End-product control of expression of branched-chain amino acid biosynthesis genes in Streptomyces coelicolor A3(2): paradoxical relationships betweenDNA sequence and regulatory phenotype

The branched-chain protein amino acids isoleucine, valine and leucine can p rovide precursors for synthesis of complex polyketide secondary metabolites in streptomycetes; therefore the regulation of their own synthesis is of i nterest. DNA sequences upstream of ilvBNC. ilvD, leuA, leuB, ilvE and leuCD in Streptomyces coelicolor A3(2) have been obtained in this laboratory or as part of the S. coelicolor genome sequencing project. Upstream of ilvB an d leuA, typical features of classical attenuator systems can be discerned, in particular hypothetical short ORFs with runs of Ile/Val/Leu and Leu codo ns, respectively. No such features are apparent upstream of other genes or gene clusters present. All five upstream regions were fused to xylE (encodi ng catechol dioxygenase. CO) as a reporter gene in the SCP2*-based low-copy -number vector pIJ2839. All wild-type regions showed strong depression of C O activity in the presence of all three branched-chain amino acids whether or not the attenuation features were present. By site-directed mutagenesis, the Ile/Val/Leu and Leu triplets in the putative attenuator peptides for i lvB and leuA were replaced by ones for other amino acids. In the case of il vB, this had no effect at all; for leuA, the wild-type regulatory phenotype persisted in at least some experiments. It was concluded that (i) an unkno wn regulatory mechanism must be operating in the ilv/leu system of S. coeli color A3(2) in place of classical attenuation; and (ii) it is unsafe to inf er the functioning of a regulatory mechanism from sequence homologies alone .