Nutrition of Bacillus brevis ATCC 9999, the Producer of Gramicidin S

Abstract

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, 1)-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, ]-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% Lphenylalanine specifically stimulated GS production. Growth kinetics in this defined medium showed two phases: early and rapid growth at the expense ofthe 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 (, = 0.53 h-') 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.The biosynthesis of the cyclic decapeptide antibiotic gramicidin S (GS) by certain strains of Bacillus brevis has been the subject of extensive research in the last decade (15,16,18). These studies have led to the discovery of a nonribosomal process for peptide biosynthesis. GS, which consists of two identical pentapeptides (DPhe-LPro-LVal-LOrn-LLeu), is synthesized by two enzymes in the presence of adenosine 5'-triphosphate and Mg2+. Synthetase II (molecular weight, 100,000) activates and racemizes L-phenylalanine, whereas synthetase I (molecular weight, 280,000) activates L-proline, L-valine, L-ornithine, and ileucine. In sequence, each amino acid is translocated from its I Contribution no.

Fermentation and Cost-Effective 13 C/ 15 N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

“…As the next step, we therefore compared urea, ammonium chloride, and ammonium sulfate as a nitrogen source in a glycerol-containing minimal medium for A. migulanus strain DSM 5759. Only ammonium sulfate was able to support GS production, in agreement with previous observations (22) for the type strain ATCC 9999. Notably, the authors reported a yield of 100 mg/liter GS with 12 g/liter ammonium sulfate, but we also checked results with 10 and 5 g/liter and obtained the same 100-mg/liter yield independent of concentration.…”

“…Therefore, glucose-containing minimal media, which are generally used for uniform labeling, cannot be employed to produce GS. A. migulanus can utilize only fructose, glycerol, or inositol as a single carbon source (22); hence, only these substrates in the isotope-enriched form can be used. In the chemically defined medium with glycerol, the best nitrogen sources are ammonium phosphate, ammonium sulfate (22), or ammonium succinate (21).…”

“…A. migulanus can utilize only fructose, glycerol, or inositol as a single carbon source (22); hence, only these substrates in the isotope-enriched form can be used. In the chemically defined medium with glycerol, the best nitrogen sources are ammonium phosphate, ammonium sulfate (22), or ammonium succinate (21). However, these salts, especially ammonium sulfate, increase the acidity of the medium, which could be a reason for the low GS yields noted earlier (36).…”

Manipulation of the tyrothricin production profile of Bacillus aneurinolyticus

“…The diauxic growth observed in the cultures supplemented with amino acids is similar to that observed by Vandamme & Demain (1976) in B. brevis ATCC 9999, the GS producer strain. They found B. brevis initially grew at the expense of amino acids supplemented in the growth medium; only after a diauxic lag was the available carbon source utilized and antimicrobial peptide production initiated.…”

Bacillus spp.; perspectiva de su efecto biocontrolador mediante antibiosis en cultivos afectados por fitopatógenos

“…El glicerol y la D-fructosa mostraron ser inhibidores de la producción de GS, de igual forma se observó que aminoá-cidos especialmente la fenilalanina, así como otros sustratos permiten fases aceleradas en la cinética de crecimiento y producción del antimicrobiano (16).…”