J. Bacteriol., Nov 1996, 6266-6274, Vol 178, No. 21
Copyright © 1996, American Society for Microbiology

Molecular analysis of a beta-lactam resistance gene encoded within the cephamycin gene cluster of Streptomyces clavuligerus

AS Paradkar, KA Aidoo, A Wong and SE Jensen
Department of Biological Sciences, University of Alberta, Edmonton, Canada.

A Streptomyces clavuligerus gene (designated pcbR) which is located immediately downstream from the gene encoding isopenicillin N synthase in the cephamycin gene cluster was characterized. Nucleotide sequence analysis and database searching of PcbR identified a significant similarity between PcbR and proteins belonging to the family of high- molecular-weight group B penicillin-binding proteins (PBPs). Eight of nine boxes (motifs) conserved within this family of proteins are present in the PcbR protein sequence in the same order and with approximately the same spacing between them. When a mutant disrupted in pcbR was constructed by gene replacement, the resulting pcbR mutant exhibited a significant decrease in its resistance to benzylpenicillin and cephalosporins, indicating that pcbR is involved in beta-lactam resistance in this organism. Western blot (immunoblot) analysis of S. clavuligerus cell membranes using PcbR-specific antibodies suggested that PcbR is a membrane protein. PcbR was also present in cell membranes when expressed in Escherichia coli and was able to bind radioactive penicillin in a PBP assay, suggesting that PcbR is a PBP. When genomic DNAs from several actinomycetes were probed with pcbR, hybridization was observed to some but not all beta-lactam-producing actinomycetes.

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