Myxococcus xanthus contains two genes (lonV and lonD) homologous to the Escherichia coli Ion gene for an ATP-dependent protease. We found that the lonD gene encodes a 90-kDa protein consisting of 827 amino acid residues. The lonD gene product shows 49, 48, and 52% sequence identity to the products of the M. xanthus lonV, E. coil Ion, and Bacillus brevis Ion genes, respectively. When a lonD-lacZ fusion was used, lonD was expressed during both vegetative growth and development. However, while lonD-disrupted strains were able to grow normally vegetatively, the development ofM. xanthus was found to be arrested at an early stage in these strains. The mutant strains were able to form neither fruiting bodies nor myxospores.
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 lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.
During lysogenization of myxophage Mx8, phage DNA can be integrated into the attB site of the Myxococcus xanthus chromosome through site-specific recombination. We previously demonstrated that the Mx8 attP site is located within the coding sequence of the Mx8 intP gene. Hence, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the 112-amino-acid C-terminal segment of the IntP protein into a 13-amino-acid C-terminal segment of a new protein, IntR. To examine whether IntR is active for Mx8 excision, we have constructed a series of plasmids carrying various lengths of the intP-attP or intR-attR regions as well as the lacZ gene. The integrated Mx8 was excised at a high frequency, indicating that IntR is active for the excision. For Mx8 excision, a gene designated xis was shown to be required in addition to intR.During lysogenization of most temperate phages, their genomic DNA is integrated into the specific attachment site of the host chromosome by a mechanism originally proposed by Campbell (2). For example, phage is integrated into the attB site of the Escherichia coli chromosome by conservative sitespecific recombination between the attP site and host attB site (1). The product of the int gene and the host integration host factor protein are required for integration. Upon induction of the SOS response phage is excised from the host chromosome. In this case the product of the xis gene is also required in addition to Int and integration host factor. On the genome, the xis and int genes overlap by 23 nucleotides and the attP site follows the int gene.While the various site-specific recombinases of the integrase family have evolved diversely, all carry the conserved box I and box II motifs (13, 18) (see Fig. 5). An arginine residue in box I and histidine, arginine, and tyrosine residues in box II are completely conserved, and they form an active center for phosphotransfer reaction. The conserved tyrosine residue in box II forms a phosphodiester bond with the 3Ј end of the recombining DNA. The processes of site-specific recombination were analyzed by X-ray crystallography for the Cre-loxP system (6-8). The loxP Holliday junction intermediate bound to four Cre molecules has been demonstrated.Myxococcus xanthus is a unique gram-negative bacterium that can undergo multicellular development involving cell-tocell interactions (for a review, see reference 4). When depleted of nutrients, cells on a solid medium aggregate to form mounds, which then convert to fruiting bodies. Rod-shaped vegetative cells change to round or ovoid myxospores. Myxophage Mx8 is a generalized transducing phage of M. xanthus (17, 23). Mx8 can be integrated into the M. xanthus chromosome by site-specific recombination during lysogeny (19). This recombination system has been used to introduce recombinant plasmids into the M. xanthus chromosome (22).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 a...
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