CHARACTERISATION OF STREPTOMYCES CLAVULIGERUS MUTANTS DEFECTIVE IN ?-LACTAM ANTIBIOTIC PRODUCTION

Abstract

Six mutants of Streptomyces clavuligerus (NP3, NP41, NP41/15, NP25, NP52 and NP7) defective in ?-lactam antibiotic production were characterised after nitrosoguanidine mutagenesis. All the mutants examined, except mutant NP52 were unable to produce any detectable levels of ?-lactam antibiotics in both complete media (CM) agar and Trytone Soya Broth (TSB) liquid cultures. Although mutant NP52 was unable to produce ?-lactam antibiotics on solid agar, it could produce about 25-37% of the cephamycin C produced by the wild-type strain in liquid media. The extracted proteins from cell-free extracts of the mutants were subjected to sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to the wild-type strain. The results obtained showed that the protein pofiles of the mutants were essentially the same as the wild-type strain except mutant NP52 which produced a protein of 35 kDa in amounts exceeding those of wild-type and mutant NP7 which produced a higher amount of a protein corresponding to about 150 kDa. Cell-free extracts of the mutants were tested for the activities of the three initial enzymes of the ?-lactam antibiotic biosynthesis pathway viz. ?-aminotransferase (LAT), ?-(L-?-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and Isopenicillin N synthase (IPNS). All the mutant examined suffered partial losses in at least one of the enzyme activities compared to the wild-type strain. Mutant NP3 was severely impaired in both ACVS and IPNS activities and mutants NP41 and NP41/15 had no IPNS activity. The defects and regulatory mechanisms governing the impairment of ?-lactam antibiotic production in these mutants are discussed.