SummaryRT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000-to 10 000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.

SummaryA protein highly overrepresented in the proteome of Streptomyces clavuligerus oppA2::aph was characterized by MS/MS as a rhodanese‐like enzyme. The rhlA gene, encoding this protein, was deleted from strains S. clavuligerus ATCC 27064 and S. clavuligerus oppA2::aph to characterized the RhlA enzyme activity, growth on different sulfur sources and antibiotic production by the mutants. Whereas total thiosulfate sulfurtransferase activity in cell extracts was not affected by the rhlA deletion, growth, cephamycin C and clavulanic acid production were impaired in the rhlA mutants. Holomycin production was drastically reduced (66–90%) in the rhlA mutants even when using S. clavuligerusΔrhlA pregrown cells, suggesting that this enzyme might be involved in the formation of the cysteine precursor for this sulfur‐containing antibiotic. While growth on thiosulfate as the sole sulfur source was particularly low the volumetric and specific antibiotic production of the three antibiotics increased in all the strains in the presence of thiosulfate. This stimulatory effect of thiosulfate on antibiotic production was confirmed by addition of thiosulfate to pre‐grown cells and appears to be a general effect of thiosulfate on oxidative stress as was also evident in the production of staurosporin by S. clavuligerus.