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The DNA fragment encoding the integrase and excisionase genes involved in site-specffic recombination of staphylococcal bacteriophage +11 was cloned and sequenced. The int and xis genes and the recombination site, attP, were highly clustered in a 1.7-kilobase DNA fragment with the gene order attP-int-xis. The int and xis genes were transcribed divergently, with the int gene transcribed toward the attp site and the xis gene transcribed away from the attP site. The deduced Int is a basic protein of 348 residues with an estimated molecular weight of 41,357. In contrast, the deduced Xis is an acidic protein containing 66 amino acids with an estimated molecular weight of 7,621. The site-specific recombination system of +11 was compared with that of a closely related bacteriophage, L54a.Staphylococcal bacteriophage 411 is a group B temperate bacteriophage originally isolated from Staphylococcus aureus 8325 (27). It is the best genetically characterized among all staphylococcal bacteriophages. The 411 DNA is a linear, double-stranded, circularly permuted, and terminally redundant molecule of about 45 kilobases (kb) (1,4,23,24). The extent of terminal repetition is about 5% of the genome length (25). The circular genetic and restriction maps have been established (2,17,25). As a temperate bacteriophage, 411 is capable of both lytic and lysogenic life cycles. During lysogeny, the viral DNA recombines with the host chromosome between the viral attachment site (attP) and the bacterial attachment site (attB). Integration generates two additional sites, attR and attL, at the right and left junctions between the prophage and the host chromosome, respectively. The attB site is located between the purB and metA loci (30). Recently, DNA sequencing revealed that all four att sites shared a 10-base-pair (bp) common core sequence (21).We have previously studied the site-specific recombination system of another staphylococcal bacteriophage, L54a (18,21,37). 411 and L54a are closely related phages; they belong to the same serogroup B and lytic group III, they are homoimmune, and their DNAs cross-hybridize extensively (25; unpublished data). Integration and excision of L54a follow the same model as that of 411. The att sites of L54a also share a short (18-bp) core sequence. The core sequences of the attachment sites of both phages have 6 bp of nucleotide homology. In addition, an 11-bp direct repeat with four repetitions in the 411 attP site is arranged similarly to that of a 12-bp direct repeat in the L54a attP site (20,21). Thus, the site-specific recombination systems of both phages may be machanistically similar. However, the two phages differ in their bacterial attachment sites. To compare the two systems at the molecular level and to begin to understand the molecular nature of the specificity, we cloned and sequenced two 411 recombination genes, int and xis, which are located adjacent to the attP site. We have previously isolated, mapped, and sequenced the recombination genes of L54a (20, 37). Here we compare the site-specific r...

We characterized the minimum length of the DNA sequence of the attachment sites involved in the integrative recombination of staphylococcal bacteriophage L54a. A DNA fragment carrying the functional viral attachment site (aiP) or the bacterial attachment site (altO) was sequentially trimmed, redoned, and tested for integrative recombination in vivo. The size of the functional aitd site was at least 228 base pairs (bp) but no more than 235 bp. The left endpoint of the aIdP site was located to between positions -142 and -140, whereas the right endpoint was located to between positions +86 and +93 with respect to the center of the core sequence. The atOB site was located to within a 27-bp sequence, from position -15 to + 12, which included the 18-bp core sequence.Staphylococcal bacteriophage L54a is a temperate phage that was originally discovered as one of the two prophages in Staphylococcus aureus PS54 (4). Its genome size is about 45 kilobases (kb), and the genome is circularly permuted (unpublished data). During lysogeny, bacteriophage L54a inserts its genome into the host chromosome. Integrative recombination occurs between the specific viral attachment site (attP) on the bacteriophage genome and the attL54a site (attB) on the host chromosome. As a result of integration, two additional attachment sites, attL and attR, are generated on the left and the right sides of the prophage genome, respectively. In excision, attL and attR recombine and regenerate attB and attP. The attB site is located at the 3' end of the lipase (geh) structural gene. As a consequence, lysogens of L54a are lipase negative because of insertional inactivation of the lipase structural ;ene (11,12). Curing of the prophage restores the lipase activity of the host. We have previously cloned and sequenced the DNA fragments containing the four attachment sites (13). All four att sites share an identical 18-base-pair (bp) core sequence. Apparently, recombination requires the core sequence. However, the question as to how much of the DNA sequence outside the core sequence is required for the function of the attachment sites attP and attB remains unanswered. Here we report the minimum sizes of the attB and attP sites. MATERIALS AND METHODSPhage and bacterial strains. The isolation and propagation of staphylococcal bacteriophage L54a were done as described previously (11). S. aureus RN4220 (10), a restrictiondeficient variant of S. aureus 8325-4, was used as the recipient in protoplast transformations. Protoplast transformation was performed by the procedure of Chang and Cohen (2). Staphylococcal bacteriophage transductions were performed at a multiplicity of infection of 0.1, as described previously (25). Escherichia coli LE392 was used for transformation with cloned DNA and for the preparation of plasmid DNA.Chenmcals and media. Media used for routine cultivation and detection of lipase activity have been described previously (11 Recombinant DNA methods. General DNA methods were carried out as described by Maniatis et al. (15). Rapid small-scale D...