The antibiotic fosfomycin was produced as a secondary metabolite in a glucose-asparagine medium containing citrate, l -methionine, and l -glutamate. The citrate requirement for antibiotic synthesis was related to its requirement for growth. In contrast, l -methionine and l -glutamate caused a marked stimulation of fosfomycin production and had no effect on growth. l -Methionine had to be added early to effect maximal antibiotic synthesis later in the fermentation. The l -glutamate requirement was not specific, since several tricarboxylic acid cycle intermediates could replace this amino acid. l -Asparagine was the most effective nitrogen source for growth and production of fosfomycin. Glycine, an alternate nitrogen source, supported fosfomycin synthesis only when added in excess of that needed for growth. Cobalt and inorganic phosphate were required also for antibiotic production at concentrations exceeding those supporting maximal growth. Radioactive incorporation studies showed that the methyl carbon of methionine was the precursor of the methyl of fosfomycin. Carbon 1 of fosfomycin was derived from glucose carbons 1 and 6, whereas glucose- 2 - 14 C labeled fosfomycin carbon 2. Radioactivity from acetate- 2 - 14 C was distributed equally between fosfomycin carbons 1 and 2. No incorporation of acetate- 1 - 14 C , asparagine- U - 14 C , citrate- 1,5 - 14 C , or glutamate- U - 14 C occurred. The labeling pattern of fosfomycin carbons 1 and 2 was similar to that found in 2-aminoethylphosphonate from Tetrahymena .

The characteristics of the biotin transport mechanism of Saccharomyces cerevisiae were investigated in nonproliferating cells. Microbiological and radioisotope assays were employed to measure biotin uptake. The vitamin existed intracellularly in both free and bound forms. Free biotin was extracted by boiling water. Chromatography of the free extract showed it to consist entirely of d-biotin. Cellular bound biotin was released by treating cells with 6 N H2SO4. The rate of biotin uptake was linear with time for 10 min, reaching a maximum at about 20 min followed by a gradual loss of accumulated free vitamin from the cells. Biotin was not degraded or converted to vitamers during uptake. Transport was temperature-and pH-dependent, optimum conditions for uptake being 30 C and pH 4.0. Glucose markedly stimulated biotin transport. In its presence, large intracellular free-biotin concentration gradients were established. Iodoacetate inhibited the glucose stimulation of biotin uptake. The rate of vitamin transport increased in a linear fashion with increasing cell mass. The transport system was saturated with increasing concentrations of the vitamin. The apparent Km for uptake was 3.23 X 10-7 M. Uptake of radioactive biotin was inhibited by unlabeled biotin and a number of analogues including homobiotin, desthiobiotin, oxybiotin, norbiotin, and biotin sulfone. Proline, hydroxyproline, and 7,8-diaminopelargonic acid did not inhibit uptake. Unlabeled biotin and desthiobiotin exchanged with accumulated intracellular "4C-biotin, whereas hydroxyproline did not. 1 Presented in part at the 68th Annual Meeting of the American Society for Microbiology, Detroit, Mich., 5-10 May 1968. Taken from a dissertation submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree in Microbiology. measuring exchange reactions and analogue inhibition patterns. In the experiments reported, vitamin uptake was measured in nonproliferating yeast cells. MATERIALS AND METHODS Chemicals. Crystalline d-biotin was purchased from the Sigma Chemical Co., St. Louis, Mo. Carbonyl-labeled 14C-biotin (57.5 mc/mmole) was obtained from Amersham-Searle Co., Des Plaines, Ill. The purity of the labeled biotin was checked by bioand radioautography. dl-Desthiobiotin was purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. The biotin analogues d-homobiotin, dl-oxybiotin, and d-norbiotin were gifts from Hoffmann

The metabolic control of biotin transport in Saccharomyces cerevisiae was investigated. Nonproliferating cells harvested from cultures grown in excess biotin (25 ng/ml) took up small amounts of biotin, whereas cells grown in biotin-sufficient medium (0.25 ng/ml) accumulated large amounts of the vitamin. Transport was inhibited maximally in cells grown in medium containing 9 ng (or more) of biotin per ml. When avidin was added to biotin-excess cultures, the cells developed the ability to take up large amounts of biotin. Boiled avidin was without effect, as was treatment of cells with avidin in buffer. Avidin did not relieve transport inhibition when added to biotin-excess cultures treated with cycloheximide, suggesting that protein synthesis was required for cells to develop the capacity to take up biotin after removal of extracellular vitamin by avidin. Cycloheximide did not inhibit the activity of the preformed transport system in biotin-sufficient cells. The presence of high intracellular free biotin pools did not inhibit the activity of the transport system. The characteristics of transport in biotin-excess cells (absence of temperature or pH dependence, no stimulation by glucose, absence of iodoacetate inhibition, independence of uptake on cell concentration, and nonsaturation kinetics) indicated that biotin entered these cells by diffusion. The results suggest that the synthesis of the biotin transport system in S. cerevisiae may be repressed during growth in medium containing high concentrations of biotin.