Plasmid transformation of Streptomyces venezuelae: modified procedures used to introduce the gene(s) for p-aminobenzoate synthase.
Sucrose was unsuitable as an osmotic stabilizer in buffer solutions and media used for transformation of Streptomyces venezuelae ISP5230. Its replacement with NaCl, together with other modifications in the procedure, allowed efficient formation and regeneration of protoplasts but did not support transformation of S. venezuelae ISP5230 by vectors pIJ41 and pIJ941. With pIJ702, transformants with a low plasmid-copy-number and altered growth characteristics were obtained. Both pIJ702 and pIJ941, but not pIJ41, transformed S. venezuelae 13s; when pIJ941 was used, the plasmid in 18 of 20 transformants contained a deletion in the region reported to code for replication and transfer. The modified plasmid transformed S. venezuelae ISP5230 efficiently and was used to introduce a fragment of DNA from the pab locus of the wild-type into a Cml-1 mutant of ISP5230 blocked in chloramphenicol formation. Transformants that overproduced p-aminobenzoic acid were obtained but they remained blocked in chloramphenicol production; thus, the cloned pab fragment did not contain genes able to complement the cml-1 mutation. The results also suggest that the Cml-1 phenotype is not due to a defective reaction common to the biosynthesis of p-aminobenzoic acid and chloramphenicol.
Aidoo
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Biology Department, Dalhousie University, Halifax, Nova Scota, Canada.
Barrett
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Vining
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D010129:4-Aminobenzoic Acid / Q000378:metabolism; D002701:Chloramphenicol / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011523:Protoplasts / Q000378:metabolism; D015183:Restriction Mapping; D013302:Streptomyces / Q000235:genetics*; D013395:Sucrose; D000637:Transaminases / Q000235:genetics*; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D013395:Sucrose; D002701:Chloramphenicol; C022309:PABA synthase; D000637:Transaminases; D010129:4-Aminobenzoic Acid