Strategy for gene replacement in Streptomyces. (A) A gene replacement cassette containing aac ()IV (ApraR), oriT of RK2, and two FRT sites was amplified by PCR by using primers containing 39-nt 5′ homology extensions corresponding to regions flanking the S. coelicolor target gene (dotted lines a and b) and 20-nt 3′ homology to the unique priming sites P1 and P2 of the disruption cassette. (B) The PCR product from A was used to transform E. coli BW25113/pIJ790 (expressing the λ-Red recombination functions), containing the Supercos-1 cosmid with the cloned target gene (cyc2). (C) ApraR transformants were selected, and the recombinant cosmid was identified by PCR and restriction analysis. (D) The potent methyl-specific restriction system of S. coelicolor was circumvented by introducing the recombinant cosmid into the methylation-deficient E. coli host ET12567/pUZ8002. The cosmid was mobilized in trans by pUZ8002 to Streptomyces by conjugation. (E) Selected exconjugants (ApraR) were screened for KanS (loss of the Kan resistance gene neo), and the double cross-over allelic exchange was confirmed by PCR and Southern blot analysis. (F) E. coli DH5α cells containing pCP20 were transformed with the mutagenized cosmid DNA, and FLP synthesis was induced. (G) The loss of the disruption resistance marker was tested, and the cosmid was introduced by polyethylene glycol (PEG)-mediated transformation into the S. coelicolor mutant carrying a marked deletion within the gene of interest. (H) KanR transformants arising from single cross-over events were restreaked nonselectively and screened for the loss of both KanR and ApraR, indicating a successful replacement by the in-frame deletion marked only by a “scar” sequence of 81 bp containing priming sites P1 and P2 (underlined), and one FRT site.