Connecting primary and secondary metabolism: AreB, an IclR-like protein, binds the ARE(ccaR) sequence of S. clavuligerus and modulates leucine biosynthesis and cephamycin C and clavulanic acid production.

A protein binding to the autoregulatory element (ARE) upstream of the regulatory ccaR gene of Streptomyces clavuligerus was isolated previously by DNA affinity binding. The areB gene, encoding this protein, is located upstream and in opposite orientation to the leuCD operon of S. clavuligerus; it encodes a 239-amino-acid protein of the IclR family with a helix-turn-helix motif at the N-terminal region. An areB-deleted mutant, S. clavuligerusDeltaareB, has been constructed by gene replacement. This strain requires leucine for optimal growth in defined media. Expression of the leuCD operon is retarded in S. clavuligerusDeltaareB, because AreB binds the areB-leuCD intergenic region acting as a positive modulator. Clavulanic acid and cephamycin C production are improved in the DeltaareB mutant although no drastic difference in ccaR expression was observed. Pure recombinant AreB protein does not bind the ARE(ccaR) sequence (as shown by EMSA) unless filtered extracts from S. clavuligerus ATCC 27064-containing molecules of Mr lower than 10 kDa are added to the binding reaction. Restoration of binding to the ARE(ccaR) sequence is not observed when filtered extracts are obtained from the DeltaareB mutant, suggesting that biosynthesis of the small-molecular-weight effector is also controlled by AreB.