Cloning and expression in Escherichia coli of a Streptomyces coelicolor A3(2) argCJB gene cluster.
From a partial Sau3AI 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Hindle
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Department of Genetics, University of Leeds, UK.
Callis
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Dowden
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Rudd
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Baumberg
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D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics*; D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D051046:Amino-Acid N-Acetyltransferase; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D005810:Multigene Family; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D000445:Aldehyde Oxidoreductases; C018722:N-acetyl-gamma-glutamyl-phosphate reductase; D000123:Acetyltransferases; D051046:Amino-Acid N-Acetyltransferase; C020646:glutamate acetyltransferase