Cell density influences antibiotic biosynthesis in Streptomyces clavuligerus

Production of cephamycin C and clavulanic acid by Streptomyces clavuligerus took place during the exponential phase of growth in a defined medium. Both antibiotic biosynthetic pathways were activated shortly after spore germination, but the timing and kinetics of activation were affected by inoculum density. Rapid activation was favoured by high inoculum density or by growth in medium conditioned by previous incubation of S. clavuligerus spores or mycelium. A heat-resistant conditioning factor able to accelerate the acquisition of antibiotic-biosynthetic capacity when added to low-density cultures was released in suspensions of spores in water. Conditioning factor was also obtained in suspensions of spores from different Streptomyces species or of Bacillus cells, indicating that the signal was not produced specifically by S. clavuligerus. Fractionation of conditioning factor showed that its effect was not due to a single molecule. The fractions contained amino acids (as free amino acids and oligopeptides) in amounts that roughly correlated with their respective conditioning power. Furthermore, the conditioning effect was reproduced by supplementing defined medium with amino acids and peptides in concentration that mimicked those found in conditioning factor. When individually tested at concentrations in the micromolar range, only some amino acids were able to stimulate antibiotic biosynthetic capacity. This stimulation ws also promoted by low concentrations (less than 1 microgram ml-1) of peptide mixtures obtained with different proteolytic enzymes. The results suggest that both amino acids and peptides are responsible for the effects of conditioning factor released by spores. Possible implications of intercellular signalling on activation of secondary metabolism are discussed.