Cloning and expression in Escherichia coli of a Streptomyces coelicolor A3 (2) argCJB gene cluster

Department of Genetics, University of Leeds, Leeds LS2 9JT, UK Glaxo Group Research Ltd, Greenford Road, Greenford, Middlesex UB6 0HE, UK ABSTRACT Summary: From a partial Sau 3AI library of Streptomyces coelicolor A3(2) DNA in pIJ916, two hybrid plasmids pGX1 and pGX2 were isolated that complemented S. coelicolor A3(2) or S. lividans arginine auxotrophs. Subcloning DNA from pGX1 in the Escherichia coli expression vector pRK9 containing the Serratia marcescens trp promoter gave rise to one plasmid, pZC2, that complemented E. coli argB, C, E and H auxotrophs, and another, pZC1, that complemented only the first three. The plasmids were markedly unstable in the various complemented hosts, to varying extents; pZC1 was characterized further as providing the stablest host/plasmid combinations. In vitro deletion of part of the vector's trp promoter did not affect complementation of the argB and C auxotrophs, implying that the S. coelicolor A3(2) arg genes may be expressed from their own promoter. The trp promoter-less plasmids included isolates, such as pZC177, that had suffered extensive further deletion without loss of complementing ability. Extracts of an E. coli argE auxotroph carrying pZC177 showed ornithine acetyltransferase activity, indicating that the complementing gene is of the argJ type. The complementation properties of in vitro deletion derivatives of pZC177 indicated the gene order argC-J-B. Part of argC and the upstream region were sequenced; an ORF was identified whose predicted product showed appreciable homology with the E. coli and Bacillus subtilis ArgC polypeptide. Upstream of this ORF a consensus-type promoter and ribosome binding site could be discerned; overlapping its promoter was a sequence with homology to arginine operators in these two other organisms. An in vitro frameshift in argC had a polar effect on expression in E. coli of argJ and B , suggesting that the three genes are transcribed in the same direction, possibly as an operon. Author for correspondence: Simon Baumberg. Tel: +44 532 333080. Fax: +44 532 441175. Keywords: Streptomyces coelicolor , argCJB gene cluster, cloning, expression, arginine auxotrophy Present address: Department of Biochemistry and Applied Molecular iology, UMIST, Manchester M60 1QD, UK. he EMBL accession number for the sequence reported in this paper is 66783.