Biomedical subjects
Publications and source records attributed to A L Demain.
The production of the new mycotoxin malformin C by a solid substrate fermentation is described. Malformin C is highly toxic (mean lethal dose = 0.9 mg/kg) and exerts antibacterial activity against a variety of gram-positive and gram-negative organisms.
Compounds structurally related to lysine were tested against Penicillium chrysogenum Wis. 54-1255 for inhibition of growth, sporulation, and penicillin formation. This strain is relatively resistant to lysine analogs. The compounds that were the more active inhibitors of growth and whose activities were reversed by L-lysine were diaminohexynoic acid, N-epsilon-methyllysine, N-alpha-methyllysine, and diaminopimelic acid. These four compounds also inhibited sporulation, which was more sensitive to inhibition than growth was. Analogs strongly inhibiting benzyl-penicillin formation by resting mycelia were diaminohexynoic acid and N-epsilon-methyllysine. The action of the most active analog (diaminohexynoic acid) on penicillin synthesis was reversed by DL-alpha-aminoadipic acid.
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Incorporation of L-[14C]ornithine into gramicidin S by crude, unfractionated lysozyme extracts of Bacillus brevis ATCC 9999 was shown to represent the activity of the gramicidin synthetase complex. Frozen-thawed cells were the source of active extracts, but when cells were shaken in air at 37 degrees C, they rapidly lost activity in a first-order reaction with a half-life of 13 min. Protease inhibitors and inhibitors of energy metabolism had no effect on the inactivation process in frozen-thawed cells. Stabilization was achieved when the cells were shaken in nitrogen or helium instead of air. The addition of dithiothreitol produced a moderate degree of stabilization. The L-ornithine- and D-phenylalanine-activating activities of the gramicidin S synthetase complex were also lost during aeration of the cells. Crude cell-free extracts also lost activity when they were shaken in oxygen, but, in this case, inactivation was slower (half-life of 80 min). Nitrogen also stabilized these cell-free extracts.
Unlabeled adenine brought about a (delayed) decrease in radioactivity that had been taken up by phosphate-limited resting cells of Streptomyces griseus from [14C]adenine-labeled adenosine 5'-monophosphate (AMP). Inorganic phosphate, on the other hand, stimulated adenine uptake from AMP, presumably by activating an energy-dependent active transport mechanism. Unlabeled phosphate rapidly diluted the uptake of radioactivity from [32P]AMP. Adenine inhibited uptake of [32P]AMP but not that of [32P]orthophosphate; adenine is thought to act by inhibiting the cleavage of AMP. The uptake of 32P and 14C from double-labeled AMP showed marked differences; 32P was taken up much faster into both cells and nucleic acids. These data indicate that uptake of AMP components takes place after extracellular dephosphorylation of the nucleotide.
Addition of cyclic-AMP (c-AMP) to Streptomyces griseus fermentations inhibited candicidin formation. In a phosphate-free resting cell system, c-AMP inhibited net candicidin formation and incorporation of labeled propionate and p-aminobenzoic acid into the antibiotic but did not inhibit protein synthesis. All nucleotides tested, regardless of the position of the phosphate ester, were effective inhibitors; nucleosides and free bases were not. Inhibition occurred whether the nucleotide was added early or late. The results indicate that inhibition of antibiotic formation by exogenous nucleotides, including cyclic nucleotides, is similar to the effect produced by inorganic phosphate.
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Homocitrate reverses the lysine inhibition of penicillin bisynthesis. Since lysine is known to inhibit homocitrate synthase, reversal by the reaction product identifies this enzyme as the crucial site of the lysine effect.
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The effects of different nutrient limitations on the production of the two enzymes of gramicidin S biosynthesis were studied during continuous culture of Bacillus brevis. Gramicidin S synthetases I and II were produced in the chemostat under carbon, nitrogen, phosphorus or sulphur limitation. The growth rate, rather than the nature of the limitation, was the major controlling factor in regulating the level of the gramicidin S synthetases. Synthetase production was low at high dilution rates (0.45 to 0.50 h-1) but increased as the dilution rate was lowered. The highest specific activities occurred at dilution rates that were different for each type of limitation: 0.40 h-1 for nitrogen, 0.32 h-1 for carbon, 0.24 h-1 for sulphur and 0.20 h-1 for phosphorus. Phosphorus limitation gave the highest specific activities. At low dilution rates (0.10 to 0.15 h-1), enzyme activities were again low. Sporulation occurred under carbon limitation, but at a lower dilution rate than that which supported optimal gramicidin S synthetase formation. The specific productivity of the synthetases in the chemostat was higher than the highest productivity obtained in batch growth.
The nutrition of the gramicidin S (GS) producer Bacillus brevis ATCC 9999 was studied with respect to growth and antibiotic production. In a complex medium containing yeast extract and peptone, only glycerol, d-fructose, and meso-inositol contributed to growth, whereas other hexoses, pentoses, polyols, polysaccharides, and organic acids were inactive. Glycerol and fructose were inhibitory to GS formation; inositol had no effect. In chemically defined media containing a mixture of five amino acids (glutamine, methionine, proline, arginine, and histidine) necessary for good growth, again only glycerol, d-fructose, and meso-inositol served as effective carbon sources for growth. With respect to antibiotic formation, inositol was much poorer than glycerol or fructose. In the presence of glycerol and the five amino acids, addition of 0.1% l-phenylalanine specifically stimulated GS production. Growth kinetics in this defined medium showed two phases: early and rapid growth at the expense of the amino acid mixture, followed by glycerol utilization after a diauxic lag of 2 to 3 h; inositol utilization suffered a much longer lag period. GS formation was initiated after the diauxic lag. Ammonium sulfate was the best individual nitrogen source, but the combination of the five growth-stimulatory amino acids was more potent for GS production. The level of potassium phosphate influenced GS production. High levels stimulated the growth rate but progressively inhibited GS production. Methionine was the best sulfur source. A defined medium (F3/6) that supported rapid exponential growth (mu = 0.53 h(-1)) followed by progressively slower growth over a 30- to 40-h period was devised containing fructose, six amino acids, and mineral salts. GS formation was initiated as the cells left the exponential growth phase and reached high levels (1,500 mg of GS per liter; 0.19 mg of GS/mg of dry cell weight) after 40 to 50 h of incubation.
Bacillus brevis produces the antibiotic gramicidin S, a cyclic peptide comprised of two pentapeptide units of the sequence d-Phe-l-Pro-l-Val-l-Orn-l-Leu. Synthesis of this antibiotic is carried out by two enzymes, GS synthetases I and II. By supplementing a defined minimal medium [glycerol, NH(4) (+), tris(hydroxymethyl)aminomethane, salts] with each of the constituent amino acids (in the l form), we found that only l-phenylalanine had a marked stimulatory effect on gramicidin S production. This effect was not caused by an increase in growth, by an induction of GS synthetases I or II, or by a stabilization of these enzymes. l-Phenylalanine apparently stimulates production via its role as a precursor of the d-phenylalanine moiety of the gramicidin S molecule.
This report describes an improved procedure for production of cytochalasin E and tremorgens by solid substrate, agitated fermentation of Aspergillus clavatus on pearled barley.
DL-Norleucine, which is known to replace methionine for stimulation of cephalosporin C formation, also mimics methionine's effect on arthrospore formation. Thus, hyphal fragmentation, like antibiotic biosynthesis, is divorced from a sulfur donation role.
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An early blocked sulfur amino acid auxotroph, Cephalosporium acremonium mutant 274-1 (which could be satisfied by methionine or cysteine), utilized organic sulfur compounds for cephalosporin C production in the following order of decreasing effectiveness; methionine > cystathionine > cysteine, despite the fact that cysteine is considered to be the immediate precursor of the antibiotic. When a genetic block was added to mutant 274-1 in the transsulfuration pathway from cysteine to methionine, the double mutant 11-8 (which grows on methonine but not cysteine) failed to produce cephalosporin C from cysteine even though enough methionine was added to support normal growth. Addition of the non-sulfur analogue, norleucine, resulted in antibiotic production from cysteine in the double mutant. These facts support the hypothesis that methionine stimulation of cephalosporin C production is due to a role of methionine other than that of sulfur donation.