Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a "supercluster" in Streptomyces clavuligerus. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy-based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late, clavulanic acid-specific steps in the pathway.Streptomyces clavuligerus produces an array of ␤-lactam compounds including cephamycin C, clavulanic acid, and at least four other clavam metabolites. Clavulanic acid has considerable chemotherapeutic and economic value because of its ␤-lactamaseinhibitory activity. In contrast, the other clavam metabolites are ineffective as ␤-lactamase inhibitors. These clavam metabolites have the same nuclear structure, a fused bicyclic ␤-lactam-oxazolidine ring system, as does clavulanic acid. However, the stereochemistry of the ring system is 5R in the clavams rather than 5S as in clavulanic acid, and the clavam metabolites also have different side chain substituents at C-2 (Fig.

SummaryA Streptomyces clavuligerus gene (designated claR ) located downstream from the gene encoding clavaminate synthase in the clavulanic acid biosynthetic gene cluster is involved in regulation of the late steps in clavulanic acid biosynthesis. Nucleotide sequence analysis and database searching of ClaR identified a significant similarity to the helix-turn-helix motif (HTH) region of LysR transcriptional regulators. A gene replacement mutant disrupted in claR was unable to produce clavulanic acid, suggesting that claR is essential for clavulanic acid biosynthesis. Furthermore, the accumulation of clavaminic acid in the claR mutant suggested that ClaR regulates the late steps in the clavulanic acid pathway, i.e. those involved in the conversion of clavaminic acid to clavulanic acid. Transcriptional analysis using RNA isolated from the wild type and the claR mutant showed that the expression of the putative late genes, but not the early genes, was regulated by ClaR. High-resolution S1 nuclease analysis of claR suggested that it is expressed as a monocistronic transcript and also as a bicistronic transcript along with the late gene orf-9. The transcription start site of the monocistronic claR transcript was identified as a C residue 155 nucleotides upstream from the claR start codon.

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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 ␤-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 ␤-lactam-producing actinomycetes.

Mutagenesis of Streptomyces wenezuelae ISP5230 and selection for paminobenzoic acid-dependent growth in the presence of sulfanilamide yielded pab mutants 01551 9 and VS620) that continued to produce chloramphenicol (Cm), although with increased medium dependence. Transforming the mutants with pDQlO2 or pDQ103, which carried a pab-complementing fragment from 5. venezuelae ISP5230 in alternative orientations, restored uniformly high Cm production in VS620, but did not alter the medium dependence of Cm production in VS519. The cloned S. wenezuelae DNA fragment was subcloned and trimmed to the minimum size conferring pab complementation. The resulting 2.8 kb BamHI-Sac1 fragment was sequenced. Codon preference analysis showed one complete ORF encoding a polypeptide of 670 amino acids. Comparison of the deduced amino acid sequence with database proteins indicated that the N-and C-terminal regions resembled PabA and PabB, respectively, of numerous bacteria. The gene product showed overall sequence similarity to the product of a fused pabAB gene associated with secondary metabolism in Streptomyces griseus. Insertion of an apramycin resistance gene into pabAB cloned in a segregationally unstable vector and replacement of the S. wenezuelae chromosomal pabAB with the disrupted copy lowered sulfanilamide resistance from 25 to 5 pg ml-l and blocked Cm production.