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 concentrations 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 was also promoted by low concentrations (less than 1 μg 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.
BascaránV., SánchezL., HardissonC., BrañaA.F. Stringent response and initiation of secondary metabolism in Streptomyces clavuligerus. J Gen Microbiol1991; 137:1625–1634
BradfordM.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem1976; 72:248–254
BushellM.E., FrydayA. The application of materials balancing to the characterization of sequential secondary metabolite formation in Streptomyces cattleya NRRL 8057. J Gen Microbiol1983; 129:1733–1741
Fernández MorenoM.A., Martín TrianaA.J., MartínezE., NiemiJ., KieserH.M., HopwoodD.A., MalpartidaF. aba A, a new pleiotropic regulatory locus for antibiotic production in Streptomyces coelicolor. J Bacterioi1992; 174:2958– 2967
FoulstoneM., ReadingC. Assay for amoxicillin and clavulanic acid, the components of augmentin, in biological fluids with high performance liquid chromatography. Antimicrob Agents Chemother1982; 22:753–762
FuquaW.C., WinansS.C., GreenbergE.P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol1994; 176:269–275
GeistlichM., LosickR., TurnerJ.R., RaoR.N. Characterization of a novel regulatory gene governing the expression of a polyketide synthase gene in Streptomyces ambofaciens. Mol Microbiol1992; 6:2019–2029
HillD.W., WaltersF.M., WilsonD., StuartJ.D. High performance liquid chromatographic determination of amino acids in the picomole range. Anal Chem1979; 51:1338–1341
HobbsG., FrazerC.M., GardnerD.C.J., FlettF., OliverS.G. Pigmented antibiotic production by Streptomyces coelicolor A3(2): kinetics and the influence of nutrients. J Gen Microbiol1990; 136:2291–2296
HorinouchiS., BeppuT. Regulation of secondary metabolism and cell differentiation in Streptomyces: A-factor as a microbial hormone and the AfsR protein as a component of a two-component regulatory system. Gene1992a; 115:167–172
IshizukaH., HorinouchiS., KieserH.M., HopwoodD.A., BeppuT. A putative two-component regulatory system involved in secondary metabolism in Streptomyces spp. J Bacteriol1992; 174:7585–7594
LavilleJ., VoisardC., KeelC., MaurhoferM., DgfagoG., HaasD. Global control in Pseudomonas fluorescens mediating antibiotic synthesis and suppression of black root rot of tobacco. Proc Natl Acad Sci USA1992; 89:1562–1566
MalpartidaF., HopwoodD.A. Physical and genetic characterization of the gene cluster for the antibiotic actinorhodin in Streptomyces coelicolor. Mol Gen Genet1986; 205:66–73
MatsumotoA., HongS.-K., IshizukaH., HorinouchiS., BeppuT. Phosphorylation of the AfsR protein involved in secondary metabolism in Streptomyces species by a eukaryotic-type protein kinase. Gene1994; 146:47–56
NarvaK.E., FeitelsonJ.S. Nucleotide sequence and transcriptional analysis of the redD locus of Streptomyces coelicolor A3(2). J Bacteriol1990; 172:326–333
O'SullivanJ., AplinR.T., StevensC.M., AbrahamE.P. Biosynthesis of a 7-a-methoxycephalosporin. Incorporation of molecular oxygen. Biochem J1979; 179:47–52
PeregoM., HigginsG.F., PearceS.R., GallagherM.P., HochJ.A. The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation. Mol Microbiol1991; 5:173–185
PiepersbergW., DistlerJ., EbertA., HeinzelP., MansouriK., MayerG., PissowotzkiK. Expression of genes for streptomycin biosynthesis. In Biology of Actinomycetes ’881988 Edited by OkamiY., BeppuT., OgawaraH. Tokyo: Japan Scientific Societies Press; pp 86–91
RudnerD.Z., Le DeauxJ.R., IretonK., GrossmanA.D. The spoOK locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence. J Bacteriol1991; 173:1388–1398
Stutzman-EngwallK.J., OttenS.L., HutchinsonC.R. Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J Bacteriol1992; 174:144–154
WilleyJ., SchwedockJ., LosickR. Multiple extracellular signals govern the production of a morphogenetic protein involved in aerial mycelium formation by Streptomyces coelicolor. Genes Dev1993; 7:895–903