EFFECT OF THE ANTIBIOTIC, CYCLOHEXIMIDE, ON THE METABOLISM AND GROWTH OF SACCHAROMYCES PASTORIANUS

COURSEN, B. W., and H. D. SISLER (U. Maryland, College Park.) Effect of the antibiotic, cycloheximide, on the metabolism and growth of Saccharomyces pastorianus. Amer. Jour. Bot. 47(7): 541-549. Illus. 1960.-Studies were made of the toxicity of cycloheximide and certain of its derivatives to Saccharomyces pastorianus Hansen. The ED50 values for cycloheximide, its semicarbazone derivative and its oxime derivative are 0.018, 0.37, and 12.0 p.p.m., respectively. In auxanographic and liquid culture tests involving 160 organic and biochemicals, only certain methylated ring ketones and vitamin A alcohol or acetate showed appreciable antagonistic activity to the toxicity of cycloheximide. Yeast cells exposed to 3.16 p.p.m. of cycloheximide and incubated for 30 min. with uniformly labeled 14C glucose remove about 10% less activity from the medium than untreated cells. Measurements of radioactivity in compounds extracted from cells with 80% ethanol showed the presence of appreciable activity in the glutamine from untreated cells but no measurable activity in this compound from treated cells. Activity in glutamic acid from treated cells was reduced while activity inf alanine and aspartic acid was increased when compared with the activity in these compounds from untreated cells. There were other differences, also, especially in the levels of activity in organic phosphorus compounds, but, in many cases, the activity in compounds from treated cells was similar to that in the corresponding compounds from untreated cells. It is possible that the antibiotic interferes with the metals involved in the enzymatic reaction leading to the synthesis of glutamic acid and glutamine or it may act as an inhibitory analog in the synthesis of these or similar compounds. The apparent interference of cycloheximide with the formation of a CO-NH bond in the synthesis of glutamine suggests also that peptide bond formation in protein synthesis may be similarly affected. A block of glutamine synthesis by cycloheximide may be sufficient to account for the toxicity of the antibiotic, but the failure of exogenous sources of glutamine to reverse the toxicity indicates that other reactions in cell metabolism may be as sensitive to cycloheximide as the synthesis of glutamine. ACTI-DIONE, an antifungal antibiotic, was first obtained from a streptomycin-producing strain of Streptomyces griseus (Whiffen et al., 1946). Kornfield et al. (1949) reported the compound to be beta 2 (3,5-dimethyl 2 oxocyclohexyl) 2 hydroxyethyl-glutarimide, and it has been given the common name of cycloheximide. It shows a remarkable specificity in regard to its biological activity. Whiffen (1948, 1950) tested various concentrations of cycloheximide against a variety of organisms. One thousand p.p.m. did not inhibit the growth of 11 different species of bacteria. Nine species of yeast, including Saccharomyces pastorianus, were completely inhibited by concentrations as low as 0.17 p.p.m., while 5 other species, including S. fragilis (ATCC 8635)3 were not affected by concentrations as high as 1000 p.p.m. Seven of 12 fungi tested which are pathogenic to man were found to be insensitive to cyclo1 Received for publication October 22, 1959. Portion of a thesis presented by the senior author to the Graduate School, University of Maryland, in partial fulfillment of the reauirement for the degree of Doctor of Philosophy, July 1959. Scientific publication No. 810, Contribution No. 3083, of the University of Maryland Agricultural Experiment Station, Department of Botany. This investigation was supported in part by research grants E225 (C5) and (C6) froni tlle National Institutes of Health, Public Health Service. 2 Present address: Department of Biology, Lawrence College, Appleton, Wisconsin. heximide. Yet, growth of 33 species and strains of phytopathogenic fungi was inhibited by concentrations of 0.125-100 p.p.m. Felber and Hamner (1948) first reported control of a fungus disease of plants with this antibiotic. The use of cycloheximide as an agent for controlling plant disease has been limited because of the narrow range between phytotoxicity and fungitoxicity. It has been used successfully, however, for control of specific plant diseases and it shows promise as a chemotherapeutic agent. Whiffen (1951) reported that sub-lethal concentrations of the antibiotic in agar induced the sporophyte of Allomyces arbuscula to form male and female gametangia. She suggested that this might result from an interference with mitotic division but indicated that cycloheximide might be acting only to intensify a tendency already present in the organism. Others (Wilson, 1950; Hawthorne, 1952) have shown that cycloheximide readily and consistently induces specific aberrations in mitotic behavior in cells of root tips of Allium cepa. Berliner and Olive (1953) have observed an effect on meiosis in Gymnosporangium suggesting that the 3 The organism designated as ATCC 8635 and referred to as S. lactis by A. Whiff en, 1948, Jour. Bact. 56: 283-291, is referred to here as S. fragilis. This organism was originally listed by the American Type Culture Collection as S. lactis (ATCC 8635) buthas since been identified by Dr. L. J. Wickerham as S. fragilis and is now listed by the ATCC as S. fragilis.