A PATHWAY-SPECIFIC TRANSCRIPTIONAL ACTIVATOR REGULATES LATE STEPS OF CLAVULANIC ACID BIOSYNTHESIS IN STREPTOMYCES-CLAVULIGERUS

A Streptomyces clavuligerus gene (designated claR) located downstream from the gene encoding clavaminate synthase in the clavulanic acid bio synthetic gene cluster is involved in regulation of the late steps in clavulanic acid biosynthesis. Nucleotide sequence analysis and databas e searching of ClaR identified a significant similarity to the helix-t urn-helix motif (HTH) region of LysR transcriptional regulators. A gen e replacement mutant disrupted in claR was unable to produce clavulani c acid, suggesting that claR is essential for clavulanic acid biosynth esis. Furthermore, the accumulation ration of clavaminic acid in the c laR mutant suggested that ClaR regulates the late steps in the clavula nic acid pathway, i.e. those involved in the conversion of clavaminic acid to clavulanic acid. Transcriptional analysis using RNA isolated f rom the wild type and the claR mutant showed that the expression of th e putative late genes, but not the early genes, was regulated by ClaR. High-resolution S1 nuclease analysis of claR suggested that it is exp ressed as a monocistronic transcript and also as a bicistronic transcr ipt along with the late gene orf-9. The transcription start site of th e monocistronic claR transcript was identified as a C residue 155 nucl eotides upstream from the claR start codon.