Architecture of the type IVa pilus machine

Many bacteria, including important pathogens, move by projecting grappling-hook–like extensions called type IV pili from their cell bodies. After these pili attach to other cells or objects in their environment, the bacteria retract the pili to pull themselves forward. Chang et al. used electron cryotomography of intact cells to image the protein machines that extend and retract the pili, revealing where each protein component resides. Putting the known structures of the individual proteins in place like pieces of a three-dimensional puzzle revealed insights into how the machine works, including evidence that ATP hydrolysis by cytoplasmic motors rotates a membrane-embedded adaptor that slips pilin subunits back and forth from the membrane onto the pilus

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

Investigating bacteroidetes gliding motility

Bacteroidetes gliding motility is a type of surface motility in which rod-shaped bacteria move up to 2 µm s in a corkscrewing motion. Flavobacterium johnsoniae is the primary model organism for the study of Bacteroidetes gliding. SprB is the main adhesin in this organism and moves in a helix along the cell surface. This movement is guided by an underlying track that is anchored to the inner leaflet of the outer membrane. The essential gliding lipoprotein GldJ, which is helically arranged when visualised in fixed cells, is suggested to form this track. However, direct in vivo imaging of GldJ is yet to be achieved. Two currently outstanding questions about Bacteroidetes gliding motility are 1) how adhesion of SprB to the substratum is controlled so that binding only occurs when moving from the leading to the lagging cell pole and 2) how/if the cell discriminate between the poles. In this thesis, a fusion of the HaloTag domain to SprB enabled labelling of SprB with stable and bright dyes. The movement of SprB could then be visualised using single-particle tracking to reveal the underlying track topology. These tracking data suggest that the underlying track is not a single closed loop currently proposed, but rather a complex and potentially dynamic structure that can form multiple loops and cover most of the cell surface. SprB is encoded by the sprB operon that further encodes RemFG, Fjoh_0982, and SprCDF. In this thesis I show that all these components, except fjoh_0982, are required for gliding motility but only sprF are required for SprB helical movement. All the sprB operon components required for gliding are also required for SprB-mediated attachment to glass, indicating that they regulate adhesion of SprB. RemG and SprCD move in a helix reminiscent of the SprB movement pattern. The helical movement does not depend on SprF or SprB, but rather on the SprFhomologous N-terminal domain of SprD. Observations of gliding cells with fluorescently labelled SprC revealed accumulation of SprC near the leading cell pole. This polar accumulation correlated with the direction of movement and was not observed in cells that did not move. Furthermore, a mutant lacking the C-terminal 50 residues of SprD was unable to accumulate SprC at the leading pole. SprB did not show a similar asymmetric distribution in gliding cells. Fluorescence microscopy shows that helically moving sprB operon proteins accumulate at midcell in dividing cells in a GldJ dependent manner. Cross-linking mass spectrometry indicates that GldJ interacts with the sprB operon proteins as well as GldKNO, essential outer membrane components of the type 9 secretion system which is a pre-requisite for Bacteroidetes gliding motility

Reverse-Engineering Self-Organized Behavior in Myxococcus xanthus Biofilms

Myxococcus xanthus ( M. xanthus ) is a gram-negative, rod-shaped soil-dwelling predatory bacterium. It can move on solid surfaces forming cooperative single-species biofilm in which various self-organizing patterns are observed. Under distinct environmental conditions, these bacteria can swarm outward, form travelling waves or aggregate into fruiting bodies as a result of diverse intercellular interactions, signaling and coordinated cell motility. M. xanthus colony actively expands when food is plentiful, but stops this under nutritional stress and thereafter aggregates into fruiting bodies where individual cells transform into spores. When in direct contact with their prey, M. xanthus cells form traveling cell-density waves called ripples to facilitate their predation. These patterns play an important role in maximizing M. xanthus adaption to the changing environment. While these phenomena have been studied using traditional experimental microbiology and genetics, recently it is becoming clear that system biology approach greatly complements traditional laboratory work. This thesis shows my effort to deepen the understanding of self-organization in microorganisms using statistical image processing techniques and agent-based modeling. Statistical image processing results illustrate that aggregation into fruiting bodies is a highly non-monotonic yet spontaneous process without long-range signal transduction. The agent-based model of aggregation accurately reproduces the final steady states of an aggregation process but fails to reproduce the experimental dynamics. The agent-based modeling for predatory ripples quantitatively reproduces all observed patterns based on three simple experimentally observed rules: regular cellular reversals, side-to-side contact induced early reversals and refractory period after each cellular reversal. Moreover, the agent-based model predicts that predatory ripples speed up the swarm expansion into the prey region and keep individual M. xanthus cells in the prey region longer. These predictions are all quantitatively verified by experimental observations. The combination of statistical image analysis and agent-based modeling brings greater understanding of self-organizing patterns in M. xanthus and will be essential for further research on similar patterns in other microorganisms and higher organisms

The mechanism of force transmission at bacterial focal adhesion complexes

Various rod-shaped bacteria mysteriously glide on surfaces in the absence of appendages such as flagella or pili. In the deltaproteobacterium Myxococcus xanthus, a putative gliding motility machinery (the Agl–Glt complex) localizes to so-called focal adhesion sites (FASs) that form stationary contact points with the underlying surface. Here we show that the Agl–Glt machinery contains an inner-membrane motor complex that moves intracellularly along a right-handed helical path; when the machinery becomes stationary at FASs, the motor complex powers a left-handed rotation of the cell around its long axis. At FASs, force transmission requires cyclic interactions between the molecular motor and the adhesion proteins of the outer membrane via a periplasmic interaction platform, which presumably involves contractile activity of motor components and possible interactions with peptidoglycan. Our results provide a molecular model of bacterial gliding motility

Understanding morphogenesis in myxobacteria from a theoretical and experimental perspective

Several species of bacteria exhibit multicellular behaviour, with individuals cells cooperatively working together within a colony. Often this has communal benefit since multiple cells acting in unison can accomplish far more than an individual cell can and the rewards can be shared by many cells. Myxobacteria are one of the most complex of the multicellular bacteria, exhibiting a number of different spatial phenotypes. Colonies engage in multiple emergent behaviours in response to starvation culminating in the formation of massive, multicellular fruiting bodies. In this thesis, experimental work and theoretical modelling are used to investigate emergent behaviour in myxobacteria. Computational models were created using FABCell, an open source software modelling tool developed as part of the research to facilitate modelling large biological systems. The research described here provides novel insights into emergent behaviour and suggests potential mechanisms for allowing myxobacterial cells to go from a vegetative state into a fruiting body. A differential equation model of the Frz signalling pathway, a key component in the regulation of cell motility, is developed. This is combined with a three-dimensional model describing the physical characteristics of cells using Monte Carlo methods, which allows thousands of cells to be simulated. The unified model explains how cells can ripple, stream, aggregate and form fruiting bodies. Importantly, the model copes with the transition between stages showing it is possible for the important myxobacteria control systems to adapt and display multiple behaviours

Iron regulation in the myxobacterium Myxococcus xanthus DK1622

The myxobacterium Myxococcus xanthus DK1622 is a reliable producer of different secondary metabolites with partially unknown bioactivities. In the present work the response of iron availibility were evaluated, concerning effects on growth, proteome profile and secondary metabolite production. The production of the siderophore myxochelin A was increased by the factor 81, myxochelin B by the factor 678. Unexpectedly, several other secondary metabolite production rates were found influenced, as e.g. myxochromids und cittilins. In proteome analysis, 1979 protein spots were detected in average, whereof 172 exhibited an iron-induced change in expression. A subsequent analysis by tandem mass spectrometry identified 169 of these spots as 131 individual proteins, some with up to 3 protein-phosphorylations. Furthermore, the functions of some, interesting proteins were investigated by knockout of the respective coding gene. At all, 12 single crossover mutants were generated and compared in iron-rich environment concerning effects on growth and rates of iron uptake or secondary metabolite production to the wild type strain. Typically, mutant strains show variations in all three parameters. An in-frame deletion mutant in one of the two fur genes (MXAN_6967) exhibited reduced growth and a decrease in iron uptake (ca. 49 % of the wild type). Additionally, production of all seven monitored secondary metabolites cannot be explained with the traditional Fur model, which suggests a new, unexpected regulation in M. xanthus.Das Myxobakterium Myxococcus xanthus DK1622 ist ein verlässlicher Produzent verschiedenster Sekundärmetabolite mit teilweise unbekannten biologische Aktivitäten. In der vorliegenden Arbeit wurde der Einfluss der Verfügbarkeit von Eisen auf Wachstum, Proteom-Muster und Sekundärmetabolit-Produktion untersucht. Die Produktion der Siderophore Myxochelin A wurde bei Eisenlimitierung um den Faktor 81 gesteigert, Myxochelin B um den Faktor 678. Unerwarteterweise wurden auch weitere Sekundärmetabolite-Produktionen stark beeinflusst, wie z.B. Myxochromid und Cittilin. In Proteomexperimenten konnten von durchschnittlich 1979 detektierten Proteinspots für 172 eine eiseninduzierte Konzentrationsveränderung gezeigt werden. Hiervon wurden 169 Spots mittels Tandem-Massenspektrometrie identifiziert als 131 individuelle Proteine mit bis zu 3 Phosphorylierungen. Des Weiteren wurde die Rolle von interessanten Proteinen durch Knockout entsprechender, codierender Genom-Bereiche untersucht. Insgesamt konnten 12 single-crossover Mutanten generiert werden, welche in eisenreicher Umgebung bezüglich Wachstum, Eisenaufnahme-Raten und Sekundärmetabolit-Produktionsraten mit dem Wildtyp-Stamm verglichen wurden, wobei die Mehrzahl Abweichungen vom Wildtyp in allen drei Parametern zeigten. Eine in-frame Deletionsmutante von einem der beiden fur-Gene (MXAN_6967) zeigte im Verglich mit dem Wildtyp reduziertes Wachstum. Die Verminderung der Eisenaufnahmeraten (49 % des Wildtyps) und Abnahme der Produktionsraten alle sieben Sekundärmetabolte kann mit dem traditionellen Fur-Model nicht erklärt werden, was eine neue, unerwartete Regulation bei M. xanthus nahe legt

Shared behavioral mechanisms underlie <i>C. elegans</i> aggregation and swarming

In complex biological systems, simple individual-level behavioral rules can give rise to emergent group-level behavior. While collective behavior has been well studied in cells and larger organisms, the mesoscopic scale is less understood, as it is unclear which sensory inputs and physical processes matter a priori. Here, we investigate collective feeding in the roundworm C. elegans at this intermediate scale, using quantitative phenotyping and agent-based modeling to identify behavioral rules underlying both aggregation and swarming—a dynamic phenotype only observed at longer timescales. Using fluorescence multi-worm tracking, we quantify aggregation in terms of individual dynamics and population-level statistics. Then we use agent-based simulations and approximate Bayesian inference to identify three key behavioral rules for aggregation: cluster-edge reversals, a density-dependent switch between crawling speeds, and taxis towards neighboring worms. Our simulations suggest that swarming is simply driven by local food depletion but otherwise employs the same behavioral mechanisms as the initial aggregation

Rules and Exceptions: The Role of Chromosomal ParB in DNA Segregation and Other Cellular Processes

Abstract: The segregation of newly replicated chromosomes in bacterial cells is a highly coordinated spatiotemporal process. In the majority of bacterial species, a tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target(s) parS sequence(s), facilitates the initial steps of chromosome partitioning. ParB nucleates around parS(s) located in the vicinity of newly replicated oriCs to form large nucleoprotein complexes, which are subsequently relocated by ParA to distal cellular compartments. In this review, we describe the role of ParB in various processes within bacterial cells, pointing out interspecies differences. We outline recent progress in understanding the ParB nucleoprotein complex formation and its role in DNA segregation, including ori positioning and anchoring, DNA condensation, and loading of the structural maintenance of chromosome (SMC) proteins. The auxiliary roles of ParBs in the control of chromosome replication initiation and cell division, as well as the regulation of gene expression, are discussed. Moreover, we catalog ParB interacting proteins. Overall, this work highlights how different bacterial species adapt the DNA partitioning ParAB-parS system to meet their specific requirements

Understanding morphogenesis in myxobacteria from a theoretical and experimental perspective

Several species of bacteria exhibit multicellular behaviour, with individuals cells cooperatively working together within a colony. Often this has communal benefit since multiple cells acting in unison can accomplish far more than an individual cell can and the rewards can be shared by many cells. Myxobacteria are one of the most complex of the multicellular bacteria, exhibiting a number of different spatial phenotypes. Colonies engage in multiple emergent behaviours in response to starvation culminating in the formation of massive, multicellular fruiting bodies. In this thesis, experimental work and theoretical modelling are used to investigate emergent behaviour in myxobacteria. Computational models were created using FABCell, an open source software modelling tool developed as part of the research to facilitate modelling large biological systems. The research described here provides novel insights into emergent behaviour and suggests potential mechanisms for allowing myxobacterial cells to go from a vegetative state into a fruiting body. A differential equation model of the Frz signalling pathway, a key component in the regulation of cell motility, is developed. This is combined with a three-dimensional model describing the physical characteristics of cells using Monte Carlo methods, which allows thousands of cells to be simulated. The unified model explains how cells can ripple, stream, aggregate and form fruiting bodies. Importantly, the model copes with the transition between stages showing it is possible for the important myxobacteria control systems to adapt and display multiple behaviours.EThOS - Electronic Theses Online ServiceEngineering and Physical Sciences Research Council (Great Britain) (EPSRC)GBUnited Kingdo