NUTRITIONAL-REQUIREMENTS FOR SYNTHESIS OF HEAT-STABLE ENTEROTOXIN BY YERSINIA-ENTEROCOLITICA

A defined medium that supported the growth of and synthesis of heat-stable enterotoxin (YST) by clinical isolates of Yersinia enterocolitica at levels equivalent to those observed in a complex Trypticase soy broth-0.6% yeast extract medium was developed. The defined medium contained four amino acids (L-methionine, L-glutamic acid, glycine, and L-histidine), inorganic salts, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, and potassium gluconate as the carbon source. Methionine was required for growth by most strains of Y. enterocolitica used in this study; thus, it was not possible to determine whether it was also required for the synthesis of YST. The other 17 amino acids commonly found in proteins did not stimulate the synthesis of YST when added to the defined medium. The yield of YST observed with other carbon sources fermented by Y. enterocolitica ranged from 4- to 26-fold lower than that obtained with potassium gluconate. The divalent cations Ca2+ and Mn2+ had no effect on the synthesis of YST; however, concentrations of Fe2+ above 10 muM inhibited the synthesis of the enterotoxin. The addition of a mixture of pyrimidines containing thymine, cytosine, and uracil, each at a concentration of 2.0 mM, stimulated the synthesis of YST by 10 to 15%, whereas a mixture of adenine and guanine, each at a similar concentration, inhibited the synthesis of YST. Vitamins had no effect on the amounts of YST produced by Y. enterocolitica strains grown in the defined medium. Vigorous aeration was necessary for the production of maximal levels of YST, and the enterotoxin was not detected in culture supernatants of Y. enterocolitica strains grown in the defined medium at >30-degrees-C.