The biosynthesis of monensin by Streptomyces cinnamonensis was studied by using '4C-labeled glucose, acetate, propionate, butyrate, and methionine. The results indicated that the antibiotic is synthesized from five acetate, seven propionate, and one butyrate molecules. The o-methyl group of monensin is derived from methionine, whereas the terminal hydroxymethyl group is incorporated from acetate.Monensin, an antibiotic produced by Streptomyces cinnamonensis (ATCC 15413), was first described by investigators from Eli Lilly and Co. (4). Initial fermentation studies were presented by Stark (7). The structure of the major component, factor A (Fig. 1), was determined by X-ray crystallographic analysis of the silver salt by Agtarap et al. (2). In addition to factor A, three additional factors have been recognized (3): in factor B the ethyl group on ring C is replaced by a methyl group, in factor C the methyl at the carboxyl end is replaced by an ethyl group, and in factor D the methyl group on the B ring is replaced by an ethyl group. The structures of factors C and D are tentative proposals. Factors B, C, and D are minor constituents of the fermentation broth. Factor A, hereafter designated monensin, and its sodium salt are only slightly soluble in water but are very soluble in organic solvents.This study is concerned with the biosynthesis of monensin and the incorporation of labeled intermediates into the antibiotic. MATERIALS AND METHODSOrganism and cultural conditions. The ingredients of the medium used during these studies with S. cinnamonensis, ATCC 15413, were (in mg/ml): glucose (16.7), L-tyrosine (3.3), L-valine (6.6), L-lysine (1.0), CaCO, (1.0), FeSO4 7H20 (0.17), K2HPO4 (0.17), KCl (0.05), MgSO4 7H20 (0.67), biotin (0.025), and folic acid (0.025). Cultures were grown at 32 C in shaken (250 rpm) flasks (100 ml of medium per 500 ml wide-mouth flask), or in 1. Isolation of monensin. The whole broth was harvested and the pH was adjusted to 9.0 with NaOH, after which it was extracted twice with one-half volume of chloroform. The extracts were combined and washed through a column containing carbon (Pittsburgh 12 by 40 mesh). The column was washed with excess chloroform, and the combined extracts were evaporated. The residue was dissolved in methanol and chilled. Cold deionized water was added until the monensin crystallized. The monensin was collected by filtration, washed with cold water, recrystallized from petroleum ether, and assayed for radioactivity.Degradation of labeled monensin. The periodate oxidation of monensin is illustrated in Fig. 2. A sample of "4C-labeled monensin (100 mg) was dissolved in t-butanol (4 ml) with stirring. To 'this solution was added 0.2 M aqueous sodium meta-periodate solution (2 ml). The final mixture was allowed to stand overnight. The reaction mixture was distilled into a receiver containing a dimedone solution (100 mg of dimedone in 2 ml of 50% ethanol-water). The distillate was allowed to stand a few minutes and then concentrated under reduced pressure to induce crystallizat...
The addition of phenoxymethylpenicillin (10 mg/ml) at any time during the penicillin fermentation inhibited further accumulation of the antibiotic in broth but had no effect on growth. Benzylpenicillin, 6-aminopenicillanic acid (6-APA), and some semisynthetic penicillins also showed this effect, but penicillin N, peniciuloic acid, cephalosporin C, and 7-aminocephalosporanic acid did not limit penicillin accretion. Incorporation of radioactive precursors (cysteine, valine, and sodium phenoxyacetate) into penicillin in the presence of inhibitory concentrations of the antibiotic indicated that penicillin synthesis continued despite the lack of accretion of the antibiotic in broth. The rates of penicillin synthesis in a 48-hr and a 136-hr culture were compared by short-term exposure to Na2"5SO4, and no significant difference in the biosynthetic rate was observed. Exogenous penicillin in the range of 1 to 15 mg/ml of culture broth had no effect on utilization of acetate or glucose by Penicillium chrysogenum. The antibiotic-synthesizing capacity of the organism was not irreversibly inhibited by exogenous penicillin. The degradation of penicillin during the fermentation was also studied. Penicillin V was stable in broth filtrate. Catabolic enzymes such as penicillinase and penicillin-acylase were not demonstrated in whole broth, nor was the accumulation of 6-APA, penicilloic acid, or other degradation products detected. An examination of the intracellular penicillin concentration and the amount of penicillin associated with the mycelium revealed that cells contained significantly more penicillin late in the fermentation than earlier in the cycle. This cell-associated antibiotic may be a regulatory factor in further penicillin synthesis.
Angeles), AND E. Z. GORDEE. Formation of bacterial flagella. I. Demonstration of a functional flagellin pool in Spirillum serpens and Bacillus subtilis. J. Bacteriol. 91:870-875. 1966-Exponentially growing cultures of Spirillum serpens and Bacillus subtilis regained motility and flagella within one generation after mechanical deflagellation. Regeneration of flagella occurred in both cultures in the presence of chloramphenicol at concentrations shown to inhibit flagellin synthesis. Cells labeled with C'4-amino acids regenerated radioactive flagella in the presence of chloramphenicol. A conditional mutant of S. serpens (T-45) was isolated. This strain did not produce flagella when grown at 45 C, but formed the organelles upon temperature shift to 30 C, even in the presence of chloramphenicol. A reduction of intracellular antibody-precipitable flagellin counts in labeled S. serpens T-45 occurred concomitant with the generation of flagella at 30 C. The data suggest that the flagella of S. serpens and B. subtilis are formed from a pool of intracellular flagellin proteins.
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