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, AND HERMAN C. LICHSTEIN. Biotin transport and accumulation by cells of Lactobacillus plantarum. I. General properties of the system. J. Bacteriol. 90:843-852. 1965.-Resting cells of Lactobacillus plantarum were saturated with bound biotin by incubation in phosphate buffer with biotin and glucose for 2 hr. This bound biotin was stable to wide changes in temperature, pH, and reaction time. Free biotin could be eluted from the cells by suspending them in cold water or saline. Immersing the cells in boiling water for 30 sec released all free biotin. Recoveries of added biotin exceeded 92%. Free biotin uptake by bound biotin-saturated cells occurred by two mechanisms. One process was independent from temperature (Qlo, 1.25), pH, cellular metabolism, and inhibition by iodoacetate. The other mechanism was dependent upon temperature (Qlo, 2.58; optimum, 37 C), pH (optimum, 7.5), and active cellular metabolism, and was inhibited by iodoacetate. Activation energies of 3,700 and 13,800 cal per mole, respectively, were observed for glucose-independent and-dependent free biotin uptake. Both processes exhibited approximately the same degree of inhibition by homobiotin. Higher concentrations of homobiotin were required to inhibit growth than to inhibit free biotin uptake. Intracellular-extracellular ratios as high as 600 were established in the absence of glucose, whereas ratios of nearly 4,000 occurred in the presence of glucose. MATERIALS AND METHODS Organism and growth conditions. L. plantarum strain 17-5, which was employed in all studies, was maintained in stab culture on APT medium (Case Laboratories, Chicago, Ill.) by serial transfer every 2 to 4 weeks, incubated at 30 C overnight, and then refrigerated. Cells for experimental use were grown in the medium of Wright and Skeggs (1944) containing 5 X 10-4,ug of biotin per ml; the medium was modified by the substitution of cysteine for cystine, and the addition of folic acid (0.5 mg per liter) to improve growth. NaCl was omitted because the Difco Vitamin Free Casamoin Acids used as a source of amino acids contains 38% NaCl. Glucose was sterilized separately by auto-843

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

Biotin auxotrophs were isolated from Escherichia coli K-12. One of the mutants was unable to grow on desthiobiotin and accumulated a large amount of a vitamer in medium when growing on an optimal concentration of biotin. The production of the vitamer was inhibited in the presence of an excess amount of biotin. The vitamer was identified as desthiobiotin on the basis of biological activities, avidin combinability, and chromatographic characteristics. The mutant lacked the ability to convert desthiobiotin to biotin. These results further support the hypothesis that desthiobiotin is a normal intermediate in the biosynthesis of biotin in E. coli.