A new type ofβ-lactamase has been isolated and characterized in Streptomyces griseus NRRL B-2682. The enzyme has membrane-bound and extracellular forms. Biochemical characterization of some of the properties of the enzyme showed that it belongs to the class A group of penicillinases. Comparison of the membrane-bound and extracellular forms of theβ-lactamases suggests that they seem to be differently processed forms of the same enzyme. The N-terminal amino acid sequence of the extracellular form of the β-lactamase showed a high degree of similarity to a D-aminopeptidase of another Streptomyces griseus strain. Secretion of the β-lactamase was affected by the differentiation state of the strain since in spontaneous non-sporulating mutants only the membrane-bound form was present. In accordance with this when sporulation of the wild-type strain was inhibited it failed to secrete extracellular β-lactamase. Addition of globomycin to the non-sporulating cells liberated the enzyme from the membrane, indicating that the protein is processed normally by signal peptidase II and a glyceride-thioether group, together with a fatty acid amide-linkage, is responsible for the attachment of the enzyme to the cellular membrane. Under sporulation-repressed conditions addition of peptidoglycan fragments and analogues or inhibition of cell wall biosynthesis by penicillin-G induced β-lactamase secretion and also restored sporulation both in solid and submerged cultures. These results confirm that β-lactamase secretion is tightly coupled to the sporulation process in S. griseus.
Previous studies showed that some lactones have beta-lactamase inhibitory or antibacterial effects, others--like A-factor (a gamma-butyrolactone) and its derivatives--stimulate sporulation in Streptomyces griseus strains. Our experiments were aimed at exploring whether synthetic gamma-lactones had such effects. None of the seven gamma-lactones studied showed antibacterial activity, but two of them inhibited beta-lactamases isolated from various bacteria. These two gamma-lactones did not reduce colony formation of murine bone marrow cells in vitro, indicating that they were not toxic to proliferating mammalian cells. Four gamma-lactones, including the two inhibiting beta-lactamase, stimulated sporulation in the non-sporulating S. griseus bald 7 mutant. Further studies of gamma-lactones as potential inhibitors of beta-lactamase seem to be warranted.
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