Gliding movements in Myxococcus xanthus.

Prokaryotic gliding motility is described as the movement of a cell on a solid surface in the direction of the cell's long axis, but its mechanics are unknown. To investigate the basis of gliding, movements of individual Myxococcus xanthus cells were monitored by employing a video microscopy method by which displacements as small as 0.03 micron could be detected and speeds as low as 1 micron/min could be resolved. Single cells were observed to glide with speeds varying between 1 and 20 microns/min. We found that speed variation was due to differences in distance between the moving cell and the nearest cell. Cells separated by less than one cell diameter (0.5 micron) moved with an average speed of 5.0 micron/min, whereas cells separated by more than 0.5 micron glided with an average speed of 3.8 microns/min. The power to glide was found to be carried separately at both ends of a cell.     

Gliding Motility Mutants of Myxococcus xanthus

Two gliding motility mutants of Myxococcus xanthus are described. The semimotile mutant (SM) originated by high-frequency segregation from the motile FB(t) strain. Segregation was enhanced by acridine dye treatment. SM cells glide only when apposed to other cells in a swarm. The nonmotile strain (NM) originated by mutation from SM. NM cells neither glide individually nor cooperatively. FB(t), SM, and NM are indistinguishable with respect to fine structure, vegetative growth rate, glycerol-induced microcyst formation, spheroplasting, bacteriophage sensitivity, and responses to light. The motility mutants are more resistant to penicillin and more sensitive to actinomycin D than is the gliding wild type. The NM mutant is also a morphogenetic mutant; it is unable to form fruiting bodies.     

Gliding motility in Myxococcus xanthus: mgl locus, RNA, and predicted protein products.

Mutants of Myxococcus xanthus that had lost the ability to glide were examined to elucidate the mechanism of gliding motility. Nonmotile mutants resulting from a single mutational step were all defective at the same locus, mgl, which implied an important role for the mgl product(s) in gliding. Deletion experiments, transposon insertion mutagenesis, and genetic rescue of mgl mutants mapped the locus to a 1.6-kilobase segment of Myxococcus DNA. Two species of RNA that hybridized with mgl DNA were found both during vegetative growth and during the starvation-induced development of fruiting bodies, which also requires cell movement. The two RNA species, of 1.5 and 1.3 kilobases, had the same 5' to 3' orientation and overlapped extensively. The DNA sequences of mgl+ and of seven mgl mutants were determined. Each mutant differed from mgl+ by a single-base-pair change in the sequence. Two adjacent open reading frames were found in the sequence hybridizing to both species of mgl RNA. Six of the single-base-pair changes, each of which would result in a single-amino-acid change, and an insertion-produced mgl mutation were located in the downstream open reading frame. This open reading frame (of 195 amino acids) is therefore an mgl gene, called mglA. The function of the upstream open reading frame is not known with certainty, although it does contain one of the mgl mutant sites and could be a second mgl gene.     

Gliding motility in slide cultures of Myxococcus xanthus in stable and steep chemical gradients.

A method was devised to construct stable and steep chemical gradients in slide cultures to study the movements of gliding cells. The movement of Myxococcus xanthus individual cells and small swarms was studied in these gradients. There was no response to gradients of Casitone and yeast extract that were previously reported to stimulate a positive chemotactic response with M. xanthus.     

From individual cell motility to collective behaviors: insights from a prokaryote, Myxococcus xanthus

In bird flocks, fish schools, and many other living organisms, regrouping among individuals of the same kin is frequently an advantageous strategy to survive, forage, and face predators. However, these behaviors are costly because the community must develop regulatory mechanisms to coordinate and adapt its response to rapid environmental changes. In principle, these regulatory mechanisms, involving communication between individuals, may also apply to cellular systems which must respond collectively during multicellular development. Dissecting the mechanisms at work requires amenable experimental systems, for example, developing bacteria. Myxococcus xanthus, a Gram-negative delatproteobacterium, is able to coordinate its motility in space and time to swarm, predate, and grow millimeter-size spore-filled fruiting bodies. A thorough understanding of the regulatory mechanisms first requires studying how individual cells move across solid surfaces and control their direction of movement, which was recently boosted by new cell biology techniques. In this review, we describe current molecular knowledge of the motility mechanism and its regulation as a lead-in to discuss how multicellular cooperation may have emerged from several layers of regulation: chemotaxis, cell-cell signaling, and the extracellular matrix. We suggest that Myxococcus is a powerful system to investigate collective principles that may also be relevant to other cellular systems.     

Exopolysaccharide-independent social motility of Myxococcus xanthus

Social motility (S motility), the coordinated movement of large cell groups on agar surfaces, of Myxococcus xanthus requires type IV pili (TFP) and exopolysaccharides (EPS). Previous models proposed that this behavior, which only occurred within cell groups, requires cycles of TFP extension and retraction triggered by the close interaction of TFP with EPS. However, the curious observation that M. xanthus can perform TFP-dependent motility at a single-cell level when placed onto polystyrene surfaces in a highly viscous medium containing 1% methylcellulose indicated that "S motility" is not limited to group movements. In an apparent further challenge of the previous findings for S motility, mutants defective in EPS production were found to perform TFP-dependent motility on polystyrene surface in methylcellulose-containing medium. By exploring the interactions between pilin and surface materials, we found that the binding of TFP onto polystyrene surfaces eliminated the requirement for EPS in EPS(-) cells and thus enabled TFP-dependent motility on a single cell level. However, the presence of a general anchoring surface in a viscous environment could not substitute for the role of cell surface EPS in group movement. Furthermore, EPS was found to serve as a self-produced anchoring substrate that can be shed onto surfaces to enable cells to conduct TFP-dependent motility regardless of surface properties. These results suggested that in certain environments, such as in methylcellulose solution, the cells could bypass the need for EPS to anchor their TPF and conduct single-cell S motility to promote exploratory movement of colonies over new specific surfaces.     

Regulation of directed motility in Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that exhibits a complex life cycle. During vegetative growth, cells move as large swarms. However, when starved, cells aggregate into fruiting bodies and sporulate. Both vegetative swarming and developmental aggregation require gliding motility, which involves the slow movement of cells on a solid surface in the absence of flagella. The frequency of cell reversals controls the direction of movement and is regulated by the frz genes, which encode the 'frizzy' signal-transduction proteins. These proteins contain domains which bear striking similarities to the major chemotaxis proteins of the enteric bacteria: CheA, CheY, CheW, CheR, CheB and Tar. However, significant differences exist between the Myxococcus Frz proteins and the enteric Che/MCP proteins. For example, the Frz system contains three CheY-like response-regulator domains: one is present on FrzE, which also contains a CheA-like domain, and two are present on FrzZ, which is a novel protein required for attractant, but not for repellent, responses. The identification of multiple CheY homologues in this system indicates a more complex regulatory pathway than that found in the enteric bacteria. While responses to repellent stimuli appear to follow the enteric paradigm, responses to attractants during vegetative swarming and development are more complex and may involve self-generated autoattractants. The Frz signal-transduction system regulates directed motility in M. xanthus and is essential for controlling both fruiting-body development and vegetative swarming.     

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.     

Multicellular development and gliding motility in Myxococcus xanthus

A great deal of progress has been made in the studies of fruiting body development and social gliding in Myxocococcus xanthus in the past few years. This includes identification of the bone fide C-signal and a receptor for type IV pili, and development of a model for the mechanism of adventurous gliding motility. It is anticipated that the next few years will see even more progress as the complete genome sequence is available and genomic and proteomic tools are applied to the study of M. xanthus social behaviors.     

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.     

Genetic circuitry controlling motility behaviors of Myxococcus xanthus

M. xanthus has a complex multicellular lifestyle including swarming, predation and development. These behaviors depend on the ability of the cells to achieve directed motility across solid surfaces. M. xanthus cells have evolved two motility systems including Type-IV pili that act as grappling hooks and a controversial engine involving mucus secretion and fixed focal adhesion sites. The necessity for cells to coordinate the motility systems and to respond rapidly to environmental cues is reflected by a complex genetic network involving at least three complete sets of chemosensory systems and eukaryotic-like signaling proteins. In this review, we discuss recent advances suggesting that motor synchronization results from spatial oscillations of motility proteins. We further propose that these dynamics are modulated by the action of multiple upstream complementary signaling systems. M. xanthus is thus an exciting emerging model system to study the intricate processes of directed cell migration.     

Chemosensory pathways, motility and development in Myxococcus xanthus

The complex life cycle of Myxococcus xanthus includes predation, swarming, fruiting-body formation and sporulation. The genome of M. xanthus is large and comprises an estimated 7,400 open reading frames, of which approximately 605 code for regulatory genes. These include eight clusters of chemotaxis-like genes that define eight chemosensory pathways, most of which have dedicated functions. Although many of these chemosensory pathways have a role in controlling motility, at least two of these pathways control gene expression during development.     

Surface tension gradients: feasible model for gliding motility of Myxococcus xanthus.

We propose that surface tension is the driving force for the gliding motility of Myxococcus xanthus. Our model requires that the cell be able to excrete surfactant in a polar and reversible fashion. We present calculations that (i) estimate the surface tension difference across a cell necessary to move the cell at the observed rate, which is less than 10(-5) dyn/cm, an extremely small value; (ii) estimate the rate of surfactant excretion necessary to produce the required surface tension difference, a rate that we conclude to be metabolically reasonable; (iii) predict the behavior of cells moving in close apposition to each other, and show that the model is consistent with observed behavior; and (iv) predict the behavior of cells moving in dense swarms. In an accompanying paper we present experimental evidence to support the surface tension model.     

Correlation of energy-dependent cell cohesion with social motility in Myxococcus xanthus.

Membrane-bound ATPase was found in membranes of the archaebacterium Methanosarcina barkeri. The ATPase activity required divalent cations, Mg2+ or Mn2+, and maximum activity was obtained at pH 5.2. The activity was specifically stimulated by HSO3- with a shift of optimal pH to 5.8, and N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. The enzyme could be solubilized from membranes by incubation in 1 mM Tris-maleate buffer (pH 6.9) containing 0.5 mM EDTA. The solubilized ATPase was purified by DEAE-Sepharose and Sephacryl S-300 chromatography. The molecular weight of the purified enzyme was estimated to be 420,000 by gel filtration through Sephacryl S-300. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two classes of subunit, Mr 62,000 (alpha) and 49,000 (beta) associated in the molar ratio 1:1. These results suggest that the ATPase of M. barkeri is similar to the F0F1 type ATPase found in many eubacteria.     

黄色粘球菌的运动机制及其多细胞发育调控的研究进展

粘细菌是原核生物中的“高等生物”,具有特殊的运动能力以及类似真核生物的复杂的多细胞发育生活史,其多细胞发育过程的调控一直是粘细菌研究的热点。近年来,许多关于粘细菌研究的新理论、新学说不断涌现,给予粘细菌研究极大的启发。本文综述了模式菌株黄色粘球菌的运动类型、运动机制以及运动的调控系统,并对其多细胞发育过程的信号传递调控模式进行初步阐释,为更深一步的研究粘细菌复杂的生命调控过程奠定基础。     

Gliding mutants of Myxococcus xanthus with high reversal frequencies and small displacements

Myxococcus xanthus cells move on a solid surface by gliding motility. Several genes required for gliding motility have been identified, including those of the A- and S-motility systems as well as the mgl and frz genes. However, the cellular defects in gliding movement in many of these mutants were unknown. We conducted quantitative, high-resolution single-cell motility assays and found that mutants defective in mglAB or in cglB, an A-motility gene, reversed the direction of gliding at frequencies which were more than 1 order of magnitude higher than that of wild type cells (2.9 min-1 for DeltamglAB mutants and 2.7 min-1 for cglB mutants, compared to 0.17 min-1 for wild-type cells). The average gliding speed of DeltamglAB mutant cells was 40% of that of wild-type cells (on average 1.9 micrometers/min for DeltamglAB mutants, compared to 4.4 micrometers/min for wild-type cells). The mglA-dependent reversals and gliding speeds were dependent on the level of intracellular MglA protein: mglB mutant cells, which contain only 15 to 20% of the wild-type level of MglA protein, glided with an average reversal frequency of about 1.8 min-1 and an average speed of 2.6 micrometers/min. These values range between those exhibited by wild-type cells and by DeltamglAB mutant cells. Epistasis analysis of frz mutants, which are defective in aggregation and in single-cell reversals, showed that a frzD mutation, but not a frzE mutation, partially suppressed the mglA phenotype. In contrast to mgl mutants, cglB mutant cells were able to move with wild-type speeds only when in close proximity to each other. However, under those conditions, these mutant cells were found to glide less often with those speeds. By analyzing double mutants, the high reversing movements and gliding speeds of cglB cells were found to be strictly dependent on type IV pili, encoded by S-motility genes, whereas the high-reversal pattern of mglAB cells was only partially reduced by a pilR mutation. These results suggest that the MglA protein is required for both control of reversal frequency and gliding speed and that in the absence of A motility, type IV pilus-dependent cell movement includes reversals at high frequency. Furthermore, mglAB mutants behave as if they were severely defective in A motility but only partially defective in S 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.