THE JOURNAL OF VITAMINOLOGY 9, 183-187 (1963) EFFECT OF SULFUR-CONTAINING AMINO ACIDS ON THE PRODUCTION OF THIAMINE BY ESCHERICHIA COLI1 MASUO AKAGI AND HIROSHI KUMAOKA2 Faculty of Pharmaceutical Science, School of Medicine, Hokkaido University, Sapporo (Received August 20, 1963) It has now been firmly established by several groups of workers (1-10) that in yeast thiamine is enzymatically synthesized from pyrimidine pyrophosphate and thiazole monophosphate. However, there is little information about the biosynthetic pathways of pyrimidine and thiazole themselves. Thiamine was found as the first natural com pound containing a thiazole ring. Recently, the presence of the thiazole ring system has been demonstrated in luciferin (11) and in antibiotics, such as micrococcin P (12, 13), bottromycin (14), thiostrepton (15, 16), and bacitracin F (17). Chemical structure similar to the thiazole ring system has been found in bacitracin A (18). It was indicated clearly that they were derived from polypeptides containing cysteine and by cyclization to a thiazoline for bacitracin A and by cyclization followed by de hydrogenation to thiazole rings for the others. The chemical structure of penicillin in which the thiazolidine ring is a dipeptide composed of a cysteine derivative and valine. It has been established that C14-, S35-, and H3-labelled cysteine gave rise to labelled penicillin (19-21). It is considered that the thiazole ring of thiamine is also syn thesized via sulfur-containing amino acid. Such concepts were already presented by several workers without reliable data (22-25). A detailed study was made on the effect of sulfur-containing amino acids and other amino acids on the production of thiamine by Escherichia coli. EXPERIMENTAL 1. Microorganism and Medium Escherichia coli ATCC 9637 was used for this work. The basal medium used in these experiments was a slight modification of the medium of Davis and Mingioli (26) and had the following composition: K2HPO4, 7.0g; KH2PO4, 3.0g; sodium citrate trihydrate, 5.0g; MgSO4 7H2O, 0.1g; (NH4)2SO4, 1.0g; glucose, 2.0g; H2O, 1,000ml; ph 7.0. 2. Cultural Conditions The cells from a 24-hour culture Escherichia coli, grown at 37 on a basal 1 Th e following abbreviations were used: pyrimidine, 2-methyl-4-amino-5-hydroxymethyl -pyrimidine; thiazole, 4-methyl-5-(2-hydroxyethyl) thiazole. 183
184 AKAGI AND KUMAOKA 1963 medium agar slant were suspended in sterile saline water and used to inoculate the medium. The inoculated medium was incubated for 10 hours at 37 on a shaker. The resulting cells were harvested under sterile conditions by centrifugation at 4,000 rpm for 10 minutes at 2. The cells were suspended in 100ml of sterile saline water and 2ml of this suspension was used to inoculate the flask containing 100ml of the growth medium. The flasks were incubated at 37 on a shaker for 2 hours. Then, 2 aliquots were withdrawn from each flask; one was used for the determination of the dry weight of the cells and the other for the determination of the thiamine content of the cells. 3. Assesment of Cell Growth To determine the dry weight of the cells, an aliquot of cell suspension was c en trifuged at 6,000 rpm for 5 minutes and the cell pellet was washed once with water. The supernatant solution was discarded and the cells were suspended in a small amount of water and transferred quantitatively to a previously weighed bottle. The cells were dried at 110 until constant weight had been attained and then weighed. 4. Extraction, and Assay of Thiamine After the addition of 1 drop of concentated HCl, the aliquot to be used for the determination of thiamine content was heated on water bath for 10 minutes. The extracts were adjusted to ph 4.5 with 0.1M acetate buffer. With the addition of Takadiastase solution, thiamine phosphates in extracts were hydrolyzed at 37 over night. After centrifugation, the extracts were subjected to assay of thiamine. Thia mine was determined by microbiological assay with Lactobacillus fermenti according to the directions described by Sarett and Cheldelin (27). 5. Chemicals Amino acids, vitamin-free casamino acid, ribose and Takadiastase were commercial preparations. RESULTS The experiments were performed to determine which substances, when added to the growth medium, might stimulate the production of thiamine by the growing culture of Escherichia coli. The effects on thiamine production of vitamin-free casamino acid, glucose, and ribose were shown in Table I. The cells grown in the basal medium were used as the basis for the comparison. It was observed that replacement of ammonium sulfate by a vitamin-free casamino acid as the nitrogen source, resulted in a decrease in cellular yield, but a slight increase in thiamine production per unit weight of cells. It may be seen that the addition of vitamin-free casamino acid to a medium decreases the production of thiamine per unit weight of cells, but this de crease is due to an increase of the cellular yield. The cells grown in a medium to which ribose had been added produced slightly more thiamine and the cells grown in a medium containing ribose and vitamin-free casamino acid as the nitrogen source produced more thiamine than the cells grown in the basal medium. Moreover, the cells grown in a glucose-free medium containing both ribose and vitamin-free casamino acid as nitrogen sources produced almost twice as much thiamine per unit weight of cells as those grown in the basal medium. Thus vitamin-free casamino acid and
Vol. 9 PRODUCTION OF THIAMINE 185 TABLE I Effect of Vitamin-free Casamino Acid, Glucose, and Ribose on the Production of Thiamine by the Growing Cells of Escherichia coli Vitamin-free casamino acid was added at 1 mg per ml of the medium. Ribose was added at 2mg per ml of the medium. ribose appeared to stimulate and glucose appeared to depress the production of thiamine by Escherichia coli. The experiments to determine which component of vitamin-free casamino acid might stimulate was undertaken. As shown in Table II, cysteine and methionine stimulated the production of thiamine per unit weight of the cells. Among the other amino acids tested, aspartic acid appears to stimulate slightly the production of thia mine, but other amino acid failed to stimulate. The effect of the concentration of sulfur-containing amino acids on the production of thiamine was shown in Table III. The lower the concentrations of sulfur-containing amino acids, the smaller the stimulating effects of these amino acids on the production of thiamine. The cells grown in a medium containing 0.09g of ammonium sulfate equivalent to 0.1g of methionine in 100ml of the medium produced nearly the equal amount of thiamine per unit weight of the cells grown in the basal medium. TABLE II Effect of Amino Acids on the Production of Thiamine by the Growing Cells of Escherichia coli Amino acid was added at 1mg per ml of the medium.
186 AKAGI AND KUMAOKA 1963 TABLE III Effect of Concentration of Sulfur-containing Amino Acid on the Production of Thiamine by the Growing Cells of Escherichia coli DISCUSSION Aspartic acid and sulfur-containing amino acids such as cysteine and methionine stimu lated the production of thiamine by the growing culture of Escherichi acoli. Except as partic acid, the other amino acids tested failed to show the stimulatory effect. Since the addition of ammonium sulfate equivalent to methionine to the basal medium showed no effect on the production of thiamine, the stimulative effect of sulfur-containing amino acids is not due to a mere increase of the sulfur source. At low concentra tions, sulfur-containing amino acids showed little stimulative effect on the production of thiamine. It is considered that at low concentrations, major quantities of sulfur -containing amino acids are used for the formation of cellular protein. Under the presence of a large amount of cysteine in a medium, the thiamine content per unit weight of cells is high but the growth of Escherichia coli is strongly inhibited. It was already indicated that antibiotics containing thiazole ring such as micrococcin P (12, 13), bottromycin (14), thiostrepton (15, 16), and bacitracin F (17) were synthesized from polypeptide containing cysteine. Moreover, it was shown that luciferin (11) isolated from the firefly contained both benzothiazole and thiazolidine rings. Taking these facts into consideration, the results of the present experiments strongly suggest that the thiazole of thiamine is also synthesized via sulfur-containing amino acid. Glucose appears to depress but, to the contrary, ribose appears to stimulate the production of thiamine by the growing culture of Escherichia coli. Similar result was reported by Ortega and Brown (28) on the production of nicotinic acid by Escherichia coli. SUMMARY The production of thiamine per unit weight of the cells by the growing culture of Escherichia coli was increased by addition of aspartic acid and sulfur-containing amino acid to the growth medium. The presence of glucose in the growth medium depressed the production of thiamine but the presence of ribose was stimulative.
Vol. 9 PRODUCTION OF THIAMINE 187 ACKNOWLEDGEMENT This work was supported by Grant-in-Aid for institutional Research from the Ministry of Education to which the authors express their gratitude. The authors also wish to thank Prof. T. Suzuki, University of Kyoto, for his kind advice. REFERENCES 1. Nose, Y., Ueda, K., and Kawasaki, T., Biochim. Biophys. Actg. 34, 277 (1959). 2. Kawasaki, T., Seikagaku 32, 31, 106 (1960). 3. Kawasaki, T., Okada, Y., and Sato, T., ibid. 32, 464 (1960). 4. Nose; Y., Ueda, K., and Kawasaki, T., J. Vitaminol. 7, 92 (1961). 5. Nose, Y., Ueda, K., Kawasaki, T., Iwashima, A., and Fujita, T., ibid. 7, 98 (1961). 6. Camiener, G. W., and Brown, G. M., J. Amer. Chem. Soc. 81, 3800 (1959). 7. Camiener, G. W., and Brown, G. M., J. Biol. Chem. 235, 2404, 2411 (1960). 8. Lwein, L. M., and Brown, G. M., ibid. 236, 2768 (1961). 9. Leder, I. G., Biochem. Biophys. Research Commons. 1, 63 (1959). 10. Leder, I. G., J. Biol. Chem. 236, 3066 (1961). 11. White, E. H., McCappa, F., and Field, G. F., J. Amer. Chem. Soc. 85, 337 (1963). 12. Brookes, P., Clark, R. J., Fuller, A. T., Mijovie, M. P. V., and Walker, J., J. Cheat. Soc. 916 (1960). 13. Brookes. P., Fuller, A. T., and Walker, J., ibid. 689 (1957). 14. Waisvisz, J. M., Van der Hoeven, M. G., and to Nijenhuis. B., J. Amer. Chem. Soc. 79, 4524 (1957). 15. Kenner, G. W., Sheppard, R. C., and Stehr, C. E., Tetrahedron Letters, No. 1, 23 (1960). 16. Bodanszky, M., Sheehan, J. T., Fried, J., Williams, N. J.. and Birkhimer, C. A., J. Amer. Chem. Soc. 82, 4747 (1960). 17. Weisiger, J. R., Hausmann, W., and Craig, L. C., ibid. 77, 3123 (1955). 18. Newton. G. G. F., and Abraham, E. P., Biochem. J. 53, 604 (1953). 19. Arnstein, H. R. V., and Grant, P. T., ibid. 57, 360 (1954). 20. Halliday. W. J., and Arnstein, H. R. V.; ibid. 64, 380 (1956). 21. Arnstein, H. R. V., and Crawhall, J. C., ibid. 67, 186 (1957). 22. Harington, C. R., and Moggridge, R. C. G., J. Chem. Soc. 443 (1939). 23. Makino, K., Morn, S., and Guo, W., Vitamins 2, 195 (1949). 24. Takada, Y., and Fukui, S., ibid. 3, 229 (1950). 25. Doudney, C. D., and Wagner. R. P., Proc. Nat. Acad. Sci. 39, 1043 (1953). 26. Davis, B. D., and Mingioli, E. S., J. Bacteriol. 60, 17 (1950). 27. Sarett, H. P., and Cheldelin, V. H.. J. Biol. Chem. 155, 153 (1944). 28. Ortega. M. V., and Brown, G. M., ibid. 235, 2939 (1960).