Intergeneric conjugation in Streptomyces peucetius and Streptomyces sp. strain C5: chromosomal integration and expression of recombinant plasmids carrying the chiC gene.
Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the phiC31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of phiC31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.
Paranthaman
Senthamaraikannan
S
Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India.
Dharmalingam
Kuppamuthu
K
D001287:Attachment Sites, Microbiological / Q000235:genetics; D001426:Bacterial Proteins; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002688:Chitinases / Q000235:genetics*; D002876:Chromosomes, Bacterial / Q000235:genetics; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000145:classification / Q000235:genetics*; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D002688:Chitinases; C101644:chitinase C-1