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The sre gene (ORF469) of the R4 phage encodes a protein similar to the resolvase-DNA invertase family proteins. Insertional gene disruption of sre prevented a lysogen from entering the lytic cycle, implying that Sre protein is a site-specific recombinase needed for excision of the R4 prophage genome (M. Matsuura, T. Noguchi, T. Aida, M. Asayama, H. Takahashi, and M. Shirai, J. Gen. Appl. Microbiol. 41:53-61, 1995). To determine whether this sre gene is also necessary for the integration reaction, we studied its function by integration plasmid analysis. When deletions, frameshifts, and site-directed mutations that caused an amino acid substitution of Ser-17 for Ala were introduced into the sre structural gene, transformation efficiency of Streptomyces parvulus 2297 with these plasmid DNAs was severely reduced. However, an adenine insertion just before the possible initiation codon of the sre gene did not significantly decrease the efficiency. These data suggest that the Sre protein is a site-specific recombinase responsible for integration of the R4 phage genome.Streptomyces strains are important organisms because they produce a variety of useful secondary metabolites, for example, antibiotics, antitumor reagents, and immunosuppressors (3). R4 is an actinophage that has been studied in depth from the viewpoint of its practical application (12-17). To improve the genetic engineering of Streptomyces strains, site-specific recombination of this R4 phage has been studied (11, 21). Our data suggested that the ORF469 gene (sre) (DDBJ accession no. D38173), which was found adjacent to the attP recombination site of the R4 phage genome, is essential for excision of the prophage genome and probably encodes a site-specific recombinase responsible for the excisive recombination (11). The protein encoded by ORF469 falls not into the integrase family but into the resolvase-DNA invertase family and is highly similar to Tn2501 resolvase and SpoIVCA of Bacillus subtilis (11). The ORF469 protein and SpoIVCA have additional C-terminal halves consisting of about 300 amino acids that typical resolvases do not have, and this kind of recombinase has been also found in Anabaena spp. (XisF) (2,20). In this study, we showed that the protein encoded by the ORF469 gene (sre) is a site-specific recombinase responsible for integration of the R4 phage genome into the Streptomyces parvulus 2297 chromosome.Construction of integration plasmids. To introduce a thiostrepton resistance gene (tsr) (6) as a selection marker into an integration plasmid, a 1.1-kb BclI fragment from pIJ702 containing the tsr gene was inserted by blunt-end ligation into the SmaI site of pAT95 (the SmaI site was in the multicloning site of the pUC18 vector), which contains a 13-kb BclI fragment carrying the ORF469 gene and the attP recombination site from the R4 phage genome (11), to generate pAT96L (data not shown). A 5.6-kb EcoRI-ScaI fragment carrying tsr, ORF469 (sre), and attP was isolated from pAT96L and inserted between the SmaI and EcoRI sites of pUC118 to create ...

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