Force and flexibility of flailing myxobacteria

Myxococcus xanthus is a common Gram-negative bacterium that moves by a process called gliding motility. In myxobacteria, two distinct mechanisms for gliding have been discovered. S-type motility requires the extension, attachment, and retraction of type IV pili. The other mechanism, designated as A-type motility, may be driven by the secretion and swelling of slime; however, experiments to confirm or refute this model are still lacking and the force exerted by this mechanism has not been measured. A previously published experiment found that when an M. xanthus cell became stuck at one end, the cell underwent flailing motions. Based on this experiment, I propose an elastic model that can estimate the force produced by the A-motility engine and the bending modulus of a single myxobacterial cell. The model estimates a bending modulus of 3 x 10(-14) erg cm and a force between 50-150 pN. This force is comparable to that predicted by slime extrusion, and the bending modulus is 30-fold smaller than that measured in Bacillus subtilis. This model suggests experiments that can further quantify this process.     

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.     

The junctional pore complex and the propulsion of bacterial cells

Gliding motility is defined as translocation in the direction of the long axis of the bacterium while in contact with a surface. This definition leaves unspecified any mechanism and, indeed, it appears that there is more than one physiological system underlying the same type of motion. Currently, two distinct mechanisms have been discovered in myxobacteria. One requires the extension, attachment, and retraction of type IV pili to pull the cell forwards. Recent experimental evidence suggests that a second mechanism for gliding motility involves the extrusion of slime from an organelle called the 'junctional pore complex'. This review discusses the role of slime extrusion and the junctional pore complex in the gliding motility of both cyanobacteria and myxobacteria.     

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.     

Nanoscale visualization of a fibrillar array in the cell wall of filamentous cyanobacteria and its implications for gliding motility

Many filamentous cyanobacteria are motile by gliding, which requires attachment to a surface. There are two main theories to explain the mechanism of gliding. According to the first, the filament is pushed forward by small waves that pass along the cell surface. In the second, gliding is powered by the extrusion of slime through pores surrounding each cell septum. We have previously shown that the cell walls of several motile cyanobacteria possess an array of parallel fibrils between the peptidoglycan and the outer membrane and have speculated that the function of this array may be to generate surface waves to power gliding. Here, we report on a study of the cell surface topography of two morphologically different filamentous cyanobacteria, using field emission gun scanning electron microscopy (FEGSEM) and atomic force microscopy (AFM). FEGSEM and AFM images of Oscillatoria sp. strain A2 confirmed the presence of an array of fibrils, visible as parallel corrugations on the cell surface. These corrugations were also visualized by AFM scanning of fully hydrated filaments under liquid; this has not been achieved before for filamentous bacteria. FEGSEM images of Nostoc punctiforme revealed a highly convoluted, not parallel, fibrillar array. We conclude that an array of parallel fibrils, beneath the outer membrane of Oscillatoria, may function in the generation of thrust in gliding motility. The array of convoluted fibrils in N. punctiforme may have an alternative function, perhaps connected with the increase in outer membrane surface area resulting from the presence of the fibrils.     

A MOLECULAR MOTOR FOR GLIDING MOTILITY IN CYANOBACTERIA

Motile microorganisms either swim, by using flagella or glide over surfaces by mechanisms that are poorly understood. In cyanobacteria, gliding motility appears as a relatively slow and smooth surface-associated translocation in the direction of the long axis of the filaments at rates up to a few micrometers a second. Many filamentous species translocate in a highly coordinated manner. Translational movements are usually accompanied by revolutions around the long axis of the filament. While moving, the cyanobacteria secrete slime which is left behind as a twisted and collapsed thin tube. The observation of the slime secretion process shows that the mucilage is formed as fine bands that emerge in close proximity to the cells cross walls. Ultrastructural studies have revealed that the cyanobacteria possess at their cross walls complex, pore-like organelles, which might be involved in slime secretion. As each cell possess two different sets of pores pointing in opposite direction, the coordinated activity of these structures could explain how the filament can reverse the direction of locomotion. Furthermore, ultrastructural studies have shown that rotating cyanobacteria possess cell surfaces formed by parallel, helically arranged surface fibrils. As the arrangement of these fibrils corresponds with the path of the filaments during locomotion, it might be imaginable that these fibrils serve as screw thread guiding the rotation of the filaments, with the necessary thrust for locomotion being derived from the secretion of slime using the pores at the cross walls.     

Cell behavior and cell-cell communication during fruiting body morphogenesis in Myxococcus xanthus

Formation of spatial patterns of cells from a mass of initially identical cells is a recurring theme in developmental biology. The dynamics that direct pattern formation in biological systems often involve morphogenetic cell movements. An example is fruiting body formation in the gliding bacterium Myxococcus xanthus in which an unstructured population of identical cells rearranges into an asymmetric, stable pattern of multicellular fruiting bodies in response to starvation. Fruiting body formation depends on changes in organized cell movements from swarming to aggregation. The aggregation process is induced and orchestrated by the cell-surface associated 17 kDa C-signal protein. C-signal transmission depends on direct contact between cells. Evidence suggests that C-signal transmission is geometrically constrained to cell ends and that productive C-signal transmission only occurs when cells engage in end-to-end contacts. Here, we review recent progress in the understanding of the pattern formation process that leads to fruiting body formation. Gliding motility in M. xanthus involves two polarly localized gliding machines, the S-machine depends on type IV pili and the A-machine seems to involve a slime extrusion mechanism. Using time-lapse video microscopy the gliding motility parameters controlled by the C-signal have been identified. The C-signal induces cells to move with increased gliding speeds, in longer gliding intervals and with decreased stop and reversal frequencies. The combined effect of the C-signal dependent changes in gliding motility behaviour is an increase in the net-distance travelled by a cell per minute. The identification of the motility parameters controlled by the C-signal in combination with the contact-dependent C-signal transmission mechanism have allowed the generation of a qualitative model for C-signal induced aggregation. In this model, the directive properties of the C-signal are a direct consequence of the contact-dependent signal-transmission mechanism, which is a local event involving direct contact between cells that results in a global organization of cells. This pattern formation process does not depend on a diffusible substance. Rather it depends on a cell-surface associated signal to direct the cells appropriately.     

Type IV pilus of Myxococcus xanthus is a motility apparatus controlled by the frz chemosensory system

Although flagella are the best-understood means of locomotion in bacteria [1], other bacterial motility mechanisms must exist as many diverse groups of bacteria move without the aid of flagella [2-4]. One unusual structure that may contribute to motility is the type IV pilus [5,6]. Genetic evidence indicates that type IV pili are required for social gliding motility (S-motility) in Myxococcus, and twitching motility in Pseudomonas and Neisseria [6,7]. It is thought that type IV pili may retract or rotate to bring about cellular motility [6,8], but there is no direct evidence for the role of pili in cell movements. Here, using a tethering assay, we obtained evidence that the type IV pilus of Myxococcus xanthus functions as a motility apparatus. Pili were required for M. xanthus cells to adhere to solid surfaces and to generate cellular movement using S-motility. Tethered cells were released from the surface at intervals corresponding to the reversal frequency of wild-type cells when gliding on a solid surface. Mutants defective in the control of directional movements and cellular reversals (frz mutants) showed altered patterns of adherence that correlate reversal frequencies with tethering. The behavior of the tethered cells was consistent with a model in which the pili are extruded from one cell pole, adhere to a surface, and then retract, pulling the cell in the direction of the adhering pili. Cellular reversals would result from the sites of pili extrusion switching from one cell pole to another and are controlled by the frz chemosensory system.     

Cell flexibility affects the alignment of model myxobacteria

Myxobacteria are social bacteria that exhibit a complex life cycle culminating in the development of multicellular fruiting bodies. The alignment of rod-shaped myxobacteria cells within populations is crucial for development to proceed. It has been suggested that myxobacteria align due to mechanical interactions between gliding cells and that cell flexibility facilitates reorientation of cells upon mechanical contact. However, these suggestions have not been based on experimental or theoretical evidence. Here we created a computational mass-spring model of a flexible rod-shaped cell that glides on a substratum periodically reversing direction. The model was formulated in terms of experimentally measurable mechanical parameters, such as engine force, bending stiffness, and drag coefficient. We investigated how cell flexibility and motility engine type affected the pattern of cell gliding and the alignment of a population of 500 mechanically interacting cells. It was found that a flexible cell powered by engine force at the rear of the cell, as suggested by the slime extrusion hypothesis for myxobacteria motility engine, would not be able to glide in the direction of its long axis. A population of rigid reversing cells could indeed align due to mechanical interactions between cells, but cell flexibility impaired the alignment.     

The motors powering A-motility in Myxococcus xanthus are distributed along the cell body

Two models have been proposed to explain the adventurous gliding motility of Myxococcus xanthus: (i) polar secretion of slime and (ii) an unknown motor that uses cell surface adhesion complexes that form periodic attachments along the cell length. Gliding movements of the leading poles of cephalexin-treated filamentous cells were observed but not equivalent movements of the lagging poles. This demonstrates that the adventurous-motility motors are not confined to the rear of the cell.     

The function of fimbriae in Myxococcus xanthus. II. The role of fimbriae in cell-cell interactions.

Anti-fimbriae antiserum specifically inhibited swarming but no gliding motility per se in Myxococcus xanthus. However, formation of motile aggregates on agar and clumps in liquid media correlated with the presence of fimbriae. Ethylenediaminetetraacetic acid which inhibited swarming also inhibited fimbriae formation. Direct electron-microscopic observations revealed that fimbriae establish contact with apposing cell surfaces. Intact but not depolymerized fimbriae exhibited hemagglutination activity against guinea pig erythrocytes. This activity was inhibited by mannose, N-acetyl-D-galactosamine, and to a lesser degree by fructose, raffinose, melibiose, and alpha-methyl-D-mannoside. It is concluded that fimbriae are organelles which function to establish and maintain intercellular contacts, perhaps by a lectin-like function, during the coordinated movement of cell aggregates' (swarming) in myxobacteria. This hypothesis is supported by the observations of other workers that genes determining movement of cells in groups also control fimbriation in 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.     

In-Situ Determination of the Mechanical Properties of Gliding or Non-Motile Bacteria by Atomic Force Microscopy under Physiological Conditions without Immobilization

We present a study about AFM imaging of living, moving or self-immobilized bacteria in their genuine physiological liquid medium. No external immobilization protocol, neither chemical nor mechanical, was needed. For the first time, the native gliding movements of Gram-negative Nostoc cyanobacteria upon the surface, at speeds up to 900 µm/h, were studied by AFM. This was possible thanks to an improved combination of a gentle sample preparation process and an AFM procedure based on fast and complete force-distance curves made at every pixel, drastically reducing lateral forces. No limitation in spatial resolution or imaging rate was detected. Gram-positive and non-motile Rhodococcus wratislaviensis bacteria were studied as well. From the approach curves, Young modulus and turgor pressure were measured for both strains at different gliding speeds and are ranging from 20±3 to 105±5 MPa and 40±5 to 310±30 kPa depending on the bacterium and the gliding speed. For Nostoc, spatially limited zones with higher values of stiffness were observed. The related spatial period is much higher than the mean length of Nostoc nodules. This was explained by an inhomogeneous mechanical activation of nodules in the cyanobacterium. We also observed the presence of a soft extra cellular matrix (ECM) around the Nostoc bacterium. Both strains left a track of polymeric slime with variable thicknesses. For Rhodococcus, it is equal to few hundreds of nanometers, likely to promote its adhesion to the sample. While gliding, the Nostoc secretes a slime layer the thickness of which is in the nanometer range and increases with the gliding speed. This result reinforces the hypothesis of a propulsion mechanism based, for Nostoc cyanobacteria, on ejection of slime. These results open a large window on new studies of both dynamical phenomena of practical and fundamental interests such as the formation of biofilms and dynamic properties of bacteria in real physiological conditions.     

Biochemical characterization of a mutant of the yeast Pichia anomala derepressed for malic acid utilization in the presence of glucose

Abstract Myxococcus xanthus cells move over surfaces by gliding motility. The frz signal transduction system is used to control the reversal frequency, and thus the overall direction of movement of M. xanthus cells. We analyzed the behavior of wild-type and frz mutant cells in response to prey bacteria ( Escherichia coli ). Wild-type cells of M. xanthus did not respond to microcolonies of E. coli until they made physical contact. Cells which penetrated a colony remained in the colony until all of the prey cells were digested. Cells of frz mutants also penetrated E. coli microcolonies and digested some of the E. coli cells, but they invariably abandoned the microcolony leaving their food source behind. These observations illustrate the importance of the frz system of signal transduction for the feeding behavior of M. xanthus cells.     

Developmental sensory transduction in Myxococcus xanthus involves methylation and demethylation of FrzCD.

Myxococcus xanthus is a bacterium that moves by gliding motility and exhibits multicellular development (fruiting body formation). The frizzy (frz) mutants aggregate aberrantly and therefore fail to form fruiting bodies. Individual frz cells cannot control the frequency at which they reverse direction while gliding. Previously, FrzCD was shown to exhibit significant sequence similarity to the enteric methyl-accepting chemotaxis proteins. In this report, we show that FrzCD is modified by methylation and that frzF encodes the methyltransferase. We also identify a new gene, frzG, whose predicted product is homologous to that of the cheB (methylesterase) gene from Escherichia coli. Thus, although M. xanthus is unflagellated, it appears to have a sensory transduction system which is similar in many of its components to those found in flagellated bacteria.     

Taxing questions in development

Bacteria use taxis-controlled movement to reach their optimum environment. Chemotaxis is probably the best understood behavioural system in biology, biasing the normal random movement of bacteria using a phospho-relay pathway from receptors to the motility organelles. The pathways are typified by signal recognition and receptor adaptation, enabling bacteria to sense and respond to changing environments. Models have been derived from the single chemosensory pathway of Escherichia coli but the sequencing of an increasing number of bacterial genomes is revealing genes that apparently encode multiple chemosensory pathways. Recently, data have accumulated suggesting that some of these pathways might not control motility, although the mechanisms by which this might happen remain unclear. Information from the soil bacterium Myxococcus xanthus could lead the way to an understanding of such mechanisms.     

Flagella-dependent gliding motility inChlamydomonas

Summary Flagella are generally recognized as organelles of motility responsible for the ability ofChlamydomonas to swim through its environment. However, the same flagella are also responsible for an alternative form of whole cell locomotion, termed gliding. Use of paralyzed flagella mutants demonstrates that gliding is independent of axonemal bend propagation. Gliding motility results from an interaction of the flagellar surface with a solid substrate. Gliding is characterized by bidirectional movements at 1.6±0.3 m/second and occurs when the cell is in a characteristic gliding configuration, where the two flagella are oriented at 180° to one another. A variety of observations suggest that the leading flagellum is responsible for the force transduction resulting in cell locomotion, although both flagella have the capacity to function as the active flagellum. The characteristics of gliding motility have been compared with theChlamydomonas flagellar surface motility phenomenon defined as surface translocation of polystyrene microspheres.     

Cyanobacterial response regulator PatA contains a conserved N-terminal domain (PATAN) with an alpha-helical insertion

The cyanobacterium Anabaena (Nostoc) PCC 7120 responds to starvation for nitrogen compounds by differentiating approximately every 10th cell in the filament into nitrogen-fixing cells called heterocysts. Heterocyst formation is subject to complex regulation, which involves an unusual response regulator PatA that contains a CheY-like phosphoacceptor (receiver, REC) domain at its C-terminus. PatA-like response regulators are widespread in cyanobacteria; one of them regulates phototaxis in Synechocystis PCC 6803. Sequence analysis of PatA revealed, in addition to the REC domain, a previously undetected, conserved domain, which we named PATAN (after PatA N-terminus), and a potential helix-turn-helix (HTH) domain. PATAN domains are encoded in a variety of environmental bacteria and archaea, often in several copies per genome, and are typically associated with REC, Roadblock and other signal transduction domains, or with DNA-binding HTH domains. Many PATAN domains contain insertions of a small additional domain, termed alpha-clip, which is predicted to form a four-helix bundle. PATAN domains appear to participate in protein-protein interactions that regulate gliding motility and processes of cell development and differentiation in cyanobacteria and some proteobacteria, such as Myxococcus xanthus and Geobacter sulfurreducens.     

Study of elastic collisions of Myxococcus xanthus in swarms

In very low density situations where a single myxobacterial cell is isolated from direct contact with other cells, the slime capsule interaction with the substrate or slime tracks on the substrate produce a viscous drag that results in a smooth gliding motion. Viscoelastic interactions of myxobacteria cells in a low-density domain close to the edge of a swarm are studied using a combination of a cell-based three-dimensional computational model and cell-tracking experiments. The model takes into account the flexible nature of Myxococcus xanthus as well as the effects of adhesion between cells arising from the interaction of the capsular polysaccharide covering two cells in contact with each other. New image and dynamic cell curvature analysis algorithms are used to track and measure the change in cell shapes that occur as flexible cells undergo significant bending during collisions resulting in direct calibration of the model parameters. Like aspect-ratio and directional reversals, the flexibility of cells and the adhesive cell-cell and cell-substrate interactions of M. xanthus play an important role in smooth gliding and more efficient 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.