Soil Bacterium Myxococcus Xanthus Research Articles

Cell behaviors underlying Myxococcus xanthus aggregate dispersal.

The soil bacterium Myxococcus xanthus is a model organism with a set of diverse behaviors. These behaviors include the starvation-induced multicellular development program, in which cells move collectively to assemble multicellular aggregates. After initial aggregates have formed, some will disperse, with smaller aggregates having a higher chance of dispersal. Initial aggregation is driven by two changes in cell behavior: cells slow down inside of aggregates and bias their motion by reversing direction less frequently when moving toward aggregates. However, the cell behaviors that drive dispersal are unknown. Here, we use fluorescent microscopy to quantify changes in cell behavior after initial aggregates have formed. We observe that after initial aggregate formation, cells adjust the bias in reversal timings by initiating reversals more rapidly when approaching unstable aggregates. Using agent-based modeling, we then show dispersal is predominantly generated by this change in bias, which is strong enough to overcome slowdown inside aggregates. Notably, the change in reversal bias is correlated with the nearest aggregate size, connecting cellular activity to previously observed correlations between aggregate size and fate. To determine if this connection is consistent across strains, we analyze a second M. xanthus strain with reduced levels of dispersal. We find that far fewer cells near smaller aggregates modified their bias. This implies that aggregate dispersal is under genetic control, providing a foundation for further investigations into the role it plays in the life cycle of M. xanthus.IMPORTANCEUnderstanding the processes behind bacterial biofilm formation, maintenance, and dispersal is essential for addressing their effects on health and ecology. Within these multicellular communities, various cues can trigger differentiation into distinct cell types, allowing cells to adapt to their specific local environment. The soil bacterium Myxococcus xanthus forms biofilms in response to starvation, marked by cells aggregating into mounds. Some aggregates persist as spore-filled fruiting bodies, while others disperse after initial formation for unknown reasons. Here, we use a combination of cell tracking analysis and computational simulations to identify behaviors at the cellular level that contribute to aggregate dispersal. Our results suggest that cells in aggregates actively determine whether to disperse or persist and undergo a transition to sporulation based on a self-produced cue related to the aggregate size. Identifying these cues is an important step in understanding and potentially manipulating bacterial cell-fate decisions.

Allopatric divergence of cooperators confers cheating resistance and limits effects of a defector mutation

BackgroundSocial defectors may meet diverse cooperators. Genotype-by-genotype interactions may constrain the ranges of cooperators upon which particular defectors can cheat, limiting cheater spread. Upon starvation, the soil bacterium Myxococcus xanthus cooperatively develops into spore-bearing fruiting bodies, using a complex regulatory network and several intercellular signals. Some strains (cheaters) are unable to sporulate effectively in pure culture due to mutations that reduce signal production but can exploit and outcompete cooperators within mixed groups.ResultsIn this study, interactions between a cheater disrupted at the signaling gene csgA and allopatrically diversified cooperators reveal a very small cheating range. Expectedly, the cheater failed to cheat on all natural-isolate cooperators owing to non-cheater-specific antagonisms. Surprisingly, some lab-evolved cooperators had already exited the csgA mutant’s cheating range after accumulating fewer than 20 mutations and without experiencing cheating during evolution. Cooperators might also diversify in the potential for a mutation to reduce expression of a cooperative trait or generate a cheating phenotype. A new csgA mutation constructed in several highly diverged cooperators generated diverse sporulation phenotypes, ranging from a complete defect to no defect, indicating that genetic backgrounds can limit the set of genomes in which a mutation creates a defector.ConclusionsOur results demonstrate that natural populations may feature geographic mosaics of cooperators that have diversified in their susceptibility to particular cheaters, limiting defectors’ cheating ranges and preventing them from spreading. This diversification may also lead to variation in the phenotypes generated by any given cooperation-gene mutation, further decreasing the chance of a cheater emerging which threatens the persistence of cooperation in the system.

The predatory soil bacterium Myxococcus xanthus combines a Tad- and an atypical type 3-like protein secretion system to kill bacterial cells.

Predatory Myxobacteria employ a multilayered predation strategy to kill and lyse soil microorganisms. Aiming to dissect the mechanism of contact-dependent killing of bacteria, we analyze four protein secretion systems in Myxococcus xanthus and investigate the predation of mutant strains on different timescales. We find that a Tad-like and a type 3-like secretion system (Tad and T3SS∗) fulfill distinct functions during contact-dependent prey killing: the Tad-like system is necessary to induce prey cell death, while the needle-less T3SS∗ initiates prey lysis. Fluorescence microscopy reveals that components of both systems interdependently localize to the predator-prey contact site prior to killing. Swarm expansion assays show that both Tad and T3SS∗ are required to handle live prey and that nutrient extraction from prey bacteria is sufficient to power M.xanthus motility. In conclusion, our observations indicate the functional interplay of two types of secretion systems for killing and lysis of bacterial cells.

Bacterial Glycocalyx Integrity Impacts Tolerance of Myxococcus xanthus to Antibiotics and Oxidative-Stress Agents

The presence of an exopolysaccharide (EPS) layer surrounding bacterial cells, termed a “glycocalyx”, confers protection against toxic molecules. However, the effect of glycocalyx integrity on the tolerance to such agents is poorly understood. Using a modified disc-diffusion assay, we tested the susceptibility to a panel of antibiotics and oxidative stress-inducing compounds of various mutant strains of the social predatory Gram-negative soil bacterium Myxococcus xanthus; the selected mutants were those that manifest different physical states of their respective EPS glycocalyces. While the overall presence of an EPS layer was indeed beneficial for tolerance, the integrity of this layer was also found to affect the susceptibility of the bacterium to killing; however, this finding was not universal, and instead was dependent on the specific compound tested. Thus, the integrity of the cell-surface EPS glycocalyx plays an important role in the tolerance of M. xanthus to harmful compounds.

Topological defects promote layer formation in Myxococcus xanthus colonies

The soil bacterium Myxococcus xanthus lives in densely packed groups that form dynamic three-dimensional patterns in response to environmental changes, such as droplet-like fruiting bodies during starvation. The development of these multicellular structures begins with the sequential formation of cell layers in a process that is poorly understood. Using confocal three-dimensional imaging, we find that motile rod-shaped M. xanthus cells are densely packed and aligned in each layer, forming an active nematic liquid crystal. Cell alignment is nearly perfect throughout the population except at point defects that carry half-integer topological charge. We observe that new cell layers preferentially form at the position of +1/2 defects, whereas holes preferentially open at -1/2 defects. To explain these findings, we model the bacterial colony as an extensile active nematic fluid with anisotropic friction. In agreement with our experimental measurements, this model predicts an influx of cells toward +1/2 defects, and an outflux of cells from -1/2 defects. Our results suggest that cell motility and mechanical cell-cell interactions are sufficient to promote the formation of cell layers at topological defects, thereby seeding fruiting bodies in colonies of M. xanthus.

An ambruticin-sensing complex modulates Myxococcus xanthus development and mediates myxobacterial interspecies communication

Starvation induces cell aggregation in the soil bacterium Myxococcus xanthus, followed by formation of fruiting bodies packed with myxospores. Sporulation in the absence of fruiting bodies can be artificially induced by high concentrations of glycerol through unclear mechanisms. Here, we show that a compound (ambruticin VS-3) produced by a different myxobacterium, Sorangium cellulosum, affects the development of M. xanthus in a similar manner. Both glycerol (at millimolar levels) and ambruticin VS-3 (at nanomolar concentrations) inhibit M. xanthus fruiting body formation under starvation, and induce sporulation in the presence of nutrients. The response is mediated in M. xanthus by three hybrid histidine kinases (AskA, AskB, AskC) that form complexes interacting with two major developmental regulators (MrpC, FruA). In addition, AskB binds directly to the mrpC promoter in vitro. Thus, our work indicates that the AskABC-dependent regulatory pathway mediates the responses to ambruticin VS-3 and glycerol. We hypothesize that production of ambruticin VS-3 may allow S. sorangium to outcompete M. xanthus under both starvation and growth conditions in soil.

Peripheral rods: a specialized developmental cell type in Myxococcus xanthus

In response to nutrient deprivation, the ubiquitous Gram-negative soil bacterium Myxococcus xanthus undergoes a well-characterized developmental response, resulting in the formation of a multicellular fruiting body. The center of the fruiting body consists of myxospores; surrounding this structure are rod-shaped peripheral cells. Unlike spores, the peripheral rods are a metabolically active cell type that inhabits nutrient-deprived environments. The survival characteristics exhibited by peripheral rods, protection from oxidative stress and heat shock, are common survival characteristics exhibited by cells in stationary phase including modifications to morphology and metabolism. Vegetative M. xanthus cells undergo a number of physiological changes during the transition into stationary phase similar to other proteobacteria. In M. xanthus, stationary-phase cells are not considered a component of the developmental response and occur when cells are grown on nutrient-rich plates or in dispersed aqueous media. However, this cell type is not routinely studied and little of its physiology is known. Similarities between these two stress-induced cell types led to the question of whether peripheral rods are actually a distinct developmental cell type or simply cells in stationary phase. In this study, we examine the transcriptome of peripheral rods and its relationship to development. This work demonstrates that peripheral rods are in fact a distinct developmentally differentiated cell type. Although peripheral rods and stationary phase cells display similar characteristics, each transcriptomic pattern is unique and quite different from that of any other M. xanthus cell type.

Kin discrimination and outer membrane exchange in Myxococcus xanthus: A comparative analysis among natural isolates.

Genetically similar cells of the soil bacterium Myxococcus xanthus cooperate at multiple social behaviours, including motility and multicellular development. Another social interaction in this species is outer membrane exchange (OME), a behaviour of unknown primary benefit in which cells displaying closely related variants of the outer membrane protein TraA transiently fuse and exchange membrane contents. Functionally incompatible TraA variants do not mediate OME, which led to the proposal that TraA incompatibilities determine patterns of intercellular cooperation in nature, but how this might occur remains unclear. Using natural isolates from a centimetre-scale patch of soil, we analyse patterns of TraA diversity and ask whether relatedness at TraA is causally related to patterns of kin discrimination in the form of both colony-merger incompatibilities (CMIs) and interstrain antagonisms. A large proportion of TraA functional diversity documented among global isolates is predicted to be contained within this cm-scale population. We find evidence of balancing selection on the highly variable PA14-portion of TraA and extensive transfer of traA alleles across genomic backgrounds. CMIs are shown to be common among strains identical at TraA, suggesting that CMIs are not generally caused by TraA dissimilarity. Finally, it has been proposed that interstrain antagonisms might be caused by OME-mediated toxin transfer. However, we predict that most strain pairs previously shown to exhibit strong antagonisms are incapable of OME due to TraA dissimilarity. Overall, our results suggest that most documented patterns of kin discrimination in a natural population of M.xanthus are not causally related to the TraA sequences of interactants.

Chimeric Synergy in Natural Social Groups of a Cooperative Microbe

Many cooperative species form internally diverse social groups in which individual fitness depends significantly on group-level productivity from cooperation [1-4]. For such species, selection is expected to often disfavor within-group diversity that reduces cooperative productivity [5, 6]. While diversity within social groups is known to enhance productivity in some animals [7-9], diversity within natural groups of social microbes is largely unexamined in this regard. Cells of the soil bacterium Myxococcus xanthus respond to starvation by constructing multicellular fruiting bodies within each of which a subpopulation of cells transforms into stress-resistant spores [10]. Fruiting bodies isolated from soil often harbor substantial endemic diversity [11] that is, nonetheless, lower than between-group diversity, which increases with distance from millimeter to global scales [12-14]. We show that M.xanthus clones isolated from the same fruiting body often collectively produce more viable spores in chimeric groups than expected from sporulation in genetically homogeneous groups. In contrast, chimerism among clones derived from different fruiting bodies tends toreduce group productivity, and it does so increasingly as a function of spatial distance between fruiting-body sample sites. For one fruiting body examined in detail, chimeric synergy-a positive quantitative effect of chimerism on group productivity-is distributed broadly across an interaction network rather than limited to a few interactions. We propose that these results strengthen the plausibility of the hypothesis that selection may operate not only within Myxococcus groups, but also between kin groups to disfavor within-group variation that reduces productivity while allowing some forms of diversity that generate chimeric synergy to persist.

Pervasive, yet idiosyncratic, epistatic pleiotropy during adaptation in a behaviourally complex microbe.

Understanding how multiple mutations interact to jointly impact multiple ecologically important traits is critical for creating a robust picture of organismal fitness and the process of adaptation. However, this is complicated by both environmental heterogeneity and the complexity of genotype-to-phenotype relationships generated by pleiotropy and epistasis. Moreover, little is known about how pleiotropic and epistatic relationships themselves change over evolutionary time. The soil bacterium Myxococcus xanthus employs several distinct social traits across a range of environments. Here, we use an experimental lineage of M.xanthus that evolved a novel form of social motility to address how interactions between epistasis and pleiotropy evolve. Specifically, we test how mutations accumulated during selection on soft agar pleiotropically affect several other social traits (hard agar motility, predation and spore production). Relationships between changes in swarming rate in the selective environment and the four other traits varied greatly over time in both direction and magnitude, both across timescales of the entire evolutionary lineage and individual evolutionary time steps. We also tested how a previously defined epistatic interaction is pleiotropically expressed across these traits. We found that phenotypic effects of this epistatic interaction were highly correlated between soft and hard agar motility, but were uncorrelated between soft agar motility and predation, and inversely correlated between soft agar motility and spore production. Our results show that 'epistatic pleiotropy' varied greatly in magnitude, and often even in sign, across traits and over time, highlighting the necessity of simultaneously considering the interacting complexities of pleiotropy and epistasis when studying the process of adaptation.

Stigmergy co-ordinates multicellular collective behaviours during Myxococcus xanthus surface migration.

Surface translocation by the soil bacterium Myxococcus xanthus is a complex multicellular phenomenon that entails two motility systems. However, the mechanisms by which the activities of individual cells are coordinated to manifest this collective behaviour are currently unclear. Here we have developed a novel assay that enables detailed microscopic examination of M. xanthus motility at the interstitial interface between solidified nutrient medium and a glass coverslip. Under these conditions, M. xanthus motility is characterised by extensive micro-morphological patterning that is considerably more elaborate than occurs at an air-surface interface. We have found that during motility on solidified nutrient medium, M. xanthus forges an interconnected furrow network that is lined with an extracellular matrix comprised of exopolysaccharides, extracellular lipids, membrane vesicles and an unidentified slime. Our observations have revealed that M. xanthus motility on solidified nutrient medium is a stigmergic phenomenon in which multi-cellular collective behaviours are co-ordinated through trail-following that is guided by physical furrows and extracellular matrix materials.

A barrier to homologous recombination between sympatric strains of the cooperative soil bacterium Myxococcus xanthus.

The bacterium Myxococcus xanthus glides through soil in search of prey microbes, but when food sources run out, cells cooperatively construct and sporulate within multicellular fruiting bodies. M. xanthus strains isolated from a 16 × 16-cm-scale patch of soil were previously shown to have diversified into many distinct compatibility types that are distinguished by the failure of swarming colonies to merge upon encounter. We sequenced the genomes of 22 isolates from this population belonging to the two most frequently occurring multilocus sequence type (MLST) clades to trace patterns of incipient genomic divergence, specifically related to social divergence. Although homologous recombination occurs frequently within the two MLST clades, we find an almost complete absence of recombination events between them. As the two clades are very closely related and live in sympatry, either ecological or genetic barriers must reduce genetic exchange between them. We find that the rate of change in the accessory genome is greater than the rate of amino-acid substitution in the core genome. We identify a large genomic tract that consistently differs between isolates that do not freely merge and therefore is a candidate region for harbouring gene(s) responsible for self/non-self discrimination.

Predation by Myxococcus xanthus induces Bacillus subtilis to form spore-filled megastructures.

Biofilm formation is a common mechanism for surviving environmental stress and can be triggered by both intraspecies and interspecies interactions. Prolonged predator-prey interactions between the soil bacterium Myxococcus xanthus and Bacillus subtilis were found to induce the formation of a new type of B. subtilis biofilm, termed megastructures. Megastructures are tree-like brachiations that are as large as 500 μm in diameter, are raised above the surface between 150 and 200 μm, and are filled with viable endospores embedded within a dense matrix. Megastructure formation did not depend on TasA, EpsE, SinI, RemA, or surfactin production and thus is genetically distinguishable from colony biofilm formation on MSgg medium. As B. subtilis endospores are not susceptible to predation by M. xanthus, megastructures appear to provide an alternative mechanism for survival. In addition, M. xanthus fruiting bodies were found immediately adjacent to the megastructures in nearly all instances, suggesting that M. xanthus is unable to acquire sufficient nutrients from cells housed within the megastructures. Lastly, a B. subtilis mutant lacking the ability to defend itself via bacillaene production formed megastructures more rapidly than the parent. Together, the results indicate that production of the megastructure facilitates B. subtilis escape into dormancy via sporulation.

Growth of Myxococcus xanthus in Continuous-Flow-Cell Bioreactors as a Method for Studying Development

Nutrient sensors and developmental timers are two classes of genes vital to the establishment of early development in the social soil bacterium Myxococcus xanthus. The products of these genes trigger and regulate the earliest events that drive the colony from a vegetative state to aggregates, which ultimately leads to the formation of fruiting bodies and the cellular differentiation of the individual cells. In order to more accurately identify the genes and pathways involved in the initiation of this multicellular developmental program in M. xanthus, we adapted a method of growing vegetative populations within a constant controllable environment by using flow cell bioreactors, or flow cells. By establishing an M. xanthus community within a flow cell, we are able to test developmental responses to changes in the environment with fewer concerns for effects due to nutrient depletion or bacterial waste production. This approach allows for greater sensitivity in investigating communal environmental responses, such as nutrient sensing. To demonstrate the versatility of our growth environment, we carried out time-lapse confocal laser scanning microscopy to visualize M. xanthus biofilm growth and fruiting body development, as well as fluorescence staining of exopolysaccharides deposited by biofilms. We also employed the flow cells in a nutrient titration to determine the minimum concentration required to sustain vegetative growth. Our data show that by using a flow cell, M. xanthus can be held in a vegetative growth state at low nutrient concentrations for long periods, and then, by slightly decreasing the nutrient concentration, cells can be allowed to initiate the developmental program.

MasABK Proteins Interact with Proteins of the Type IV Pilin System to Affect Social Motility of Myxococcus xanthus

Gliding motility is critical for normal development of spore-filled fruiting bodies in the soil bacterium Myxococcus xanthus. Mutations in mgl block motility and development but one mgl allele can be suppressed by a mutation in masK, the last gene in an operon adjacent to the mgl operon. Deletion of the entire 5.5 kb masABK operon crippled gliding and fruiting body development and decreased sporulation. Expression of pilAGHI, which encodes type IV pili (TFP) components essential for social (S) gliding, several cryptic pil genes, and a LuxR family protein were reduced significantly in the Δmas mutant while expression of the myxalamide operon was increased significantly. Localization and two-hybrid analysis suggest that the three Mas proteins form a membrane complex. MasA-PhoA fusions confirmed that MasA is an integral cytoplasmic membrane protein with a ≈100 amino acid periplasmic domain. Results from yeast two-hybrid assays showed that MasA interacts with the lipoprotein MasB and MasK, a protein kinase and that MasB and MasK interact with one another. Additionally, yeast two-hybrid analysis revealed a physical interaction between two gene products of the mas operon, MasA and MasB, and PilA. Deletion of mas may be accompanied by compensatory mutations since complementation of the Δmas social gliding and developmental defects required addition of both pilA and masABK.

NMR structure note: N-terminal domain of Thermus thermophilus CdnL

The CarD_CdnL_TRCF protein family (PF02559 in the protein family database; http://pfam.sanger.ac.uk) consists of members that are widely (and exclusively) distributed in bacteria, and for many of these proteins the functions remain to be fully characterized. The family is defined by the *180-residue N-terminal domain of CarD, a global transcriptional regulator in the Gram-negative soil bacterium Myxococcus xanthus required in the activation of lightand starvation-induced genes as well as in other processes (Padmanabhan et al. 2001; Cayuela et al. 2003; Garcia-Moreno et al. 2010). This domain in CarD interacts with CarG, a zinc-associated factor essential in every CarDdependent process (Penalver-Mellado et al. 2006), and with a specific domain in the RNA polymerase (RNAP) b-subunit (Garcia-Moreno et al. 2010). CarD can also bind to DNA via an intrinsically unfolded, *140-residue C-terminal domain resembling eukaryotic high mobility group A (HMGA) proteins (Padmanabhan et al. 2001). Besides CarD and its orthologs (found so far only in myxobacteria; Elias-Arnanz et al. 2010), the CarD_CdnL_TRCF family consists of two other classes of proteins. One class corresponds to that formed by the RNAP-interacting domain, RID, that spans an *70-residue segment of the much larger ([1,000 residues) transcription-repair coupling factor or TRCF, a widely conserved multidomain protein that mediates transcription-coupled repair of DNA lesions encountered by the transcribing complex in bacteria (Selby and Sancar 1995; Deaconescu et al. 2006). The other class within the CarD_CdnL_TRCF family includes several proteins that are typically 150–200 residues long (and so considerably smaller than CarD or TRCF orthologs) and have been denoted as CdnL (for CarD N-terminus-Like) to distinguish them from CarD (Cayuela et al. 2003; GarciaMoreno et al. 2010). CdnL has been found to be essential for cell viability and growth in nearly all of the few bacterial species where it has been examined (Garcia-Moreno et al. 2010; Stallings et al. 2009). However, the functions of CdnL and its mode of action remain enigmatic. In M. xanthus, CdnL has been shown to be functionally distinct from CarD, and to interact with the same domain of RNAP-b as CarD but not with CarG. Despite lacking the ability to directly bind DNA, CdnL localizes to the nucleoid in vivo and directly or indirectly affects cell division (Garcia-Moreno et al. 2010). Mycobacterial CdnL has been reported to also interact with RNAP-b and has been implicated in regulating ribosomal RNA transcription (Stallings et al. 2009). In bacteria where it is present, CdnL often coexists with TRCF and, in myxobacteria, with CarD as well. Since all three proteins interact with the same RNAP-b domain, understanding the molecular details for these interactions would provide significant insights into the interplay among them and their functional differences. Electronic supplementary material The online version of this article (doi:10.1007/s10858-012-9648-z) contains supplementary material, which is available to authorized users.

Identification and Characterization of a Putative Arginine Kinase Homolog from Myxococcus xanthus Required for Fruiting Body Formation and Cell Differentiation

Arginine kinases catalyze the reversible transfer of a high-energy phosphoryl group from ATP to l-arginine to form phosphoarginine, which is used as an energy buffer in insects, crustaceans, and some unicellular organisms. It plays an analogous role to that of phosphocreatine in vertebrates. Recently, putative arginine kinases were identified in several bacterial species, including the social Gram-negative soil bacterium Myxococcus xanthus. It is still unclear what role these proteins play in bacteria and whether they have evolved to acquire novel functions in the species in which they are found. In this study, we biochemically purified and characterized a putative M. xanthus arginine kinase, Ark, and demonstrated that it has retained the ability to catalyze the phosphorylation of arginine by using ATP. We also constructed a null mutation in the ark gene and demonstrated its role in both certain stress responses and development.

MglB Fills a GAP in Bacterial Polarity and Motility

MglB Fills a GAP in Bacterial Polarity and Motility

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Developmental expression of dnaA is required for sporulation and timing of fruiting body formation in Myxococcus xanthus

Inhibition of DNA replication within the first 6 h of development results in a block in the developmental programme in the social soil bacterium Myxococcus xanthus. We have interpreted these data to imply that M. xanthus requires a new round of DNA replication early in the developmental programme. To further understand the role of DNA replication during development in M. xanthus we focused on the regulation of dnaA which encodes the initiator protein of DNA replication. In this work, we demonstrate that immediately upon nutrient deprivation dnaA (MXAN1001) transcript levels decrease to 10-15% of vegetative levels and then transiently increase between 4 and 6 h post initiation. This expression is dependent on several early developmental regulators, including relA (MXAN3204), sigD (MXAN2957) and sdeK (MXAN1014). It is also dependent upon an 85 bp region located just upstream to the dnaA promoter. Our data suggest that while developmental dnaA expression is not essential for development, its expression allows for the proper timing and maximum efficiency of the sporulation process. In addition, we speculate that developmental control of dnaA expression may provide a mechanism for predetermination of cell fate during the differentiation process.