Triple mutants uncover three new genes required for social motility in Myxococcus xanthus

The bacterium Myxococcus xanthus glides over surfaces using two different locomotive mechanisms, called S (social) and A (adventurous) motility that enable cells to move both as groups and as individuals. Neither mechanism involves flagella. The functions of these two motors are coordinated by the activity of a small Ras-like protein, encoded by the mglA gene. The results of previous studies of a second-site suppressor of the mglA-8 missense mutation masK-815 indicate that MglA interacts with a protein tyrosine kinase, MasK, to control social motility. Sequence analysis of the sites of 12 independent insertions of the transposon magellan-4 that result in the loss of motility in an M. xanthus mglA-8 masK-815 double mutant shows that nine of these 12 insertions are in genes known to be required for S gliding motility. This result confirms that the masK-815 suppressor restores S but not A motility. Three of the 12 insertions define three new genes required for S motility and show that the attachment of heptose to the lipopolysaccharide inner core, an ortholog of the CheR methyltransferase, and a large protein with YD repeat motifs, are required for S motility. When these three insertions are backcrossed into an otherwise wild-type genetic background, their recombinants are found to have defects in S, but not, A motility. The spectrum of magellan-4 insertions that lead to the loss of S motility in the mglA-8 masK-815 double mutant background is different than that resulting from a previous mutant hunt starting with a different (A mutant) genetic background, suggesting that the number of genes required for S motility in M. xanthus is quite large.     

The Myxococcus xanthus wbgB gene encodes a glycosyltransferase homologue required for lipopolysaccharide O-antigen biosynthesis

Myxococcus xanthus is a gram-negative soil bacterium that initiates a complex developmental program in response to starvation. A transposon insertion (Tn5-lac omega109) mutant with developmental deficiencies was isolated and characterized in this study. A strain containing this insertion mutation in an otherwise wild-type background showed delayed developmental aggregation for about 12 h and sporulated at 1-2% of the wild-type level. Tn5-lac omega109 was found to have disrupted the M. xanthus wbgB gene, which is located 2.1 kb downstream of the M. xanthus lipopolysacharide (LPS) O-antigen biosynthesis genes wzm wzt wbgA. The deduced polypeptide sequence of WbgB shares significant similarity with bacterial glycosyltransferases including M. xanthus WbgA. The wbgB::Tn5-lac omega109 mutant was found to be defective in LPS O-antigen synthesis by immunochemical analysis. Further mutational analysis indicated that the defects of the wbgB::Tn5-lac omega109 mutant were not the result of polar effects on downstream genes. Various motility assays demonstrated that the Tn5-lac omega109 mutation affected both social (S) and adventurous (A) gliding motility of M. xanthus cells. The pleiotrophic effects of wbgB mutations indicate the importance of LPS O-antigen biosynthesis for various cellular functions in M. xanthus.     

"Frizzy" mutants: a new class of aggregation-defective developmental mutants of Myxococcus xanthus

During fruiting-body formation in Myxococcus xanthus, cells aggregate into raised mounds, where they sporulate. A new class of aggregation-defective developmental mutants was identified within a collection of nonfruiting mutants of M. xanthus. The mutants failed to aggregate into discrete mounds, but rather aggregated into "frizzy" filaments. Many cells within the filaments sporulated normally. Pairwise mixtures of representative frizzy mutants were unable to stimulate each other to aggregate normally. Two strains of M. xanthus were isolated which contained transposon Tn5 insertions mapping near one frizzy mutation. A search through 36 mutants exhibiting the frizzy phenotype showed that all were linked to the same Tn5 insertion sites. Three-factor cross-analysis of 22 of these mutants allowed the mapping of these mutations into many loci. The localization of Tn5 inserts adjacent to this region make possible further manipulation of these genes.     

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.     

A CheW homologue is required for Myxococcus xanthus fruiting body development,social gliding motility,and fibril biogenesis

In bacteria with multiple sets of chemotaxis genes, the deletion of homologous genes or even different genes in the same operon can result in disparate phenotypes. Myxococcus xanthus is a bacterium with multiple sets of chemotaxis genes and/or homologues. It was shown previously that difA and difE, encoding homologues of the methyl-accepting chemoreceptor protein (MCP) and the CheA kinase, respectively, are required for M. xanthus social gliding (S) motility and development. Both difA and difE mutants were also defective in the biogenesis of the cell surface appendages known as extracellular matrix fibrils. In this study, we investigated the roles of the CheW homologue encoded by difC, a gene at the same locus as difA and difE. We showed that difC mutations resulted in defects in M. xanthus developmental aggregation, sporulation, and S motility. We demonstrated that difC is indispensable for wild-type cellular cohesion and fibril biogenesis but not for pilus production. We further illustrated the ectopic complementation of a difC in-frame deletion by a wild-type difC. The identical phenotypes of difA, difC, and difE mutants are consistent and supportive of the hypothesis that the Dif chemotaxis homologues constitute a chemotaxis-like signal transduction pathway that regulates M. xanthus fibril biogenesis and S motility.     

Regulation of cohesion-dependent cell interactions in Myxococcus xanthus.

Myxococcus xanthus has two nearly independent genetic systems, A and S, which appear to mediate adventurous (single-cell) movement and social (group) movement, respectively. In addition to a notable reduction in group movement, social motility mutants exhibit decreased biofilm formation, cell cohesion, dye binding, fibril production, and fruiting body formation. The stk-1907 allele, containing transposon Tn5 insertion omega DK1907, was introduced into wild-type cells and many social motility mutants. This allele, which was epistatic to most social motility mutations, caused wild-type and most mutant cells to exhibit increased group movement, cell cohesion, dye binding, and production of cell surface fibrils. The presence of the stk-1907 allele in dsp mutants, which almost completely lack cell surface fibrils, did not result in these phenotypic changes; therefore, stk-1907 is hypostatic to dsp mutations. Those mutants which exhibited increased group movement and cell cohesion with the stk-1907 allele also had increased fruiting body formation, but no significant changes in spore production were observed. These results suggest that fibrils may mediate cell cohesion, dye binding, and group movement. Additionally, the results suggest that the dsp locus contains genes involved in subunit synthesis, transport, and/or assembly of fibrils. The wild-type and mutant alleles of stk were cloned and studied in merodiploids. The mutant allele is recessive, suggesting that Tn5 omega DK1907 caused a null mutation in a gene which acts as a negative regulator of fibril synthesis. The stk-1907 allele appears to cause utilization of the A motility system for group movement, possibly because of increased fibril production.     

Effect of mechanical removal of pili on gliding motility of Myxococcus xanthus.

Gliding motility of Myxococcus xanthus is governed by both the adventurous (A) and the social (S) motility gene systems. The presence of pili has previously been shown to be correlated with a genetically intact S-motility system (D. Kaiser, Proc. Natl. Acad. Sci. USA 76:5952-5956, 1979). The purpose of the present work was to study the direct effect of mechanical removal of pill on the social motility of M. xanthus. Depiliation resulted in (i) a loss of streaming motility of A- S+ mutants, i.e., strains which are able to move by virtue of the S-motility system only, (ii) no effect on motility in A+ S- mutants, i.e., strains capable of movement by the A-motility system only, and (iii) a retardation of streaming speed in the wild-type strain (A+ S+). Cell-cell cohesion, another characteristic of social behavior, was not affected by mechanical removal of pill. The observation that mechanical depiliation perturbed the motility of strains which rely on the S-motility system strongly supports a role for pili in social motility of M. xanthus.     

Myxococcus xanthus dif genes are required for biogenesis of cell surface fibrils essential for social gliding motility

Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M. xanthus S motility. We have demonstrated here that M. xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacterium Pseudomonas aeruginosa, the M. xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the dif genes are linked to the M. xanthus dsp region, a locus known to be crucial for M. xanthus fibril biogenesis and S gliding.     

Genetic analysis of tag mutants of Myxococcus xanthus provides evidence for two developmental aggregation systems

Temperature-dependent aggregation mutants (tag) of the myxobacterium Myxococcus xanthus aggregated into mounds and developed into fruiting bodies normally at 28 degrees C; however, they failed to form mounds at 34 degrees C. The timing of sporulation was unaffected by the mutations, and normal numbers of spores were produced at both permissive and restrictive temperatures. This class of mutations was originally identified through screening of ethyl methanesulfonate (EMS)-generated mutations. Subsequent work identified a linked insertion of transposon Tn5, which was used to map the EMS-generated mutations to four loci. In this paper, we describe the cloning of the tag loci and the use of transposon mutagenesis to further analyze the tag loci. Nine tag complementation groups spanning 8.5 kilobase pairs of DNA were identified through mapping of 28 independent Tn5 insertions. All insertion and deletion mutants had the same phenotype as the EMS mutants: they were temperature sensitive for mound formation. This result suggests that M. xanthus has at least two sets of genes for developmental aggregation. The tag genes constitute one set of these genes; they are required for normal development at 34 degrees C but are not required for normal development at 28 degrees C.     

A DnaK Homolog in Myxococcus xanthus Is Involved in Social Motility and Fruiting Body Formation

Myxococcus xanthus is a gram-negative soil bacterium which exhibits a complex life cycle and social behavior. In this study, two developmental mutants of M. xanthus were isolated through Tn5 transposon mutagenesis. The mutants were found to be defective in cellular aggregation as well as in sporulation. Further phenotypic characterization indicated that the mutants were defective in social motility but normal in directed cell movements. Both mutations were cloned by a transposon-tagging method. Sequence analysis indicated that both insertions occurred in the same gene, which encodes a homolog of DnaK. Unlike the dnaK genes in other bacteria, this M. xanthus homolog appears not to be regulated by temperature or heat shock and is constitutively expressed during vegetative growth and under starvation. The defects of the mutants indicate that this DnaK homolog is important for the social motility and development of M. xanthus.     

A new set of chemotaxis homologues is essential for Myxococcus xanthus social motility

Myxococcus xanthus cells aggregate and develop into multicellular fruiting bodies in response to starvation. A new M. xanthus locus, designated dif for defective in fruiting, was identified by the characterization of a mutant defective in fruiting body formation. Molecular cloning, DNA sequencing and sequence analysis indicate that the dif locus encodes a new set of chemotaxis homologues of the bacterial chemotaxis proteins MCPs (methyl-accepting chemotaxis proteins), CheW, CheY and CheA. The dif genes are distinct genetically and functionally from the previously identified M. xanthus frz chemotaxis genes, suggesting that multiple chemotaxis-like systems are required for the developmental process of M. xanthus fruiting body formation. Genetic analysis and phenotypical characterization indicate that the M. xanthus dif locus is required for social (S) motility. This is the first report of a M. xanthus chemotaxis-like signal transduction pathway that could regulate or co-ordinate the movement of M. xanthus cells to bring about S motility.     

The Che4 pathway of Myxococcus xanthus regulates type IV pilus-mediated motility

Myxococcus xanthus co-ordinates cell movement during its complex life cycle using multiple chemotaxis-like signal transduction pathways. These pathways regulate both type IV pilus-mediated social (S) motility and adventurous (A) motility. During a search for new chemoreceptors, we identified the che4 operon, which encodes homologues to a MCP (methyl-accepting chemotaxis protein), two CheWs, a hybrid CheA-CheY, a response regulator and a CheR. Deletion of the che4 operon did not cause swarming or developmental defects in either the wild-type (A(+)S(+)) strain or in a strain sustaining only A motility (A(+)S(-)). However, in a strain displaying only S motility (A(-)S(+)), deletion of the che4 operon or the gene encoding the response regulator, cheY4, caused enhanced vegetative swarming and prevented aggregation and sporulation. In contrast, deletion of mcp4 caused reduced vegetative swarming and enhanced development compared with the parent strain. Single-cell analysis of the motility of the A(-)S(+) parent strain revealed a previously unknown inverse correlation between velocity and reversal frequency. Thus, cells that moved at higher velocities showed a reduced reversal frequency. This co-ordination of reversal frequency and velocity was lost in the mcp4 and cheY4 mutants. The structural components of the S motility apparatus were unaffected in the che4 mutants, suggesting that the Che4 system affects reversal frequency of cells by modulating the function of the type IV pilus.     

Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.     

Defects in motility and development of Myxococcus xanthus lipopolysaccharide mutants.

Five transposon Tn5 mutants of the procaryote Myxococcus xanthus had been shown previously to be defective in lipopolysaccharide biosynthesis (J. M. Fink,-M. Kalos, and J. F. Zissler, J. Bacteriol. 171:2033-2041, 1989). These mutants were studied for possible defects in gliding motility and multicellular development. Wild-type M. xanthus cells glide both as single cells and as groups of cells. We found that the Tn5 lipopolysaccharide O-antigen mutants were defective in single-cell motility but were unaltered in group motility. These mutant strains were slow to develop but eventually gave rise to normal, spore-filled fruiting bodies. We also had shown previously that 56 (ethyl methanesulfonate-induced and spontaneous) phage-resistant mutants were defective in lipopolysaccharide biosynthesis. We found that many of these lipopolysaccharide O-antigen mutants were defective in single-cell motility but were unaltered in group motility. These mutants also gave rise to normal, spore-filled fruiting bodies. We also studied several phage-resistant mutants which were lacking a side-chain carbohydrate on the lipopolysaccharide core. These mutants possessed both single-cell motility and group motility but were altered in the magnitude of gliding. These mutants were blocked early in development and could not form multicellular fruiting bodies. Several of the mutations in the developmentally aberrant strains were mapped to a single locus by using a collection of genetically linked transposons as genetic markers.     

Mutations in two new loci that impair both extracellular protein production and development in Myxococcus xanthus.

Two transposon insertion mutants of Myxococcus xanthus altered in the secretion of protein as determined by the hydrolytic activities of several enzymes during vegetative growth were also unable to complete fruiting body formation and were severely impaired in sporulation. The insertions were located in the same part of the M. xanthus chromosome but were unlinked by transduction and therefore define two distinct loci, called excA and excB. Since both Exc +/- mutants were able to rescue development of an asgB mutation, they do not belong to the Asg- group, despite of the fact that asg mutants are also Exc +/-. Our results sustain the hypothesis of a possible relationship between protein secretion during vegetative growth and development or sporulation.     

Insertions of Tn5 near genes that govern stimulatable cell motility in Myxococcus

Insertions of transposon Tn5 were used to examine the genetics of motility mutants in Myxococcus. Fifteen independent insertions of Tn5 were isolated that were linked to seven different loci that govern motility. Among the motility mutants that can be stimulated to move transiently by contact with other cells, a one-to-one correspondence was confirmed between specificity of stimulation and genetic locus. There are six different specificities and six corresponding loci, as if each locus governs a different protein required for gliding motility.     

Exopolysaccharide biosynthesis genes required for social motility in Myxococcus xanthus

Social (S)-motility in Myxococcus xanthus is a flagellum-independent gliding motility system that allows bacteria to move in groups on solid surfaces. S-motility has been shown to require type IV pili (TFP), exopolysaccharide (EPS; a component of fibrils) and lipopolysaccharide (LPS). Previously, information concerning EPS biogenesis in M. xanthus was lacking. In this study, we screened 5000 randomly mutagenized colonies for defects in S-motility and EPS and identified two genetic regions essential for EPS biogenesis: the EPS synthesis (eps) region and the EPS-associated (eas) region. Mutants with insertions in the eps and eas regions were defective in S-motility and fruiting body formation. These mutants failed to bind the dye calcofluor white, indicating that they lacked EPS; however, they retained normal TFP and LPS. Analysis of the eps locus showed several open reading frames (ORFs) that encode homologues to glycosyltransferases, glucanases and EPS transporters as well as regulatory proteins; the eas locus contains two ORFs: one exhibits homology to hypothetical proteins with a conserved domain of unknown function and the other displays no apparent homology to other proteins in the database. Further genetic mutagenesis analysis indicates that the whole eps region is involved in the biosynthesis of fibrils and fibril EPS. The operon at the proximal end of the eps region was analysed by generating in-frame deletion mutations. These mutants showed varying degrees of defects in the bacterium's ability to produce EPS or perform EPS-related functions, confirming the involvement of these genes in M. xanthus EPS biogenesis.