Replacement vectors for localized gene cloning in the specified region of Streptomyces lividans 66 and model method for the screening of desired recombinants via counter-selection.
Two DNA fragments (3.5 kb and 3.8 kb in size) flanking both ends of the phi HAU3R gene in the genome of Streptomyces lividans 66 were determined and cloned in their natural relative orientations in pIJ653, a cosmid vector derived from the multi-copy Streptomyces plasmid pIJ101, resulting in pHZ806. After insertion of spectinomycin/streptomycin (spc/str) resistance gene into the pIJ101 replication region in pHZ806 and insertion of a hygromycin (hyg) resistance gene between 3.5 kb and 3.8 kb DNA fragments, a new vector with a non-functional Streptomyces replicon, pHZ808, was obtained. In principle, any DNA fragment cloned between 3.5 kb and 3.8 kb fragments of this vector can be stably integrated between the two corresponding regions after introduction into the wild-type S.lividans strains, with synchronous replacement of the DNA between the two regions of the chromosome within which phi HAU3R gene is located. The resultant recombinant strains will thus become phi HAU3-sensitive (phi HAU3S). This phenotype could serve as a good indication that the desired gene replacement has occurred. This principle was demonstrated to be successful using pHZ808 as vector. phi HAU3-resistance gene (phi HAU3R) from the genome of S.lividans 66 was substituted by the hygromycin resistance gene (hyg) from pHZ808. An additional advantage of using pHZ808 as the vector to clone foreign genes is that hyg could be served as a reportor gene to imply that foreign DNA fragment has been co-integrated with hyg. The recombinants will have both hygR and phi HAU3S.
Zhou
X
X
Huazhong Agricultural University, Wuhan, China.
Bai
L
L
Deng
Z
Z
Tobias
K
K
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002876:Chromosomes, Bacterial / Q000235:genetics; D002934:Cinnamates; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D005798:Genes, Bacterial; D005818:Genetic Engineering; D005822:Genetic Vectors; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013302:Streptomyces / Q000235:genetics*
D000900:Anti-Bacterial Agents; D002934:Cinnamates; D006921:Hygromycin B; C026273:hygromycin A