Integration of bacteriophage Mx8 into the Myxococcus xanthus chromosome causes a structural alteration at the C-terminal region of the IntP protein

Abstract

Mx8 is a generalized transducing phage that infects Myxococcus xanthus cells. This phage is lysogenized inM. xanthus cells by the integration of its DNA into the host chromosome through site-specific recombination. Here, we characterize the mechanism of Mx8 integration into the M. xanthus chromosome. The Mx8 attachment site, attP, the M. xanthus chromosome attachment site, attB, and two phage-host junctions, attL and attR, were cloned and sequenced. Sequence alignments of attP, attB, attL, and attR sites revealed a 29-bp segment that is absolutely conserved in all four sequences. The intP gene of Mx8 was found to encode a basic protein that has 533 amino acids and that carries two domains conserved in site-specific recombinases of the integrase family. Surprisingly, the attP site was located within the coding sequence of the intP gene. Hence, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the intP gene to a new gene designated intR. As a result of this conversion, the 112-residue C-terminal sequence of the intP protein is replaced with a 13-residue sequence. A 3-base deletion within the C-terminal region had no effect on Mx8 integration into the chromosome, while a frameshift mutation with the addition of 1 base at the same site blocked integration activity. This result indicates that the C-terminal region is required for the enzymatic function of the intP product.Myxococcus xanthus is a unique gram-negative bacterium living in soil. M. xanthus cells can undergo multicellular development involving cell-to-cell interactions (for a review, see reference 8). Upon nutritional starvation on a solid surface, cells aggregate to form mounds called fruiting bodies within which rod-shaped cells are converted into spherical or ovoid myxospores.Several bacteriophages that infect M. xanthus cells are known (17). Myxophage Mx8 is a generalized transducing phage of M. xanthus (22). Purified phage particles have 56-kb linear double-stranded DNA molecules with an average terminal redundancy of 4.3 kb (31). Restriction analyses showed that Mx8 phage DNA is circularly permuted (see Fig. 1A). This phage can be lysogenized in M. xanthus cells by integrating its DNA into the host chromosome through site-specific recombination between the attP site on the phage DNA and the attB site on the host chromosome (4, 25). This recombination system has been used to introduce recombinant plasmids into the M. xanthus chromosome, since various plasmids containing a fragment of Mx8 DNA have been shown to stably integrate into the chromosomal attB site (15,29,31). In spite of the effectiveness and wide utilization of the Mx8 attP-mediated integration of plasmids, the integration mechanism is not well understood at present.To reveal the mechanism of Mx8 site-specific recombination in M. xanthus, we analyzed the intP-attP region of Mx8 phage.Comparison of attP, attB, attL, and attR sequences revealed a 29-bp segment that is absolutely conserved. The Mx8 intP gene was shown to encode a basic protein with 533 amino a...

The IntP C-Terminal Segment Is Not Required for Excision of Bacteriophage Mx8 from theMyxococcus xanthusChromosome

“…P1clr100 Cm was used for the introduction of various plasmids from E. coli into M. xanthus cells (22). The Mx8 TM1 strain was from laboratory stock (25). pMC1403 and pSI1403 (3,11) were used as vectors.…”

“…pMC1403 and pSI1403 (3,11) were used as vectors. Various segments of intP-attP and intR-attR were obtained from pMP001 and pMP004, respectively (25). pP1inc and pMXL101 (24) were also used.…”

“…As a result, the 112-residue C-terminal segment of the IntP protein is replaced by a 13-residue sequence of the IntR protein after Mx8 integration. The entire region of the intP gene, including the convertible C-terminal segment, was shown to be essential for the integration of Mx8 into the M. xanthus chromosome (25).…”

“…In a previous study (25), we have investigated the mechanism of Mx8 integration into the M. xanthus chromosome. The Mx8 attP site, M. xanthus chromosome attB site, and attL and attR phage-host junctions were cloned and sequenced.…”

Lysogenic bacteriophage M1 from Selenomonas ruminantium: isolation, characterization and DNA sequence analysis of the integration site

“…In most bacteriophages, site-specific recombination functions (attP, int and xis) are tightly clustered on a relatively small stretch of DNA (Tojo et at., 1996). Therefore, we sequenced the cloned 2.3 kb EcoRIEcoRV fragment which includes the predicted attP site (Fig.…”