein Commun. 13: 319, 1950), the small amount of glucose facilitated the growth of galstrains. On testing random isolates from platings of known mixtures of the gal+ and gat strains, no false-reacting colonies were observed. This medium greatly facilitated subsequent experiments as it eliminated the necessity of testing each individual colony for its ability to ferment galactose. The minimal medium used for selection was similar to that of A. G. Moat et al. (J. Bacteriol. 77:673,1959), except that 2% galactose was substituted for glucose and 40 mg of adenine per liter was included for strain 173-1/a. Tryptophan and histidine were added (40 mg/liter) when strain S-732-C was employed. This medium supported the growth of gal+ cells but not that of gabcells. All gat colonies observed on the EMB medium were retested for galactose fermentation by repeated transfer on EMB-agar and on minimal broth medium with galactose as the sole carbon source. In addition, they were tested for respiratory deficiency by the technique of M. R. Ogur et al. (Science 125:928, 1957) and for their nutritional requirements. Known mixtures of gal+ and gabcells were inoculated into the defined medium containing galactose as the sole source of carbohydrate. Amphotericin B (20 ,ug/ml) was added at the beginning of the exponential growth phase (180 min for the wild type). The wild-type cells were rapidly killed by the antibiotic while the nongrowing gat mutants remained in constant numbers. This indicated that gal+ cells could be selectively eliminated from gatcells and that no cross-feeding occurred under the conditions employed. Amphotericin B was tested, under identical conditions, for its effectiveness in selectively eliminating gal+ cells from a random population. In the absence of mutagenic treatment, the spontaneous mutation frequency was of the order of 1:140,000 or less (Table 1). When 9.0 ml of a washed cell suspension of strain 173-1/a (1.32 ± 0.2 x 107 cells per ml) was treated with f-propiolactone (final concentration, 0.15%), it was observed that a 20-min exposure resulted in a 75% reduction in viable cells, with a frequency of galmutants of 1:6,300. A 25-min exposure to the