title: Cloning and expression of a p-aminobenzoic acid synthetase gene of the candicidin-producing Streptomyces griseus.
abstract: 4.5-kb BamHI fragments of DNA coding for p-aminobenzoic acid (PABA) synthetase from the candicidin-producing Streptomyces griseus IMRU 3570 and from a sulphonamide resistant mutant of it were cloned on the plasmid vector pIJ41 into Streptomyces lividans 66. The cloned DNA restored prototrophy to a pab auxotroph of S. lividans; when the S. griseus donor was a sulphonamide resistant, PABA-overproducing mutant, the S. lividans clone was sulphonamide resistant as well as Pab+. Sub-cloning the 4.5-kb fragment of S. griseus DNA into Escherichia coli pabA- or pabB- mutants by insertion at the BamHI site of pBR322 did not yield prototrophic clones directly. However, when the cloned fragment had the proper orientation relative to the tet promoter, but not the opposite one, it was possible to select Pab+ colonies, which arose by deletion in vivo of approx. 1 kb of the S. griseus inserted DNA. These results, and those of studies in which Tn5 abolished the Pab+ phenotype by insertion in vivo in the tet promoter or downstream of it, indicated that the S. griseus pab promoter was not expressed in E. coli but that the pab gene could be expressed by transcriptional readthrough from the vector. Experiments in which the cloned DNA was transferred back from E. coli to S. lividans suggested, but did not prove, that the Streptomyces pab promoter had been deleted by loss of the approx. 1-kb segment. These experiments showed expression of both the tet (of pBR322) and kan (of Tn5) promoters in S. lividans.
mesh_terms: D000935:Antifungal Agents / Q000096:biosynthesis*; D002174:Candicidin / Q000096:biosynthesis*; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D014176:Protein Biosynthesis; D013305:Streptomyces griseus / Q000235:genetics* / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D000935:Antifungal Agents; D013449:Sulfonamides; D002174:Candicidin; C022309:PABA synthase; D000637:Transaminases