Methylation of FrzCD Defines a Discrete Step in the Developmental Program of Myxococcus xanthus

Myxococcus xanthus is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The frz signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized asg, bsg, csg, and esg mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of M. xanthus and that social motility mutants are not blocked in these early developmental steps.     

A Chaperone in the HSP70 Family Controls Production of Extracellular Fibrils in Myxococcus xanthus

Three independent Tn5-lac insertions in the S1 locus of Myxococcus xanthus inactivate the sglK gene, which is nonessential for growth but required for social motility and multicellular development. The sequence of sglK reveals that it encodes a homologue of the chaperone HSP70 (DnaK). The sglK gene is cotranscribed with the upstream grpS gene, which encodes a GrpE homologue. Unlike sglK, grpS is not required for social motility or development. Wild-type M. xanthus is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein. Mutations in sglK inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that sglK mutants do not make fibrils. The fibR gene, located immediately upstream of the grpS-sglK operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida. Inactivation of fibR leads to the overproduction of fibrillin, suggesting that M. xanthus fibril production and Pseudomonas alginate production are regulated in analogous ways. M. xanthus and Pseudomonas exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria.     

Exopolysaccharides promote Myxococcus xanthus social motility by inhibiting cellular reversals

The biofilm‐forming bacterium Myxococcus xanthus moves on surfaces as structured swarms utilizing type IV pili‐dependent social (S) motility. In contrast to isolated cells that reverse their moving direction frequently, individual cells within swarms rarely reverse. The regulatory mechanisms that inhibit cellular reversal and promote the formation of swarms are not well understood. Here we show that exopolysaccharides (EPS), the major extracellular components of M. xanthus swarms, inhibit cellular reversal in a concentration‐dependent manner. Thus, individual wild‐type cells reverse less frequently in swarms due to high local EPS concentrations. In contrast, cells defective in EPS production hyper‐reverse their moving direction and show severe defects in S‐motility. Surprisingly, S‐motility and wild‐type reversal frequency are restored in double mutants that are defective in both EPS production and the Frz chemosensory system, indicating that EPS regulates cellular reversal in parallel to the Frz pathway. Here we clarify that besides functioning as the structural scaffold in biofilms, EPS is a self‐produced signal that coordinates the group motion of the social bacterium M. xanthus.     

Synergism between morphogenetic mutants of Myxococcus xanthus.

Myxococcus xanthus, a social procaryotic microorganism, forms fruiting bodies and myxospores. We have isolated a collection of mutants of M. xanthus that are defective in fruiting morphogenesis and have studied synergistic interaction in pairwise mixtures of these mutants. Certain pairs of these fruiting-defective mutants can fruit when mixed together. Similarly, certain mutants that cannot sporulate under standard fruiting conditions can form myxospores in the presence of wildtype or other nonsporulating mutants. The pattern of synergism between pairs of conditional nonsporulating mutants defines at least three and probably four groups of mutants, such that members of a group cannot synergize with each other but can synergize with members of other groups.     

PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae

Neisseria gonorrhoeae, the Gram-negative aetiological agent of gonorrhoea, is one of many mucosal pathogens of man that expresses competence for natural transformation. Expression of this phenotype by gonococci appears to rely on the expression of type IV pili (Tfp), but the mechanistic basis for this relationship remains unknown. During studies of gonococcal pilus biogenesis, a homologue of the PilT family of proteins, required for Tfp-dependent twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was discovered. Like the findings in these other species, we show here that gonococcal pilT mutants constructed in vitro no longer display twitching motility. In addition, we demonstrate that they have concurrently lost the ability to undergo natural transformation, despite the expression of structurally and morphologically normal Tfp. These results were confirmed by the findings that two classes of spontaneous mutants that failed to express twitching motility and transformability carried mutations in pilT. Piliated pilT mutants and a panel of pilus assembly mutants were found to be deficient in sequence-specific DNA uptake into the cell, the earliest demonstrable step in neisserial competence. The PilT-deficient strains represent the first genetically defined mutants that are defective in DNA uptake but retain Tfp expression.     

Genes required for both gliding motility and development in Myxococcus xanthus

Myxococous xanthus cells can glide both as individual cells, dependent on A dventurous motility (A motility), and as groups of cells, dependent upon S ocial motility (S motility), Tn5-lac mutagenesis was used to generate 16 new A^- and nine new S^- mutations. In contrast with previous results, we find that subsets of A^- mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S^- mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S^- mutants produce a wild-type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5–lac insertions result in surprising phenotypes. Colonies of two S^- mutants glide on ‘swim’ (0.35% agar) plates to form fractal patterns. These S^- mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild-type strain with one S^- insertion resembles the frz^- sglA1^- mutants upon development, suggesting that this S^- gene defines a new chemotaxis component in M. xanthus.     

Functional Organization of a Multimodular Bacterial Chemosensory Apparatus

Chemosensory systems (CSS) are complex regulatory pathways capable of perceiving external signals and translating them into different cellular behaviors such as motility and development. In the δ-proteobacterium Myxococcus xanthus, chemosensing allows groups of cells to orient themselves and aggregate into specialized multicellular biofilms termed fruiting bodies. M. xanthus contains eight predicted CSS and 21 chemoreceptors. In this work, we systematically deleted genes encoding components of each CSS and chemoreceptors and determined their effects on M. xanthus social behaviors. Then, to understand how the 21 chemoreceptors are distributed among the eight CSS, we examined their phylogenetic distribution, genomic organization and subcellular localization. We found that, in vivo, receptors belonging to the same phylogenetic group colocalize and interact with CSS components of the respective phylogenetic group. Finally, we identified a large chemosensory module formed by three interconnected CSS and multiple chemoreceptors and showed that complex behaviors such as cell group motility and biofilm formation require regulatory apparatus composed of multiple interconnected Che-like systems.     

Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Genes controlling movement of single cells

Summary M. xanthus is a gliding bacterium whose motility is subject to intercellular control. Strain DK101 of M. xanthus gives rise to 6 distinct types of nonmotile mutants and transduction of motility between mutants, mediated by the generalized transducing phage Mx8, identifies the gene loci that underlie the six types. Five of the types, B, C, D, E, and F, are contitional mutants that can be stimulated to move by wild-type cells or by cells of a different mutant type. Mutants of each stimulation type lie in separate and distinct loci, cglB, cglC, cglD, cglE and cglF. The sixth mutant type can stimulate any of the five other types to move, never moves itself, and is produced by mutations in at least 17 loci.     

Type IV Pilus Proteins Form an Integrated Structure Extending from the Cytoplasm to the Outer Membrane

The bacterial type IV pilus (T4P) is the strongest biological motor known to date as its retraction can generate forces well over 100 pN. Myxococcus xanthus, a δ-proteobacterium, provides a good model for T4P investigations because its social (S) gliding motility is powered by T4P. In this study, the interactions among M. xanthus T4P proteins were investigated using genetics and the yeast two-hybrid (Y2H) system. Our genetic analysis suggests that there is an integrated T4P structure that crosses the inner membrane (IM), periplasm and the outer membrane (OM). Moreover, this structure exists in the absence of the pilus filament. A systematic Y2H survey provided evidence for direct interactions among IM and OM proteins exposed to the periplasm. For example, the IM lipoprotein PilP interacted with its cognate OM protein PilQ. In addition, interactions among T4P proteins from the thermophile Thermus thermophilus were investigated by Y2H. The results indicated similar protein-protein interactions in the T4P system of this non-proteobacterium despite significant sequence divergence between T4P proteins in T. thermophilus and M. xanthus. The observations here support the model of an integrated T4P structure in the absence of a pilus in diverse bacterial species.     

Contact- and Protein Transfer-Dependent Stimulation of Assembly of the Gliding Motility Machinery in Myxococcus xanthus

Bacteria engage in contact-dependent activities to coordinate cellular activities that aid their survival. Cells of Myxococcus xanthus move over surfaces by means of type IV pili and gliding motility. Upon direct contact, cells physically exchange outer membrane (OM) lipoproteins, and this transfer can rescue motility in mutants lacking lipoproteins required for motility. The mechanism of gliding motility and its stimulation by transferred OM lipoproteins remain poorly characterized. We investigated the function of CglC, GltB, GltA and GltC, all of which are required for gliding. We demonstrate that CglC is an OM lipoprotein, GltB and GltA are integral OM β-barrel proteins, and GltC is a soluble periplasmic protein. GltB and GltA are mutually stabilizing, and both are required to stabilize GltC, whereas CglC accumulate independently of GltB, GltA and GltC. Consistently, purified GltB, GltA and GltC proteins interact in all pair-wise combinations. Using active fluorescently-tagged fusion proteins, we demonstrate that GltB, GltA and GltC are integral components of the gliding motility complex. Incorporation of GltB and GltA into this complex depends on CglC and GltC as well as on the cytoplasmic AglZ protein and the inner membrane protein AglQ, both of which are components of the gliding motility complex. Conversely, incorporation of AglZ and AglQ into the gliding motility complex depends on CglC, GltB, GltA and GltC. Remarkably, physical transfer of the OM lipoprotein CglC to a ΔcglC recipient stimulates assembly of the gliding motility complex in the recipient likely by facilitating the OM integration of GltB and GltA. These data provide evidence that the gliding motility complex in M. xanthus includes OM proteins and suggest that this complex extends from the cytoplasm across the cell envelope to the OM. These data add assembly of gliding motility complexes in M. xanthus to the growing list of contact-dependent activities in bacteria.     

Dual Biochemical Oscillators May Control Cellular Reversals in Myxococcus xanthus

Myxococcus xanthus is a Gram-negative, soil-dwelling bacterium that glides on surfaces, reversing direction approximately once every 6 min. Motility in M. xanthus is governed by the Che-like Frz pathway and the Ras-like Mgl pathway, which together cause the cell to oscillate back and forth. Previously, Igoshin et al. (2004) suggested that the cellular oscillations are caused by cyclic changes in concentration of active Frz proteins that govern motility. In this study, we present a computational model that integrates both the Frz and Mgl pathways, and whose downstream components can be read as motor activity governing cellular reversals. This model faithfully reproduces wildtype and mutant behaviors by simulating individual protein knockouts. In addition, the model can be used to examine the impact of contact stimuli on cellular reversals. The basic model construction relies on the presence of two nested feedback circuits, which prompted us to reexamine the behavior of M. xanthus cells. We performed experiments to test the model, and this cell analysis challenges previous assumptions of 30 to 60 min reversal periods in frzCD, frzF, frzE, and frzZ mutants. We demonstrate that this average reversal period is an artifact of the method employed to record reversal data, and that in the absence of signal from the Frz pathway, Mgl components can occasionally reverse the cell near wildtype periodicity, but frz- cells are otherwise in a long nonoscillating state.     

Experimental social evolution with Myxococcus xanthus

Genetically-based social behaviors are subject to evolutionary change in response to natural selection. Numerous microbial systems provide not only the opportunity to understand the genetic mechanisms underlying specific social interactions, but also to observe evolutionary changes in sociality over short time periods. Here we summarize experiments in which behaviors of the social bacterium Myxococcus xanthus changed extensively during evolutionary adaptation to two relatively asocial laboratory environments. M. xanthus moves cooperatively, exhibits cooperative multicellular development upon starvation and also appears to prey cooperatively on other bacteria. Replicate populations of M. xanthus were evolved in both structured (agar plate) and unstructured (liquid) environments that contained abundant resources. The importance of social cooperation for evolutionary fitness in these habitats was limited by the absence of positive selection for starvation-induced spore production or predatory efficiency. Evolved populations showed major losses in all measured categories of social proficiency- motility, predation, fruiting ability, and sporulation. Moreover, several evolved genotypes were observed to exploit the social behavior of their ancestral parent when mixed together during the developmental process. These experiments that resulted in both socially defective and socially exploitative genotypes demonstrate the power of laboratory selection experiments for studying social evolution at the microbial level. Results from additional selection experiments that place positive selection pressure on social phenotypes can be integrated with direct study of natural populations to increase our understanding of principles that underlie the evolution of microbial social behavior. This revised version was published online in August 2006 with corrections to the Cover Date.     

Myxococcus xanthus Gliding Motors Are Elastically Coupled to the Substrate as Predicted by the Focal Adhesion Model of Gliding Motility

Myxococcus xanthus is a model organism for studying bacterial social behaviors due to its ability to form complex multi-cellular structures. Knowledge of M. xanthus surface gliding motility and the mechanisms that coordinated it are critically important to our understanding of collective cell behaviors. Although the mechanism of gliding motility is still under investigation, recent experiments suggest that there are two possible mechanisms underlying force production for cell motility: the focal adhesion mechanism and the helical rotor mechanism, which differ in the biophysics of the cell–substrate interactions. Whereas the focal adhesion model predicts an elastic coupling, the helical rotor model predicts a viscous coupling. Using a combination of computational modeling, imaging, and force microscopy, we find evidence for elastic coupling in support of the focal adhesion model. Using a biophysical model of the M. xanthus cell, we investigated how the mechanical interactions between cells are affected by interactions with the substrate. Comparison of modeling results with experimental data for cell-cell collision events pointed to a strong, elastic attachment between the cell and substrate. These results are robust to variations in the mechanical and geometrical parameters of the model. We then directly measured the motor-substrate coupling by monitoring the motion of optically trapped beads and find that motor velocity decreases exponentially with opposing load. At high loads, motor velocity approaches zero velocity asymptotically and motors remain bound to beads indicating a strong, elastic attachment.     

Genetic Population Structure of the Soil Bacterium Myxococcus xanthus at the Centimeter Scale

Myxococcus xanthus is a gram-negative soil bacterium best known for its remarkable life history of social swarming, social predation, and multicellular fruiting body formation. Very little is known about genetic diversity within this species or how social strategies might vary among neighboring strains at small spatial scales. To investigate the small-scale population structure of M. xanthus, 78 clones were isolated from a patch of soil (16 by 16 cm) in Tübingen, Germany. Among these isolates, 21 genotypes could be distinguished from a concatemer of three gene fragments: csgA (developmental C signal), fibA (extracellular matrix-associated zinc metalloprotease), and pilA (the pilin subunit of type IV pili). Accumulation curves showed that most of the diversity present at this scale was sampled. The pilA gene contains both conserved and highly variable regions, and two frequency-distribution tests provide evidence for balancing selection on this gene. The functional domains in the csgA gene were found to be conserved. Three instances of lateral gene transfer could be inferred from a comparison of individual gene phylogenies, but no evidence was found for linkage equilibrium, supporting the view that M. xanthus evolution is largely clonal. This study shows that M. xanthus is surrounded by a variety of distinct conspecifics in its natural soil habitat at a spatial scale at which encounters among genotypes are likely.     

Branched-chain fatty acids: the case for a novel form of cell-cell signalling during Myxococcus xanthus development

The esg locus is required for the formation of muiti-cellular fruiting bodies and spores by the developmental bacterium Myxococcus xanthus Studies have suggested that esg mutants are defective in the production of an essential signal (E-signal) used in cell-cell communication and that E-signalling is required for the expression of many developmental genes. Recently we have determined that the esg locus encodes components of a branched-chain keto acid dehydrogenase. a multienzyme complex involved in branched-chain amino acid metabolism in many bacteria and higher organisms. During vegetative growth in M. xanthus. this enzyme complex appears to participate in the production of the branched-chain fatty acids found in this organism. M. xanthus fatty acids (including the branched-chain fatty acids) have been observed to have a variety of effects on developing cells. These effects include; (i) the lysis of M. xanthus cells (autocide activity), (ii) acceleration of the rate of sporulation and (iii) rescue of sporulation by certain development-defective mutants. These and other results suggest a model in which the branched-chain fatty acids. Synthesized during growth, are released from cellular phospholipid by a developmentally regulated phospholipase during fruiting-body formation. This model proposes that one or more of the branched-chain fatty acids that are released constitutes the E-signal which must be transmitted between cells to complete M. xanthus development.     

Killing of Escherichia coli by Myxococcus xanthus in Aqueous Environments Requires Exopolysaccharide-Dependent Physical Contact

Nutrient or niche-based competition among bacteria is a widespread phenomenon in the natural environment. Such interspecies interactions are often mediated by secreted soluble factors and/or direct cell–cell contact. As ubiquitous soil bacteria, Myxococcus species are able to produce a variety of bioactive secondary metabolites to inhibit the growth of other competing bacterial species. Meanwhile, Myxococcus spp. also exhibit sophisticated predatory behavior, an extreme form of competition that is often stimulated by close contact with prey cells and largely depends on the availability of solid surfaces. Myxococcus spp. can also be isolated from aquatic environments. However, studies focusing on the interaction between Myxococcus and other bacteria in such environments are still limited. In this study, using the well-studied Myxococcus xanthus DK1622 and Escherichia coli as model interspecies interaction pair, we demonstrated that in an aqueous environment, M. xanthus was able to kill E. coli in a cell contact-dependent manner and that the observed contact-dependent killing required the formation of co-aggregates between M. xanthus and E. coli cells. Further analysis revealed that exopolysaccharide (EPS), type IV pilus, and lipopolysaccharide mutants of M. xanthus displayed various degrees of attenuation in E. coli killing, and it correlated well with the mutants' reduction in EPS production. In addition, M. xanthus showed differential binding ability to different bacteria, and bacterial strains unable to co-aggregate with M. xanthus can escape the killing, suggesting the specific nature of co-aggregation and the targeted killing of interacting bacteria. In conclusion, our results demonstrated EPS-mediated, contact-dependent killing of E. coli by M. xanthus, a strategy that might facilitate the survival of this ubiquitous bacterium in aquatic environments.     

Type IV‐pili dependent motility is co‐regulated by PilSR and PilS2R2 two‐component systems via distinct pathways in Myxococcus xanthus

Myxococcus xanthus is an environmental bacterium with two forms of motility. One type, known as social motility, is dependent on extension and retraction of Type‐IV pili (T4P) and production of extracellular polysaccharides (EPS). Several signaling systems have been linked to regulation of T4P‐dependent motility. In particular, expression of the pilin subunit pilA requires the PilSR two‐component signaling system (TCS). A second TCS, PilS2R2, encoded within the same locus that encodes PilSR, has also been linked to M. xanthus T4P‐dependent motility. We demonstrate that PilSR and PilS2R2 regulate M. xanthus T4P‐dependent motility through distinct pathways. Consistent with known roles of PilSR, our results indicate that the primary function of PilSR is to regulate expression of pilA. In contrast, PilS2 and PilR2 have little to no affect on PilA protein levels. However, deletion of pilR2 resulted in a reduction of assembled pili, significant decreases in EPS production and loss of T4P‐dependent motility. Furthermore, the pilR2 mutation led to increased production of outer membrane vesicles (OMV). Collectively, we propose that PilS2R2 is required for proper assembly of T4P and regulation of OMV production, and hypothesize that production of these vesicles is related to M. xanthus motility.     

Mechanism for Collective Cell Alignment in Myxococcus xanthus Bacteria

Myxococcus xanthus cells self-organize into aligned groups, clusters, at various stages of their lifecycle. Formation of these clusters is crucial for the complex dynamic multi-cellular behavior of these bacteria. However, the mechanism underlying the cell alignment and clustering is not fully understood. Motivated by studies of clustering in self-propelled rods, we hypothesized that M. xanthus cells can align and form clusters through pure mechanical interactions among cells and between cells and substrate. We test this hypothesis using an agent-based simulation framework in which each agent is based on the biophysical model of an individual M. xanthus cell. We show that model agents, under realistic cell flexibility values, can align and form cell clusters but only when periodic reversals of cell directions are suppressed. However, by extending our model to introduce the observed ability of cells to deposit and follow slime trails, we show that effective trail-following leads to clusters in reversing cells. Furthermore, we conclude that mechanical cell alignment combined with slime-trail-following is sufficient to explain the distinct clustering behaviors observed for wild-type and non-reversing M. xanthus mutants in recent experiments. Our results are robust to variation in model parameters, match the experimentally observed trends and can be applied to understand surface motility patterns of other bacterial species.     

SigF, a new sigma factor required for a motility system of Myxococcus xanthus

A new sigma factor, SigF, was identified from the social and developmental bacterium Myxococcus xanthus. SigF is required for fruiting body formation during development as well as social motility during vegetative growth. Analysis of gene expression indicates that it is possible that the sigF gene is involved in regulation of an unidentified gene for social motility.