Identification of genes required for adventurous gliding motility in Myxococcus xanthus with the transposable element mariner

Myxococcus xanthus glides over solid surfaces without the use of flagella, dependent upon two large sets of adventurous (A) and social (S) genes, using two different mechanisms of gliding motility. Myxococcus xanthus A-S- double mutants form non-motile colonies lacking migratory cells at their edges. We have isolated 115 independent mutants of M. xanthus with insertions of transposon magellan-4 in potential A genes by screening for insertions that reduce the motility of a mutant S- parental strain. These insertions are found not only in the three loci known to be required for A motility, mglBA, cglB, and aglU, but also in 30 new genes. Six of these new genes encode different homologues of the TolR, TolB, and TolQ transport proteins, suggesting that adventurous motility is dependent on biopolymer transport. Other insertions which affect both A and S motility suggest that both systems share common energy and cell wall determinants. Because the spectrum of magellan-4 insertions in M. xanthus is extraordinarily broad, transposon mutagenesis with this eukaryotic genetic element permits the rapid genetic analysis of large sets of genes that contribute to a complex microbial behaviors such as A motility.     

Gliding motility in bacteria: insights from studies of Myxococcus xanthus.

Gliding motility is observed in a large variety of phylogenetically unrelated bacteria. Gliding provides a means for microbes to travel in environments with a low water content, such as might be found in biofilms, microbial mats, and soil. Gliding is defined as the movement of a cell on a surface in the direction of the long axis of the cell. Because this definition is operational and not mechanistic, the underlying molecular motor(s) may be quite different in diverse microbes. In fact, studies on the gliding bacterium Myxococcus xanthus suggest that two independent gliding machineries, encoded by two multigene systems, operate in this microorganism. One machinery, which allows individual cells to glide on a surface, independent of whether the cells are moving alone or in groups, requires the function of the genes of the A-motility system. More than 37 A-motility genes are known to be required for this form of movement. Depending on an additional phenotype, these genes are divided into two subclasses, the agl and cgl genes. Videomicroscopic studies on gliding movement, as well as ultrastructural observations of two myxobacteria, suggest that the A-system motor may consist of multiple single motor elements that are arrayed along the entire cell body. Each motor element is proposed to be localized to the periplasmic space and to be anchored to the peptidoglycan layer. The force to glide which may be generated here is coupled to adhesion sites that move freely in the outer membrane. These adhesion sites provide a specific contact with the substratum. Based on single-cell observations, similar models have been proposed to operate in the unrelated gliding bacteria Flavobacterium johnsoniae (formerly Cytophaga johnsonae), Cytophaga strain U67, and Flexibacter polymorphus (a filamentous glider). Although this model has not been verified experimentally, M. xanthus seems to be the ideal organism with which to test it, given the genetic tools available. The second gliding motor in M. xanthus controls cell movement in groups (S-motility system). It is dependent on functional type IV pili and is operative only when cells are in close proximity to each other. Type IV pili are known to be involved in another mode of bacterial surface translocation, called twitching motility. S-motility may well represent a variation of twitching motility in M. xanthus. However, twitching differs from gliding since it involves cell movements that are jerky and abrupt and that lack the organization and smoothness observed in gliding. Components of this motor are encoded by genes of the S-system, which appear to be homologs of genes involved in the biosynthesis, assembly, and function of type IV pili in Pseudomonas aeruginosa and Neisseria gonorrhoeae. How type IV pili generate force in S-motility is currently unknown, but it is to be expected that ongoing physiological, genetic, and biochemical studies in M. xanthus, in conjunction with studies on twitching in P. aeruginosa and N. gonorrhoeae, will provide important insights into this microbial motor. The two motility systems of M. xanthus are affected to different degrees by the MglA protein, which shows similarity to a small GTPase. Bacterial chemotaxis-like sensory transduction systems control gliding motility in M. xanthus. The frz genes appear to regulate gliding movement of individual cells and movement by the S-motility system, suggesting that the two motors found in this bacterium can be regulated to result in coordinated multicellular movements. In contrast, the dif genes affect only S-system-dependent swarming.     

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.     

New locus important for Myxococcus social motility and development

The mts locus in salt-tolerant Myxococcus fulvus HW-1 was found to be critical for gliding motility, fruiting-body formation, and sporulation. The homologous genes in Myxococcus xanthus are also important for social motility and fruiting-body development. The mts genes were determined to be involved in cell-cell cohesion in both myxobacterial species.     

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.     

The Myxococcus xanthuspilT locus is required for social gliding motility although pili are still produced

Social gliding motility in Myxococcus xanthus depends on the presence of Type IV pili. To begin to examine the role of pili in social motility, 17 mutants were identified which had lost social motility, but still expressed pili. Four of these mutants carry point mutations which mapped to a locus upstream of the recently identified pilS , pilR , and pilA genes. Sequencing of this locus revealed a gene with homology to pilT from Pseudomonas aeruginosa . Sequencing of the four point mutations revealed that they occurred within the M. xanthus pilT locus. A markerless deletion within M. xanthus pilT , similar to the four point mutations, disrupted social gliding behaviour but did not interfere with pilus formation or pilus-dependent cell–cell agglutination. Using time-lapse videomicroscopy, residual social motility was observed in dsp ⁻ strains (known to be deficient in fibril but not pilus production); this was not observed in a Δ pilT dsp ⁻ double mutant. Two genes flanking pilT  were also sequenced, and found to have homology to pilB and pilC from P. aeruginosa . Markerless deletions within these genes caused both pilus and social-motility defects. These results indicate that M. xanthus pilB and pilC are required for pilus biogenesis, while pilT is required for assembled pili to play their role in social motility. Thus, pilB , pilT , pilC , pilS , pilR and pilA form a contiguous cluster of pil genes required for social motility.     

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.     

Phenotypic analyses of frz and dif double mutants of Myxococcus xanthus

Myxococcus xanthus is a Gram-negative gliding bacterium that aggregates and develops into multicellular fruiting bodies in response to starvation. Two chemosensory systems (frz and dif), both of which are homologous to known chemotaxis proteins, were previously identified through characterization of various developmental mutants. This study aims to examine the interaction between these two systems since both of them are required for fruiting body formation of M. xanthus. Through detailed phenotypic analyses of frz and dif double mutants, we found that both frz and dif are involved in cellular reversal and social motility; however, the frz genes are epistatic in controlling cellular reversal, whereas the dif genes are epistatic in controlling social motility. The study suggests that the integration of these two chemotaxis systems may play a central role in controlling the complicated social behaviors of M. xanthus.     

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.     

A Versatile Class of Cell Surface Directional Motors Gives Rise to Gliding Motility and Sporulation in Myxococcus xanthus

Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories.     

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.     

Polarity of motility systems in Myxococcus xanthus

Myxococcus xanthus is a gliding bacterium that contains two motility systems: S-motility, powered by polar type IV pili, and A-motility, powered by uncharacterized motors and adhesion complexes. The localization and coordination of the two motility engines is essential for directed motility as cells move forward and reverse. During cell reversals, the polarity and localization of motility proteins are rapidly inverted, rendering this system a fascinating example of dynamic protein localization.     

The mosaic genome of Anaeromyxobacter dehalogenans strain 2CP-C suggests an aerobic common ancestor to the delta-proteobacteria

Anaeromyxobacter dehalogenans strain 2CP-C is a versaphilic delta-Proteobacterium distributed throughout many diverse soil and sediment environments. 16S rRNA gene phylogenetic analysis groups A. dehalogenans together with the myxobacteria, which have distinguishing characteristics including strictly aerobic metabolism, sporulation, fruiting body formation, and surface motility. Analysis of the 5.01 Mb strain 2CP-C genome substantiated that this organism is a myxobacterium but shares genotypic traits with the anaerobic majority of the delta-Proteobacteria (i.e., the Desulfuromonadales). Reflective of its respiratory versatility, strain 2CP-C possesses 68 genes coding for putative c-type cytochromes, including one gene with 40 heme binding motifs. Consistent with its relatedness to the myxobacteria, surface motility was observed in strain 2CP-C and multiple types of motility genes are present, including 28 genes for gliding, adventurous (A-) motility and 17 genes for type IV pilus-based motility (i.e., social (S-) motility) that all have homologs in Myxococcus xanthus. Although A. dehalogenans shares many metabolic traits with the anaerobic majority of the delta-Proteobacteria, strain 2CP-C grows under microaerophilic conditions and possesses detoxification systems for reactive oxygen species. Accordingly, two gene clusters coding for NADH dehydrogenase subunits and two cytochrome oxidase gene clusters in strain 2CP-C are similar to those in M. xanthus. Remarkably, strain 2CP-C possesses a third NADH dehydrogenase gene cluster and a cytochrome cbb(3) oxidase gene cluster, apparently acquired through ancient horizontal gene transfer from a strictly anaerobic green sulfur bacterium. The mosaic nature of the A. dehalogenans strain 2CP-C genome suggests that the metabolically versatile, anaerobic members of the delta-Proteobacteria may have descended from aerobic ancestors with complex lifestyles.     

Genetic and Molecular Analysis of cglB, a Gene Essential for Single-Cell Gliding in Myxococcus xanthus

Gliding movements of individual isolated Myxococcus xanthus cells depend on the genes of the A-motility system (agl and cgl genes). Mutants carrying defects in those genes are unable to translocate as isolated cells on solid surfaces. The motility defect of cgl mutants can be transiently restored to wild type by extracellular complementation upon mixing mutant cells with wild-type or other motility mutant cells. To develop a molecular understanding of the function of a Cgl protein in gliding motility, we cloned the cglB wild-type allele by genetic complementation of the mutant phenotype. The nucleotide sequence of a 2.85-kb fragment was determined and shown to encode two complete open reading frames. The CglB protein was determined to be a 416-amino-acid putative lipoprotein with an unusually high cysteine content. The CglB antigen localized to the membrane fraction. The swarming and gliding defects of a constructed DeltacglB mutant were fully restored upon complementation with the cglB wild-type allele. Experiments with a cglB allele encoding a CglB protein with a polyhistidine tag at the C terminus showed that this allele also promoted wild-type levels of swarming and single-cell gliding, but was unable to stimulate DeltacglB cells to move. Possible functions of CglB as a mechanical component or as a signal protein in single cell gliding are discussed.     

The pilH gene encodes an ABC transporter homologue required for type IV pilus biogenesis and social gliding motility in Myxococcus xanthus

Type IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus . We report the discovery of four additional pil genes: pilD , a homologue of type IV prepilin leader peptidases; and pilG , pilH and pilI , which have no known homologues in other type IV pilus systems. pilH encodes an ATP-binding cassette (ABC) transporter homologue, the first such homologue to be required for the biogenesis of any bacterial pilus type. pilG and pilI are co-transcribed with pilH and appear to be functionally related to pilH . Null mutants of pilG , pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in the supernatant after cells were sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, in which pilI is an integral membrane protein with membrane-spanning domains, and pilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.     

Myxococcus xanthus chemotaxis homologs DifD and DifG negatively regulate fibril polysaccharide production

The extracellular matrix fibrils of Myxococcus xanthus are essential for the social lifestyle of this unusual bacterium. These fibrils form networks linking or encasing cells and are tightly correlated with cellular cohesion, development, and social (S) gliding motility. Previous studies identified a set of bacterial chemotaxis homologs encoded by the dif locus. It was determined that difA, difC, and difE, encoding respective homologs of a methyl-accepting chemotaxis protein, CheW, and CheA, are required for fibril production and therefore S motility and development. Here we report the studies of three additional genes residing at the dif locus, difB, difD, and difG. difD and difG encode homologs of chemotaxis proteins CheY and CheC, respectively. difB encodes a positively charged protein with limited homology at its N terminus to conserved bacterial proteins with unknown functions. Unlike the previously characterized dif genes, none of these three newly studied dif genes are essential for fibril production, S motility, or development. The difB mutant showed no obvious defects in any of the processes examined. In contrast, the difD and the difG mutants were observed to overproduce fibril polysaccharides in comparison with production by the wild type. The observation that DifD and DifG negatively regulate fibril polysaccharide production strengthens our hypothesis that the M. xanthus dif genes define a chemotaxis-like signal transduction pathway which regulates fibril biogenesis. To our knowledge, this is the first report of functional studies of a CheC homolog in proteobacteria. In addition, during this study, we slightly modified previously developed assays to easily quantify fibril polysaccharide production in M. xanthus.     

Cell polarity, intercellular signalling and morphogenetic cell movements in Myxococcus xanthus

In Myxococcus xanthus morphogenetic cell movements constitute the basis for the formation of spreading vegetative colonies and fruiting bodies in starving cells. M. xanthus cells move by gliding and gliding motility depends on two polarly localized engines, type IV pili pull cells forward, and slime extruding nozzle-like structures appear to push cells forward. The motility behaviour of cells provides evidence that the two engines are localized to opposite poles and that they undergo polarity switching. Several proteins involved in regulating polarity switching have been identified. The cell surface-associated C-signal induces the directed movement of cells into nascent fruiting bodies. Recently, the molecular nature of the C-signal molecule was elucidated and the motility parameters regulated by the C-signal were identified. From the effect of the C-signal on cell behaviour it appears that the C-signal inhibits polarity switching of the two motility engines. This establishes a connection between cell polarity, signalling by an intercellular signal and morphogenetic cell movements during fruiting body formation.     

FrzZ, a dual CheY-like response regulator, functions as an output for the Frz chemosensory pathway of Myxococcus xanthus

Myxococcus xanthus utilizes two distinct motility systems for movement (gliding) on solid surfaces: adventurous motility (A-motility) and social motility (S-motility). Both systems are regulated by the Frz signal transduction pathway, which controls cell reversals required for directed motility and fruiting body formation. The Frz chemosensory system, unlike the Escherichia coli chemotaxis system, contains proteins with multiple response regulator domains: FrzE, a CheA-CheY hybrid protein, and FrzZ, a CheY-CheY hybrid protein. Previously, the CheY domain of FrzE was hypothesized to act as the response regulator output of the Frz system. In this study, using a genetic suppressor screen, we identified FrzZ and showed FrzZ is epistatic to FrzE, demonstrating that FrzZ is the principal output component of the pathway. We constructed M. xanthus point mutations in the phosphoaccepting aspartate residues of FrzZ and demonstrated the respective roles of these residues in group and single cell motility. We also performed in vitro assays and showed rapid phosphotransfer between the CheA domain of FrzE and each of the CheY domains of FrzZ. These experiments showed that FrzZ plays a direct role as an output of the Frz chemosensory pathway and that both CheY domains of FrzZ are functional.