The recombination betweenattLon a plasmid andattRon a linear fragment is more efficient (by a factor of two to three) than the reverse situation (Fig.2A and B). chromosome in a site-specific manner: recombination between the attachment sitesattBandattP, located on the bacterial and phage genomes, respectively, is usually catalyzed by a phage-encoded recombinase (integrase) (14,42) and leads to formation of the hybrid attachment sitesattLandattRat the junctions between the phage and bacterial genomes. Prophage excision, which occurs during the induction of lytic growth, involves site-specific recombination between the attachment junctionsattLandattRand requires at least one phage-encoded recombination directionality factor (RDF) or excise, in addition to integrase (32,43). In the modelEscherichia coliphage and its close relatives, the recombination directionality is usually controlled directly by the RDF Xis. The genexisis located upstream from theintgene encoding the integrase. Xis is required for the excision reaction but inhibits integrative recombination (2,41). Xis determines the directionality of recombination by influencing the PD146176 (NSC168807) formation of specific protein-DNA architectures (30). To exert its effect, Xis binds cooperatively with Int and FIS to specific sites, thereby inducing and stabilizing a DNA bend that alters the intasome structure formed during recombination (1,15,44,53). In bacteriophages of the P2 family, site-specific recombination-related genes are organized differently, even though the directionality of recombination seems to be controlled as in phage . The P2 genetic switch region contains two overlapping face-to-face promoters,pcandpe(50). The leftward promoter,pc, produces the lysogenic transcript, including the C repressor and P2 integrase, whereas the rightward promoter,pe, produces the lytic transcript. The first protein encoded by the lytic operon, Cox, is a Cro-like repressor of the promoterpccontrolling lysogeny (50). However, Cox is a multifunctional protein that also acts as a recombination directionality factor (57). Site-specific recombination has been well studied for bacteriophages of Gram-positive bacteria, essentially for bacteriophages ofActinomycetales, such asMycobacteriumphage L5 (36,46-48) andStreptomycesphage C31 (52,55), but also for bacteriophages of lactic acid bacteria (3,10,11,22,49). However, only few RDFs have been characterized to date, for example, those of mycobacteriophage L5 (34,36) and of lactococcal phage TP901-1 (9). The site-specific integration system of bacteriophage mv4 PD146176 (NSC168807) has been characterized previously (6,22). The mv4 integrase belongs to the tyrosine recombinase family and has no absolute requirement for accessory factors, unlike most of the Int family recombinases (4). The mv4 integrase also functions in a wide range of Gram-positive bacteria and inEscherichia coli(6). Furthermore, a site-specific integrative Mouse monoclonal to CD3E vector derived from mv4 is stably maintained in the chromosome of several lactobacilli in the absence of selection, suggesting thatmv4Int cannot catalyze excisive recombination on its own (6). The minimal 234-bpattPsite of mv4 contains five putative integrase arm-binding sites and has a 17-bp core sequence in common with theattBsite (4). During recombination, strand exchange occurs inside the core sequence. TheattBsite is located at the 3 end of a tRNAsergene (22). The minimal size of the functionalattBsite is 16 bp, and no symmetric inverted repeats have been found in the core sequence PD146176 (NSC168807) (5). In this study, we identified the RDF genemv4xisas the second gene of the mv4 early lytic operon. This location is unusual, being reported here for the first time in a temperate bacteriophage harboring a tyrosine recombinase.mv4Xis is a 56-residue basic peptide PD146176 (NSC168807) that acts by binding to two sites withinattRorattPDNA, thereby bending the DNA. The presence ofmv4Xis is absolutely required, together withmv4Int, for excisive recombination between theattLandattRattachment sites. However, in contrast to results for the well-studied systems (, L5, HP1, and P2 phages), preliminary results suggest thatmv4Xis does PD146176 (NSC168807) not inhibit integrative recombination between theattPandattBsites. == MATERIALS AND METHODS == == Bacterial strains, bacteriophage, and growth conditions. == The bacterial strains, bacteriophage, and plasmids used in this study are listed in Table1.Lactobacillusplantarumstrains were grown at 37C in MRS medium (19) or on MRS medium solidified with 1.5% (wt/vol) agar. Erythromycin and chloramphenicol were used at concentrations of 5 g/ml and 10 g/ml, respectively.E.coliwas grown at 37C in Luria-Bertani broth (Difco Laboratories, Detroit, MI) or on LB broth solidified with 1.5% (wt/vol) agar. Antibiotics were used at the following concentrations: 100 g/ml for ampicillin, 150 g/ml for erythromycin, 10 g/ml for chloramphenicol, and 65 g/ml for spectinomycin. ==.