Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences.
We have developed a method for gene replacement in Streptomyces hygroscopicus which permits introduction of an in vitro derived mutation carried on a plasmid into the chromosome. We constructed the plasmid pMSB212 which can replicate in S. hygroscopicus and contains the step5 gene of the bialaphos biosynthetic pathway which was inactivated by a frame-shift mutation caused by filling in the cohesive ends of the EcoR I site in the structural gene. pMSB212 was introduced into a bialaphos producer strain and by protoplast regeneration of the primary thiostrepton-resistant transformants, non-producing mutants, were obtained. Biochemical and genetical analyses indicated that these mutants were specifically blocked by introduction of the frame-shift mutation in the step5 gene on the chromosome. This method will enable us to obtain isogenic mutants of known genes and to identify new genes encoded on a cloned fragment.
Anzai
H
H
Pharmaceutical Research Laboratories, Meiji Seika Kaisha, Ltd., Yokohama, Japan.
Kumada
Y
Y
Hara
O
O
Murakami
T
T
Itoh
R
R
Takano
E
E
Imai
S
S
Satoh
A
A
Nagaoka
K
K
D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D009943:Organophosphorus Compounds / Q000096:biosynthesis*; D010957:Plasmids; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D009943:Organophosphorus Compounds; C003122:bialaphos