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

Mx8 is a generalized transducing phage that infects Myxococcus xanthus cells. This phage is lysogenized in M. 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.     

Abnormal motility and fruiting behavior of Myxococcus xanthus bacteriophage-resistant strains induced by a clear-plaque mutant of bacteriophage Mx8

Myxococcus xanthus mutants resistant to a clear-plaque derivative of phage Mx8 were isolated. A significant fraction of the mutants, easily recognizable by their colony morphology, were induced by the presence of the phage and may correspond to low-frequency lysogens. They were all defective in cell motility and showed the same nonfruiting phenotype under starvation conditions.     

RasA is required for Myxococcus xanthus development and social motility

An insertion in the rasA gene entirely blocked developmental aggregation and sporulation in Myxococcus xanthus while also reducing swarm expansion on a 0.3% agar surface. Data presented here demonstrate that rasA is required for extracellular fibril formation and social gliding motility.     

Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8

Summary We have constructed a restriction map for the genome of bacteriophage MX-8 from Myxococcus xanthus using the enzymes PvuII, MboI, and EcoRI. The phage genome size, as determined by restriction analysis, is 51.7±0.6 Kb. Double digestions, redigestions of isolated fragments, and crossed-contact hybridization of partial digestion products show that the restriction map is circular. Restriction analysis and Southern hybridization show that the phage DNA molecules are packaged sequentially from a concatemer starting from a specific site which we have mapped. The DNA molecules have an average terminal redundancy of approximately 8% and are circularly permuted over at least 40% of the genome.     

Genetic Determinants of Immunity and Integration of Temperate Myxococcus xanthus Phage Mx8

An 8.1-kb fragment of the temperate Myxococcus xanthus phage Mx8 genome, when cloned into a plasmid vector, permits site-specific integration of the plasmid and confers superinfection immunity. Sequence analysis of a 9.5-kb region of Mx8 DNA containing this fragment reveals 19 densely packed open reading frames, four of which have predicted products with known or suspected activities. The Mx8 imm gene, required for superinfection immunity, has a sequence similar to that of Arabidopsis thaliana G-box-binding factor 1. Mx8 makes a DNA adenine methylase, Mox, and integrase, Int, related to other methylases and integrases. The int gene has two alternate translation initiation codons within the extensively overlapping uoi (upstream of int) gene. Comparison of the predicted product of the uoi gene with Salmonella phage P22 and Streptomyces plasmid Xis proteins shows that temperate phage excisionases may use variations of a helix-turn-helix motif to recognize specific DNA sequences.     

The asgE locus is required for cell-cell signalling during Myxococcus xanthus development

In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome-shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co-developed with wild-type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild-type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell-cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild-type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A-factor, a cell density signal. A-factor consists of a mixture of heat-stable amino acids and peptides, and at least two heat-labile extracellular proteases. The asgE mutant yielded about 10-fold less heat-labile A-factor and about twofold less heat-stable A-factor than wild-type cells, suggesting that the primary defect of asgE cells is in the production or release of heat-labile A-factor.     

Site-Specific Recombination of Temperate Myxococcus xanthus Phage Mx8: Genetic Elements Required for Integration

Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host, Myxococcus xanthus, by a mechanism of site-specific recombination. The Mx8 int-attP genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome. When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3' ends of either of two tandem tRNAAsp genes, trnD1 and trnD2, located within the attB locus of the M. xanthus genome. Although Int-mediated site-specific recombination occurs between attP and either attB1 (within trnD1) or attB2 (within trnD2), the attP x attB1 reaction is highly favored and often is accompanied by a deletion between attB1 and attB2. The int gene is the only Mx8 gene required in trans for attP x attB recombination. The int promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state. All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediate attP x attB recombination efficiently. The attP core lies within the int coding sequence, and the product of integration is a prophage in which the 3' end of int is replaced by host sequences. The prophage intX gene is predicted to encode an integrase with a different C terminus.     

Intercellular signaling is required for developmental gene expression in Myxococcus xanthus

Certain developmental mutants of Myxococcus xanthus can be complemented (extracellularly) by wild-type cells. Insertions of Tn5 lac (a transposon which couples beta-galactosidase expression to exogenous promoters) into developmentally regulated genes were used to investigate extracellular complementation of the A group mutations. A- mutations reduced developmental beta-galactosidase expression from 18 of 21 Tn5 lac insertions tested and that expression was restored to A- Tn5 lac cells by adding wild-type cells. The earliest A-dependent Tn5 lac normally expresses beta-galactosidase at 1.5 hr of development indicating a developmental block at 1-2 hr in A- mutants. A substance which can rescue the expression of this early Tn5 lac is released by wild-type (A+) but not by A- cells. This substance appears in a cell-free wash of wild-type cells or in starvation buffer conditioned by wild-type cells 1-2 hr after development is initiated. The conditioned starvation buffer also restores normal morphological development to an A- mutant.     

The Myxococcus xanthus lipopolysaccharide O-antigen is required for social motility and multicellular development

The gliding bacterium Myxococcus xanthus aggregates to form spore-filled fruiting bodies when nutrients are limiting. Defective fruiting-body formation and sporulation result from mutations in the sasA locus, which encodes the wzm wzt wbgA (formerly rfbABC ) lipopolysaccharide (LPS) O-antigen biosynthesis genes. Mutants carrying these same sasA mutations are defective in social motility and form small glossy colonies. We report here that the developmental and motility phenotypes of four mutants each containing different Tn 5 insertions in LPS O-antigen biosynthesis genes are similar to those of the original sasA locus mutants. All of the LPS O-antigen mutants tested exhibited defective developmental aggregation and sporulated at only 0.02–15% of the wild-type level. In addition, all of the LPS O-antigen mutants were determined by genetic analyses to be wild type for adventurous motility and defective in social motility, indicating that the LPS O-antigen is necessary for normal development and social motility. The two previously identified cell-surface components required for social motility, type IV pili and the protein-associated polysaccharide material termed fibrils, were detected on the surfaces of all of the LPS O-antigen mutants. This indicates that LPS O-antigen is a third cell-surface component required for social motility.     

Propionyl coenzyme A carboxylase is required for development of Myxococcus xanthus.

A dcm-1 mutant, obtained by transposon mutagenesis of Myxococcus xanthus, could aggregate and form mounds but was unable to sporulate under nutrient starvation. A sequence analysis of the site of insertion of the transposon showed that the insertion lies within the 3' end of a 1,572-bp open reading frame (ORF) designated the M. xanthus pccB ORF. The wild-type form of the M. xanthus pccB gene, obtained from a lambdaEMBL library of M. xanthus, shows extensive similarity to a beta subunit of propionyl coenzyme A (CoA) carboxylase, an alpha subunit of methylmalonyl-CoA decarboxylase, and a 12S subunit of transcarboxylase. In enzyme assays, extracts of the dcm-1 mutant were deficient in propionyl-CoA carboxylase activity. This enzyme catalyzes the ATP-dependent carboxylation of propionyl-CoA to yield methylmalonyl-CoA. The methylmalonyl-CoA rescued the dcm-1 mutant fruiting body and spore development. During development, the dcm-1 mutant cells also had reduced levels of long-chain fatty acids (C16 to C18) compared to wild-type cells.     

Genetic and physical characterization of lysogeny by bacteriophage MX8 in Myxococcus xanthus

Myxophage MX8 can initiate a lysogenic cycle in Myxococcus xanthus. The lysogenic phage was gentically stable in vegetative cells and persisted in the latent state through many cell generations in the absence of extracellular phage reinfection. The latent state also was stable during the host developmental cycle, since myxospores transmitted latent MX8 genetic information to future progeny cells. DNA hybridization experiments to probe the structure of the lysogenic phage provided physical evidence that MX8 formed a prophage. During lysogenization, MX8 DNA was cut at a specific site (attP) on phage DNA, and we have concluded that genetic recombination between attP and a bacterial DNA site (attB) leads to integration of MX8 DNA and formation of stable MX8 prophage. The genetic and physical properties of MX8 that we describe should make MX8 useful in the analysis of development of M. xanthus by genetic methods.     

Physical map of the Myxococcus xanthus chromosome.

The genome of Myxococcus xanthus, which is 9,454 kbp, is one of the largest bacterial genomes. The organization of the DNA and the distribution of genes encoding social and developmental behaviors were examined by using pulsed field gel electrophoresis. Intact genomic DNA was digested with AseI into 16 restriction fragments, which were separated by contour-clamped homogeneous electric field electrophoresis, purified, and radiolabeled. Each AseI fragment was hybridized to SpeI-digested DNA and to an M. xanthus genomic library contained in yeast artificial chromosomes. Some SpeI restriction fragments and yeast artificial chromosome clones contained AseI sites and hybridized with two different AseI restriction fragments, providing evidence for the juxtaposition of these AseI restriction fragments in the chromosome. The deduced AseI physical map is circular, suggesting that this bacterium contains a single, circular chromosome. Transposable elements shown by transduction to be in or near genes of interest were located on specific AseI restriction fragments by restriction analysis and Southern hybridization. Most AseI restriction fragments contained genes involved in social and developmental behaviors.     

AglZ is a filament-forming coiled-coil protein required for adventurous gliding motility of Myxococcus xanthus

The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro.     

An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus

An extracellular matrix connects bacteria that live in organized assemblages called biofilms. While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction. The extracellular matrix of the soil bacterium Myxococcus xanthus is essential for biofilm formation and fruiting body development. The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine. We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A). Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family. Disruption of fibA had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors. However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed. Chemotactic excitation of the fibA mutant was restored by incubation with purified wild-type fibrils. The results suggest that this metalloprotease is involved in sensory transduction.     

A Myxococcus xanthus bacterial tyrosine kinase, BtkA, is required for the formation of mature spores

A Myxococcus xanthus cytoplasmic bacterial tyrosine kinase, BtkA, showed phosphorylation activity in the presence of Exo. Phosphorylated BtkA was expressed late after starvation induction and early after glycerol induction. The btkA mutant was unable to complete maturation to heat- and sonication-resistant spores under both starvation- and glycerol-induced developmental conditions.     

Site-Specific Recombination of Temperate Myxococcus xanthus Phage Mx8: Regulation of Integrase Activity by Reversible, Covalent Modification

Temperate Myxococcus xanthus phage Mx8 integrates into the attB locus of the M. xanthus genome. The phage attachment site, attP, is required in cis for integration and lies within the int (integrase) coding sequence. Site-specific integration of Mx8 alters the 3' end of int to generate the modified intX gene, which encodes a less active form of integrase with a different C terminus. The phage-encoded (Int) form of integrase promotes attP x attB recombination more efficiently than attR x attB, attL x attB, or attB x attB recombination. The attP and attB sites share a common core. Sequences flanking both sides of the attP core within the int gene are necessary for attP function. This information shows that the directionality of the integration reaction depends on arm sequences flanking both sides of the attP core. Expression of the uoi gene immediately upstream of int inhibits integrative (attP x attB) recombination, supporting the idea that uoi encodes the Mx8 excisionase. Integrase catalyzes a reaction that alters the primary sequence of its gene; the change in the primary amino acid sequence of Mx8 integrase resulting from the reaction that it catalyzes is a novel mechanism by which the reversible, covalent modification of an enzyme is used to regulate its specific activity. The lower specific activity of the prophage-encoded IntX integrase acts to limit excisive site-specific recombination in lysogens carrying a single Mx8 prophage, which are less immune to superinfection than lysogens carrying multiple, tandem prophages. Thus, this mechanism serves to regulate Mx8 site-specific recombination and superinfection immunity coordinately and thereby to preserve the integrity of the lysogenic state.