Microbial production of vitamin B12 antimetabolites. III. Compound 102804 from Bacillus cereus.

Abstract

A basic compound with empirical formula C12H19N2O5 was isolated from Bacillus cereus 102804 fermentations of a soybean meal -glucose medium. The inhibitory activity of compound 102804 on growth of Gram-positive and Gram-negative bacteria growing in a chemically defined medium was reversed by vitamin B12, by L-methionine, and by n-methionine.It has no inhibitory activity for Escherichia coli (Davis 113-3) when grown in media containing L-methionine.The biosynthesis of 102804 coincides with the sporulation of the B. cereus, and the compound is not produced in the absence of sporulation.A search for microorganisms producing substances inhibiting vitamin B12-stimulated growth of Escherichia coli (Davis 113-3) resulted in selection of a strain of Bacillus cereus which had this capability.The vitamin B12 antimetabolite is produced by this culture (designated as 102804 in our collection) when grown under several conditions, and its production seems to coincide with sporulation of the bacillus. We wish to summarize in this communication our studies on the effect of fermentation conditions on production of compound 102804, and some of the chemical and biological attributes of this vitamin B12-antimetabolite. Materials and MethodsIsolation of culture 102804 A soil sample collected in Madison was diluted with sterile water and plated on nutrient agar. Discrete colonies were transferred to tubes of soybean meal -glucose -CaCO3 medium and placed on a shaker at 30°C. After several days' growth aliquots of the fermented medium were centrifuged and the supernatant liquid tested for ability to inhibit growth of E. coli (Davis 113-3) growing in an agar diffusion bioassay in the DAVIS -MINGi0LI1) medium (with added vitamin B12), and to inhibit the growth of Escherichia coli B(ATCC 11303) (growing in nutrient agar) and Staphylococcus aureus FDA 209P (ATCC 6538) (growing in nutrient agar). The supernatant solution from culture 102804 was found to inhibit the E. coli (Davis 113-3) and not the other two organisms under the test conditions, and the inhibition was competitively reversed by vitamin B12 in an agar diffusion bioassay.2) Culture 102804 was maintained for further study by periodic transfers on agar slants and by storage of cell suspensions in liquid nitrogen.Identification of culture 102804 Culture 102804 was grown in a variety of media mentioned in BERGEY'S manual3) and compared with cultures of Bacillus cereus (ATCC 14579), Bacillus mycoides (ATCC 6462), and Bacillus subtilis (ATCC 6633) grown under the same conditions. Culture 102804 is closely related to B. cereus on the basis of: Colonial morphology on glucose-containing agar; Gram staining characteristics; shape of sporangia; vegetative cell and spore size; acid production when grown on glucose and glycerol containing agars, and no acid production when grown on mannitol and xylose containing agars; a positive VOGES-PROSKAUER test and a positive test for reduction of nitrate to nitrite.