The Mechanistic Basis of Myxococcus xanthus Rippling Behavior and Its Physiological Role during Predation

Myxococcus xanthus cells self-organize into periodic bands of traveling waves, termed ripples, during multicellular fruiting body development and predation on other bacteria. To investigate the mechanistic basis of rippling behavior and its physiological role during predation by this Gram-negative soil bacterium, we have used an approach that combines mathematical modeling with experimental observations. Specifically, we developed an agent-based model (ABM) to simulate rippling behavior that employs a new signaling mechanism to trigger cellular reversals. The ABM has demonstrated that three ingredients are sufficient to generate rippling behavior: (i) side-to-side signaling between two cells that causes one of the cells to reverse, (ii) a minimal refractory time period after each reversal during which cells cannot reverse again, and (iii) physical interactions that cause the cells to locally align. To explain why rippling behavior appears as a consequence of the presence of prey, we postulate that prey-associated macromolecules indirectly induce ripples by stimulating side-toside contact-mediated signaling. In parallel to the simulations, M. xanthus predatory rippling behavior was experimentally observed and analyzed using time-lapse microscopy. A formalized relationship between the wavelength, reversal time, and cell velocity has been predicted by the simulations and confirmed by the experimental data. Furthermore, the results suggest that the physiological role of rippling behavior during M. xanthus predation is to increase the rate of spreading over prey cells due to increased side-to-side contact-mediated signaling and to allow predatory cells to remain on the prey longer as a result of more periodic cell motility

Myxobacteria: Moving, Killing, Feeding, and Surviving Together

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2016.00781Myxococcus xanthus, like other myxobacteria, is a social bacterium that moves and feeds cooperatively in predatory groups. On surfaces, rod-shaped vegetative cells move in search of the prey in a coordinated manner, forming dynamic multicellular groups referred to as swarms. Within the swarms, cells interact with one another and use two separate locomotion systems. Adventurous motility, which drives the movement of individual cells, is associated with the secretion of slime that forms trails at the leading edge of the swarms. It has been proposed that cellular traffic along these trails contributes to M. xanthus social behavior via stigmergic regulation. However, most of the cells travel in groups by using social motility, which is cell contact-dependent and requires a large number of individuals. Exopolysaccharides and the retraction of type IV pili at alternate poles of the cells are the engines associated with social motility. When the swarms encounter prey, the population of M. xanthus lyses and takes up nutrients from nearby cells. This cooperative and highly density-dependent feeding behavior has the advantage that the pool of hydrolytic enzymes and other secondary metabolites secreted by the entire group is shared by the community to optimize the use of the degradation products. This multicellular behavior is especially observed in the absence of nutrients. In this condition, M. xanthus swarms have the ability to organize the gliding movements of 1000s of rods, synchronizing rippling waves of oscillating cells, to form macroscopic fruiting bodies, with three subpopulations of cells showing division of labor. A small fraction of cells either develop into resistant myxospores or remain as peripheral rods, while the majority of cells die, probably to provide nutrients to allow aggregation and spore differentiation. Sporulation within multicellular fruiting bodies has the benefit of enabling survival in hostile environments, and increases germination and growth rates when cells encounter favorable conditions. Herein, we review how these social bacteria cooperate and review the main cell–cell signaling systems used for communication to maintain multicellularity.This work has been funded by the Spanish Government (grants CSD2009-00006 and BFU2012-33248, 70% funded by FEDER) and Junta de Andalucía (group BIO318)

The Nature of Phenotypes: Provoking and Measuring the Dynamic Processes of Swarm Expansion, Predation, and Antibiotic Resistance in Myxococcus Xanthus

The question of how genotype affects phenotype has fascinated and puzzled scientists for generations. Regardless of organism, the correlation between observation and underlying condition is necessary for scientists to understand the world around us. Myxococcus xanthus, a fascinating organism with a large genome (7,314 genes), is known for complex social behaviors and is an excellent model system for the study of this relationship. Upon starvation, M. xanthus cells condense into large multicellular aggregates to await renewed nutrient availability. M. xanthus cells swarm together on surfaces as a predatory biofilm, lysing and killing prey as a singular unit. During predation, swarms display dynamic multicellular patterns called ripples. Individual cells also retain the ability to move independently, leaving behind a slime trail for other cells to follow. This creates complex flare structures at the edge of multicellular swarms. The first part of this thesis addresses the problem of genotype-to-phenotype accuracy in complex organisms. Using 50 single gene knockout M. xanthus strains, I performed the traditional motility phenotype assay as well as a novel motility phenotype assay under magnification. Comparison of these results demonstrates not only that genes not related to motility machinery can induce a motility phenotype, but also that motility phenotypes are dynamic and can change over time. Additionally, I demonstrate that motility phenotype can change significantly when swarms move over prey, as opposed to simple agar surfaces. This more in-depth analysis allows for phenotypic classification of knockout strains previously thought to be without phenotype. The complexity of the M. xanthus genome is not only responsible for its incredibly complex multicellular phenotype, but for its potential use as a model for the study of antibiotic resistance and production of novel antibiotic compounds. The production of secondary metabolites like antibiotics is not ubiquitous in bacteria. The unique resources or building blocks required for their manufacture as well as their complicated molecular scaffolds restricts their production to more complex organisms like M. xanthus. The laboratory-friendly and non-pathogenic nature of M. xanthus adds another category to its list of interesting attributes. By sharing many qualities with pathogenic bacteria while remaining a biosafety level one organism, M. xanthus represents a unique opportunity to study how antibiotic resistance is obtained, maintained, and compensated for in a complex genome. The second part of this thesis studies M. xanthus\u27 usefulness in the fight against antibiotic resistance. I created strains of M. xanthus resistant to a wide variety of antibiotics. Some were exposed to initially low concentrations slowly building over time while others experienced higher antibiotic concentrations over a shorter period. The creation of these strains, simulating the manner in which pathogenic bacteria may gain resistance to antibiotics in vivo, allowed for a laboratory-friendly study of phenotypes and fitness costs associated with varying levels of resistance. I also supply preliminary proof of antibiotic-resistant M. xanthus\u27 utility as a general heterologous host for novel antibiotics. Upon introduction of the oxytetracycline gene cluster, resistant strains were significantly more likely to retain the plasmid than WT. These results represent an important step in both understanding how and why antibiotic resistance arises in complex bacteria as well as a potential mechanism for novel antibiotic discovery

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 coordinate 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

Myxobacteria

Myxobacteria are fascinating and important prokaryotes. They have large genomes and exhibit a wide range of interesting behaviors, including multicellular fruiting body formation, social interaction, predation, and secondary metabolite production. Substantial progress is being made in understanding their ecological roles and the evolutionary forces that have shaped their phenotypes and behaviors. Novel species of myxobacteria are regularly described, often producing unusual metabolites and enzymes which can be of significant biotechnological interest. Molecular studies, ranging in subject from individual enzymes to entire ‘omes, continue to provide rich insights into myxobacterial biology. This collected volume brings together five research articles and three reviews, to provide a snapshot of current myxobacterial research in all its diversity

Wavelength selection of rippling patterns in myxobacteria

Rippling patterns of myxobacteria appear in starving colonies before they aggregate to form fruiting bodies. These periodic traveling cell density waves arise from the coordination of individual cell reversals, resulting from an internal clock regulating them, and from contact signaling during bacterial collisions. Here we revisit a mathematical model of rippling in myxobacteria due to Igoshin et al.\ [Proc. Natl. Acad. Sci. USA {\bf 98}, 14913 (2001) and Phys. Rev. E {\bf 70}, 041911 (2004)]. Bacteria in this model are phase oscillators with an extra internal phase through which they are coupled to a mean-field of oppositely moving bacteria. Previously, patterns for this model were obtained only by numerical methods and it was not possible to find their wavenumber analytically. We derive an evolution equation for the reversal point density that selects the pattern wavenumber in the weak signaling limit, show the validity of the selection rule by solving numerically the model equations and describe other stable patterns in the strong signaling limit. The nonlocal mean-field coupling tends to decohere and confine patterns. Under appropriate circumstances, it can annihilate the patterns leaving a constant density state via a nonequilibrium phase transition reminiscent of destruction of synchronization in the Kuramoto model.Comment: Revtex 26 pages, 7 figure

Dynamic polarity control by a tunable protein oscillator in bacteria

International audienc

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.Comment: Added paragraph on high cell density simulations (new Supp. Figure S6) in Discussion section; Moved cell model and simulation procedure from Supplementary methods to Methods section in Main Tex

Developing a New Mechanical Model for Swarm Development in Myxococcus xanthus & Establishing a Genotype-to-Phenotype Correlation Between Swarm Pattern Formation and Gene Homology

The primary goal of systems biologists is to understand the mechanics underlying complex, collective, self-organizing behaviors displayed by all living systems, from biofilm formation and wound healing to embryogenesis. Myxococcus xanthus is a soil bacterium used as a model organism to study biofilm self-organization. It has a relatively large genome and a complex life cycle that involves two distinct phases. M. xanthus cells can move on agar, and a few million cells will organize to form a predatory biofilm or “swarm” that grows and expands if placed on a nutrient-rich agar surface. If placed on a nutrient-poor agar surface, the same swarm will turn inward and contract, aggregating to form spore-filled multicellular fruiting bodies designed to survive periods of starvation. Extensive progress has been made in identifying genes and genetic pathways that regulate fruiting body formation. However, an accurate description of the dynamics that underlie the process of aggregation is still lacking, and there is still debate and disagreement on the subject. This dissertation provides some explanation regarding individual M. xanthus cell behavior during fruiting body formation, as well as the behavior of aggregates. In the first part of this work, we show that the transition from individual cells to the formation of multicellular aggregates can be controlled through relatively small changes in M. xanthus cell behavior; complicated cell-to-cell signaling, stigmergy (where a trace formed by a cell on an agar surface influences the movement of nearby cells that contact the trace), and cell differentiation are not required for aggregation. We propose that M. xanthus aggregation matches a physical phenomenon that has been characterized in non-living systems, called motility induced phase separation (MIPS). By studying non-reversing mutant cells and manipulating their velocities, we show that cell movement can be made to fall within the boundary of the phase region so that cells succeed in forming aggregates. Alternately, cell movement can be made to fall outside the boundary of the phase region so that cells fail to form aggregates. After the initial stages of aggregation, an M. xanthus swarm actively rearranges the number and relative positions of aggregates by causing some of them to move, merge, or disappear. In the second part of this work, we demonstrate that equations describing Ostwald ripening within a thin liquid film were able to predict aggregate behavior with high accuracy. Consistent with this theory, both relative aggregate size and the distance between aggregates influence the likelihood that a given aggregate will disappear. In general, in neighboring aggregates, the ones that are small and in close proximity will tend to shrink and disappear, while the larger, more isolated aggregates will likely persist and become permanent. By tracking individual cells around aggregates, we show that more cells are leaving shrinking, disappearing aggregates than entering them, while more cells are entering growing, persistent aggregates than leaving them. All of these data are in good agreement with the Ostwald ripening equations. In the last part of this work, we show that aggregation can break down in only a certain number of ways by analyzing single gene disruption of four paralogous gene families representing almost 400 genes. We found that gene families correlate with phenotype, suggesting possible redundancy. In conclusion, my dissertation data provides possible answers to some of the persistent questions regarding M. xanthus developmental dynamics

Biophysics at the coffee shop: lessons learned working with George Oster

Over the past 50 years, the use of mathematical models, derived from physical reasoning, to describe molecular and cellular systems has evolved from an art of the few to a cornerstone of biological inquiry. George Oster stood out as a pioneer of this paradigm shift from descriptive to quantitative biology not only through his numerous research accomplishments, but also through the many students and postdocs he mentored over his long career. Those of us fortunate enough to have worked with George agree that his sharp intellect, physical intuition and passion for scientific inquiry not only inspired us as scientists but also greatly influenced the way we conduct research. We would like to share a few important lessons we learned from George in honor of his memory and with the hope that they may inspire future generations of scientists.Comment: 22 pages, 3 figures, accepted in Molecular Biology of the Cel