Добірка наукової літератури з теми "Bacterial cell motility"

The bacterial pathogen, Listeria monocytogenes, grows in the cytoplasm of host cells and spreads intercellularly using a form of actin-based motility mediated by the bacterial protein ActA. Tightly adherent monolayers of MDCK cells that constitutively express GFP-actin were infected with L. monocytogenes, and intercellular spread of bacteria was observed by video microscopy. The probability of formation of membrane-bound protrusions containing bacteria decreased with host cell monolayer age and the establishment of extensive cell-cell contacts. After their extension into a recipient cell, intercellular membrane-bound protrusions underwent a period of bacterium-dependent fitful movement, followed by their collapse into a vacuole and rapid vacuolar lysis. Actin filaments in protrusions exhibited decreased turnover rates compared with bacterially associated cytoplasmic actin comet tails. Recovery of motility in the recipient cell required 1–2 bacterial generations. This delay may be explained by acid-dependent cleavage of ActA by the bacterial metalloprotease, Mpl. Importantly, we have observed that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of paracytophagy.

Providencia stuartiiis the most commonProvidenciaspecies capable of causing human infections. CurrentlyP. stuartiiis involved in high incidence of urinary tract infections in catheterized patients. The ability of bacteria to swarm on semisolid (viscous) surfaces and adhere to and invade host cells determines the specificity of the disease pathogenesis and its therapy. In the present study we demonstrated morphological changes ofP. stuartiiNK cells during migration on the viscous medium and discussed adhesive and invasive properties utilizing the HeLa-M cell line as a host model. To visualize the interaction ofP. stuartiiNK bacterial cells with eukaryotic cellsin vitroscanning electron and confocal microscopy were performed. We found that bacteriaP. stuartiiNK are able to adhere to and invade HeLa-M epithelial cells and these properties depend on the age of bacterial culture. Also, to invade the host cells the infectious dose of the bacteria is essential. The microphotographs indicate that after incubation of bacterialP. stuartiiNK cells together with epithelial cells the bacterial cells both were adhered onto and invaded into the host cells.

The bacterial flagellum is a helical filamentous organelle responsible for motility. In bacterial species possessing flagella at the cell exterior, the long helical flagellar filament acts as a molecular screw to generate thrust. Meanwhile, the flagella of spirochetes reside within the periplasmic space and not only act as a cytoskeleton to determine the helicity of the cell body, but also rotate or undulate the helical cell body for propulsion. Despite structural diversity of the flagella among bacterial species, flagellated bacteria share a common rotary nanomachine, namely the flagellar motor, which is located at the base of the filament. The flagellar motor is composed of a rotor ring complex and multiple transmembrane stator units and converts the ion flux through an ion channel of each stator unit into the mechanical work required for motor rotation. Intracellular chemotactic signaling pathways regulate the direction of flagella-driven motility in response to changes in the environments, allowing bacteria to migrate towards more desirable environments for their survival. Recent experimental and theoretical studies have been deepening our understanding of the molecular mechanisms of the flagellar motor. In this review article, we describe the current understanding of the structure and dynamics of the bacterial flagellum.

ABSTRACT We tested the impact of bacterial swimming speed on the survival of planktonic bacteria in the presence of protozoan grazers. Grazing experiments with three common bacterivorous nanoflagellates revealed low clearance rates for highly motile bacteria. High-resolution video microscopy demonstrated that the number of predator-prey contacts increased with bacterial swimming speed, but ingestion rates dropped at speeds of >25 μm s−1 as a result of handling problems with highly motile cells. Comparative studies of a moderately motile strain (<25 μm s−1) and a highly motile strain (>45 μm s−1) further revealed changes in the bacterial swimming speed distribution due to speed-selective flagellate grazing. Better long-term survival of the highly motile strain was indicated by fourfold-higher bacterial numbers in the presence of grazing compared to the moderately motile strain. Putative constraints of maintaining high swimming speeds were tested at high growth rates and under starvation with the following results: (i) for two out of three strains increased growth rate resulted in larger and slower bacterial cells, and (ii) starved cells became smaller but maintained their swimming speeds. Combined data sets for bacterial swimming speed and cell size revealed highest grazing losses for moderately motile bacteria with a cell size between 0.2 and 0.4 μm3. Grazing mortality was lowest for cells of >0.5 μm3 and small, highly motile bacteria. Survival efficiencies of >95% for the ultramicrobacterial isolate CP-1 (≤0.1 μm3, >50 μm s−1) illustrated the combined protective action of small cell size and high motility. Our findings suggest that motility has an important adaptive function in the survival of planktonic bacteria during protozoan grazing.

Skin and wound infections are serious medical problems, and the diversity of bacteria makes such infections difficult to treat. Bacteria possess many virulence factors, among which motility plays a key role in skin infections. This feature allows for movement over the skin surface and relocation into the wound. The aim of this paper is to review the type of bacterial movement and to indicate the underlying mechanisms than can serve as a target for developing or modifying antibacterial therapies applied in wound infection treatment. Five types of bacterial movement are distinguished: appendage-dependent (swimming, swarming, and twitching) and appendage-independent (gliding and sliding). All of them allow bacteria to relocate and aid bacteria during infection. Swimming motility allows bacteria to spread from ‘persister cells’ in biofilm microcolonies and colonise other tissues. Twitching motility enables bacteria to press through the tissues during infection, whereas sliding motility allows cocci (defined as non-motile) to migrate over surfaces. Bacteria during swarming display greater resistance to antimicrobials. Molecular motors generating the focal adhesion complexes in the bacterial cell leaflet generate a ‘wave’, which pushes bacterial cells lacking appendages, thereby enabling movement. Here, we present the five main types of bacterial motility, their molecular mechanisms, and examples of bacteria that utilise them. Bacterial migration mechanisms can be considered not only as a virulence factor but also as a target for antibacterial therapy.

ABSTRACTWe previously demonstrated that bacterial flagellar motility is a fundamental mechanism by which host phagocytes bind and ingest bacteria. Correspondingly, loss of bacterial motility, consistently observed in clinical isolates from chronicPseudomonas aeruginosainfections, enables bacteria to evade association and ingestion ofP. aeruginosaby phagocytes bothin vitroandin vivo. Since bacterial interactions with the phagocyte cell surface are required for type three secretion system-dependent NLRC4 inflammasome activation byP. aeruginosa, we hypothesized that reduced bacterial association with phagocytes due to loss of bacterial motility, independent of flagellar expression, will lead to reduced inflammasome activation. Here we report that inflammasome activation is reduced in response to nonmotileP. aeruginosa. NonmotileP. aeruginosaelicits reduced IL-1β production as well as caspase-1 activation by peritoneal macrophages and bone marrow-derived dendritic cellsin vitro. Importantly, nonmotileP. aeruginosaalso elicits reduced IL-1β levelsin vivoin comparison to those elicited by wild-typeP. aeruginosa. This is the first demonstration that loss of bacterial motility results in reduced inflammasome activation and antibacterial IL-1β host response. These results provide a critical insight into how the innate immune system responds to bacterial motility and, correspondingly, how pathogens have evolved mechanisms to evade the innate immune system.

Phagocytosis of the bacterial pathogen Pseudomonas aeruginosa is the primary means by which the host controls bacterially induced pneumonia during lung infection. Previous studies have identified flagellar swimming motility as a key pathogen-associated molecular pattern (PAMP) recognized by phagocytes to initiate engulfment. Correspondingly, loss of flagellar motility is observed during chronic pulmonary infection with P. aeruginosa, and this likely reflects a selection for bacteria resistant to phagocytic clearance. However, the mechanism underlying the preferential phagocytic response to motile bacteria is unknown. Here we have identified a cellular signaling pathway in alveolar macrophages and other phagocytes that is specifically activated by flagellar motility. Genetic and biochemical methods were employed to identify that phagocyte PI3K/Akt activation is required for bacterial uptake and, importantly, it is specifically activated in response to P. aeruginosa flagellar motility. Based on these observations, the second important finding that emerged from these studies is that titration of the bacterial flagellar motility results in a proportional activation state of Akt. Therefore, the Akt pathway is responsive to, and corresponds with, the degree of bacterial flagellar motility, is independent of the actin polymerization that facilitates phagocytosis, and determines the phagocytic fate of P. aeruginosa. These findings elucidate the mechanism behind motility-dependent phagocytosis of extracellular bacteria and support a model whereby phagocytic clearance exerts a selective pressure on P. aeruginosa populations in vivo, which contributes to changes in pathogenesis during infections.

In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility.

Bacterial motility is a widespread characteristic that can provide several advantages for the cell, allowing it to move towards more favorable conditions and enabling host-associated processes such as colonization. There are different bacterial motility types, and their expression is highly regulated by the environmental conditions. Because of this, methods for studying motility under realistic experimental conditions are required. A wide variety of approaches have been developed to study bacterial motility. Here, we present the most common techniques and recent advances and discuss their strengths as well as their limitations. We classify them as macroscopic or microscopic and highlight the advantages of three-dimensional imaging in microscopic approaches. Lastly, we discuss methods suited for studying motility in bacterial–host interactions, including the use of the zebrafish model.

Chez les bactéries, les pôles cellulaires servent de domaines subcellulaires impliqués dans plusieurs processus cellulaires. Chez l’agent pathogène du choléra, Vibrio cholerae, en forme de bâtonnet incurvé, le pole contenant l’unique flagelle est impliqué dans la virulence. La protéine d’ancrage polaire HubP interagit avec plusieurs ATPases telles que ParA1 (ségrégation des chromosomes), ParC (localisation polaire du système de chimiotaxie) et FlhG (biosynthèse des flagelles), organisant ainsi l'identité polaire de V. cholerae. Cependant, les mécanismes moléculaires exacts de cet ancrage polaire doivent encore être élucidés. L’objectif de cette thèse est d’établir une vue d'ensemble de l'organisation de pôle cellulaire ce qui implique le mécanisme d’orchestration des différentes fonctions cellulaires par l’identification de l’ensemble des partenaires d'interaction de HubP ainsi que la cartographie fine du pôle cellulaire par microscopie à super résolution (PALM). Afin d’identifier de nouveaux partenaires d'interaction de HubP, j'ai étudié la différence de composition en protéines polaires entre les contextes HubP+ et HubP-. La composition en protéines polaires a été quantifiée de manière relative et absolue en ajoutant des Tag isobares aux protéines extraites de mini-cellules. Ces mini-cellules correspondent des petits compartiments cellulaires issus d’un évènement de division anormal proche du pole et sont enrichies en protéines polaires. Parmi ~800 protéines identifiées, ~ 80 protéines ont été considérées comme enrichies en contexte HubP+ incluant de nombreuses protéines attendues (FlhG, ParC et en aval des protéines de chimiotaxie). J'ai étudié la localisation de 14 protéines par microscopie à fluorescence et pu révéler 4 nouvelles protéines présentant une localisation polaire dépendant de HubP : VbrX, VbrY, et 2 protéines hypothétiques MotV et MotW. La délétion de motV et motW provoque un défaut significatif de propagation dans une gélose molle suggérant une implication dans la chimiotaxie et/ou la motilité. Alors que la microscopie électronique a montré que les deux mutants ont bien un flagelle polaire unique, le suivi-vidéo de leur déplacement a révélé que les deux mutants présentaient des défauts de nage assez distincts: ∆motV est plutôt affecté dans le changement de direction et ∆motW dans la vitesse de déplacement. Des expériences de microscopie fluorescente ont montré que MotV, MotW et HubP présentaient des dynamiques de localisation polaire distinctes au cours du cycle cellulaire. Pour une observation fine du pôle cellulaire par PALM, de nouveaux outils d’analyse d’image à haut débit étaient exigés. La précision des contours des petites cellules bactériennes faiblement contrastées n’est pas suffisante par l’observation en fond clair, j'ai développé une nouvelle technique de marquage avec des protéines fluorescentes photo-activables pour un tracé précis de la membrane interne ou du périplasme. En outre, nous avons créé un logiciel utilisant Matlab appelé Vibio qui intègre le contour de cellule et la liste des molécules obtenues par microscopie à super résolution. La capacité d’analyse à haut débit du logiciel permet d’étudier la distribution des molécules de l’échelle de la cellule unique à une population en orientant les cellules par leur courbure longitudinale. J’ai pu révéler que HubP est principalement localisé du côté convexe du pôle de la cellule, tandis que ses partenaires se situaient principalement au milieu du pôle. Mon travail de thèse a révélé avec succès de nouveaux partenaires d'interaction de HubP et la fonction de certaines protéines dans la motilité cellulaire. J'ai développé une nouvelle technique de microscopie pour une localisation subpolaire précise qui fonctionne bien pour l'analyse d'images PALM dans Vibio. J’ai ainsi pu faire progresser les connaissances de l’orchestration des fonctions polaires chez V. cholerae
In rod shaped bacteria, cell poles serve as important subcellular domains involved in several cellular processes including motility, chemotaxis, protein secretion, antibiotic resistance, and chromosome segregation. In the cholera pathogen Vibrio cholerae, vibrioid rod shape and single polarized flagellum involve in the virulence. Polar landmark protein HubP was shown to interact with multiple ATPases, such as ParA1 (chromosome segregation), ParC (polar localization of chemotaxis apparatus), and FlhG (flagella biosynthesis), thus organizing the polar identity of V. cholerae by tethering proteins to cell pole. However, the exact molecular mechanisms are yet to be elucidated. In this thesis, I tackled to unveil comprehensive view of the cell pole organization which implies the orchestration of different cellular functions, by identifying further interaction partners of HubP as well as drawing conceivable picture of the cell pole by super-resolution photoactivated localization microscopy. To identify new interaction partners of HubP, I used minicells in which cell poles were enriched as they derived from cell division near the cell pole. Difference in protein composition between HubP+ and HubP- minicells were examined by isobaric tags for relative and absolute quantitation. Among ~800 proteins identified, ~80 proteins were considered to be enriched in HubP+ minicells including many expected proteins (FlhG, ParC and downstream chemotaxis proteins). I chose 14 proteins to investigate their subcellular localization with fluorescent microscopy. In conclusion, I discovered 4 proteins that showed polar localization in a HubP-dependent manner. These proteins are VbrX, VbrY, and 2 hypothetical proteins MotV and MotW. ∆motV and ∆motW showed significant defect in a diameter of travel in soft agar plate that suggesting the possible involvement in chemotaxis and/or motility. Whereas electron microscopy showed that both mutants possess intact monotrichous flagellum, video-tracking revealed that the two mutants showed rather distinct defects during swimming: MotV is rather turning mutant while MotW is a speed mutant. Fluorescent microscopy experiments indicated that MotV, MotW and HubP showed distinct polar dynamics over cell cycle. For fine-scale observation of the cell pole by PALM, it was appreciated that novel tools for high-throughput analysis was demanded. Since brightfield images are not sufficient to have accurate contours of small and low contrast bacterial cells, I developed new labeling technique with photoactivatable fluorescent proteins for precise outlining at either inner membrane or periplasm. Furthermore, we created Matlab-based software called Vibio which integrates cell outline and the list of molecules obtained by super-resolution microscopy. High-throughput capability of the software enabled to analyze distribution of detected molecules from single cell to whole bunch of cells in a manner that cells are oriented by cell curvature. These allowed me to discover that HubP is mostly lopsided at the convex side of the cell pole, while its partners mostly located middle of the pole. Altogether, I successfully unveiled 4 novel interaction partners of HubP. I revealed of the function of hypothetical proteins that are involved in cell motility. I developed new labeling technique for precise polar localization that works well for PALM image analysis in Vibio. Therefore, I observed precise polar localization of HubP and other polar proteins

In the presence of a solid-liquid or liquid-air interface, bacteria can choose between a planktonic and a sessile lifestyle. Depending on environmental conditions, cells swimming in close proximity to the interface can irreversibly attach to the surface and grow into three-dimensional aggregates where the majority of cells is sessile and embedded in an extracellular polymer matrix (biofilm). We used microfluidic tools and time lapse microscopy to perform experiments with the polarly flagellated soil bacterium Pseudomonas putida (P. putida), a bacterial species that is able to form biofilms. We analyzed individual trajectories of swimming cells, both in the bulk fluid and in close proximity to a glass-liquid interface. Additionally, surface related growth during the early phase of biofilm formation was investigated. In the bulk fluid, P.putida shows a typical bacterial swimming pattern of alternating periods of persistent displacement along a line (runs) and fast reorientation events (turns) and cells swim with an average speed around 24 micrometer per second. We found that the distribution of turning angles is bimodal with a dominating peak around 180 degrees. In approximately six out of ten turning events, the cell reverses its swimming direction. In addition, our analysis revealed that upon a reversal, the cell systematically changes its swimming speed by a factor of two on average. Based on the experimentally observed values of mean runtime and rotational diffusion, we presented a model to describe the spreading of a population of cells by a run-reverse random walker with alternating speeds. We successfully recover the mean square displacement and, by an extended version of the model, also the negative dip in the directional autocorrelation function as observed in the experiments. The analytical solution of the model demonstrates that alternating speeds enhance a cells ability to explore its environment as compared to a bacterium moving at a constant intermediate speed. As compared to the bulk fluid, for cells swimming near a solid boundary we observed an increase in swimming speed at distances below d= 5 micrometer and an increase in average angular velocity at distances below d= 4 micrometer. While the average speed was maximal with an increase around 15% at a distance of d= 3 micrometer, the angular velocity was highest in closest proximity to the boundary at d=1 micrometer with an increase around 90% as compared to the bulk fluid. To investigate the swimming behavior in a confinement between two solid boundaries, we developed an experimental setup to acquire three-dimensional trajectories using a piezo driven objective mount coupled to a high speed camera. Results on speed and angular velocity were consistent with motility statistics in the presence of a single boundary. Additionally, an analysis of the probability density revealed that a majority of cells accumulated near the upper and lower boundaries of the microchannel. The increase in angular velocity is consistent with previous studies, where bacteria near a solid boundary were shown to swim on circular trajectories, an effect which can be attributed to a wall induced torque. The increase in speed at a distance of several times the size of the cell body, however, cannot be explained by existing theories which either consider the drag increase on cell body and flagellum near a boundary (resistive force theory) or model the swimming microorganism by a multipole expansion to account for the flow field interaction between cell and boundary. An accumulation of swimming bacteria near solid boundaries has been observed in similar experiments. Our results confirm that collisions with the surface play an important role and hydrodynamic interactions alone cannot explain the steady-state accumulation of cells near the channel walls. Furthermore, we monitored the number growth of cells in the microchannel under medium rich conditions. We observed that, after a lag time, initially isolated cells at the surface started to grow by division into colonies of increasing size, while coexisting with a comparable smaller number of swimming cells. After 5:50 hours, we observed a sudden jump in the number of swimming cells, which was accompanied by a breakup of bigger clusters on the surface. After approximately 30 minutes where planktonic cells dominated in the microchannel, individual swimming cells reattached to the surface. We interpret this process as an emigration and recolonization event. A number of complementary experiments were performed to investigate the influence of collective effects or a depletion of the growth medium on the transition. Similar to earlier observations on another bacterium from the same family we found that the release of cells to the swimming phase is most likely the result of an individual adaption process, where syntheses of proteins for flagellar motility are upregulated after a number of division cycles at the surface.
Bakterien sind einzellige Mikroorganismen, die sich in flüssigem Medium mit Hilfe von rotierenden Flagellen, länglichen Fasern aus Proteinen, schwimmend fortbewegen. In Gegenwart einer Grenzfläche und unter günstigen Umweltbedingungen siedeln sich Bakterien an der Oberfläche an und gehen in eine sesshafte Wachstumsphase über. Die Wachstumsphase an der Oberfläche ist gekennzeichnet durch das Absondern von klebrigen, nährstoffreichen extrazellulären Substanzen, welche die Verbindung der Bakterien untereinander und mit der Oberfläche verstärken. Die entstehenden Aggregate aus extrazellulärer Matrix und Bakterien werden als Biofilm bezeichnet. In der vorliegenden Arbeit untersuchten wir ein Bodenbakterium, Pseudomonas putida (P. putida), welches in wässriger Umgebung an festen Oberflächen Biofilme ausbildet. Wir benutzten photolithographisch hergestellte Mikrokanäle und Hochgeschwindigkeits-Videomikroskopie um die Bewegung schwimmender Zellen in verschiedenen Abständen zu einer Glasoberfläche aufzunehmen. Zusätzlich wurden Daten über das parallel stattfindende Wachstum der sesshaften Zellen an der Oberfläche aufgezeichnet. Die Analyse von Trajektorien frei schwimmender Zellen zeigte, dass sich Liniensegmente, entlang derer sich die Zellen in eine konstante Richtung bewegen, mit scharfen Kehrtwendungen mit einem Winkel von 180 Grad abwechseln. Dabei änderte sich die Schwimmgeschwindigket von einem zum nächsten Segment im Mittel um einen Faktor von 2. Unsere experimentellen Daten waren die Grundlage für ein mathematisches Modell zur Beschreibung der Zellbewegung mit alternierender Geschwindigkeit. Die analytische Lösung des Modells zeigt elegant, dass eine Population von Bakterien, welche zwischen zwei Geschwindigkeiten wechseln, signifikant schneller expandiert als eine Referenzpopulation mit Bakterien konstanter Schwimmgeschwindkeit. Im Vergleich zu frei schwimmenden Bakterien beobachteten wir in der Nähe der Oberfläche eine um 15% erhöhte Schwimmgeschwindigkeit der Zellen und eine um 90 % erhöhte Winkel-geschwindigkeit. Außerdem wurde eine signifikant höhere Zelldichte in der Nähe der Grenzfläche gemessen. Während sich der Anstieg in der Winkelgeschwindigkeit durch ein Drehmoment erklären lässt, welches in Oberflächennähe auf den rotierenden Zellkörper und die rotierenden Flagellen wirkt, kann die Beschleunigung und Akkumulation der Zellen bei dem beobachteten Abstand nicht durch existierende Theorien erklärt werden. Unsere Ergebnisse lassen vermuten, dass neben hydrodynamischen Effekten auch Kollisionen mit der Oberfläche eine wichtige Rolle spielen und sich die Rotationsgeschwindigkeit der Flagellenmotoren in der Nähe einer festen Oberfläche grundsätzlich verändert. Unsere Experimente zum Zellwachstum an Oberflächen zeigten, dass sich etwa sechs Stunden nach Beginn des Experiments größere Kolonien an der Kanaloberfläche auflösen und Zellen für ca. 30 Minuten zurück in die schwimmende Phase wechseln. Ergebnisse von mehreren Vergleichsexperimenten deuten darauf hin, dass dieser Übergang nach einer festen Anzahl von Zellteilungen an der Oberfläche erfolgt und nicht durch den Verbrauch des Wachstumsmediums bedingt wird.

Myxococcus Xanthus possède un cycle developpemental multicellulaire entièrement sous la dépendance de la capacité des cellules à se déplacer sur des surfaces solides. M. xanthus possède deux systèmes de motilité génétiquement séparé, une motilité Sociale dépendant des pili de Type IV et une motilité Aventurière dont le mécanisme est encore peu compris. Notre hypothèse de travail est que la motilité Aventurière est qu’en des points régulièrement répartis le long du corps cellulaire soient couplés adhésion et traction de ce corps par une interaction entre des moteurs moléculaire et le cytosquelette d’Actine bactérienne MreB. Mon projet est de caractériser la relation qu’il pourrait y avoir entre le cytosquelette et les points d’adhésion durant la motilité. Pour étudier l’implication du cytosquelette MreB durant le mouvement, nous avons utilisé une approche pharmaceutique utilisant l’A22, une drogue permettant la dépolymérisation rapide et spécifique du cytosquelette sans affecter la viabilité des cellules à court terme. De plus j’ai aussi étudier les interactions possible entre MreB et différentes protéines de motilité comme la petite GTPase MglA, qui est connue pour est essentielle au recrutement des machineries de motilité
Myxococcus xanthus has a multicellular developmental cycle which is dependent on the capacity of the cells to move accross solid surfaces. M. xanthus uses two motility systems: Social motility system is dependent on Type-IV pili, and the Adventurous motility system, the mechanism of which is poorly understood. Our working hypothesis is that Adventurous motility is performed by adhesion points localized along the cell body where a molecular machinery pulls the cell body by interacting with the MreB cytoskeleton. My project aims to characterize the relationship between the adhesion points and the cytoskeleton during movement. To study the involvement of MreB during motility we use A22, a drug known to rapidly and specifically depolymerise in live microscopy assays. Furthermore, I have study also the interactions between MreB and differents proteins like MglA a small GTPase, which we belive is essential for the recruitment of the machineries

Bacillus thuringiensis est capable de former un biofilm à l’interface air-liquide dans des tubes en verre en condition statique. Pendant la formation du biofilm, deux populations coexistent : une population sessile qui flotte sur le milieu de culture et une population planctonique, située dans le milieu de culture sous la pellicule. En utilisant des méthodes spectrophotométriques, nous avons suivi l’évolution de la croissance des bactéries planctoniques et du biofilm de la souche B. thuringiensis 407. Les résultats obtenus montrent qu’au moment où la biomasse sessile augmente rapidement, la population planctonique chute brutalement et descend jusqu’à une valeur proche de zéro. Cette chute de la population planctonique n’est pas observée avec le mutant spoOA de la souche 407 ou avec des souches incapables de former un biofilm, et ne peut pas être attribuée à la sédimentation ou à la lyse cellulaire. Elle est donc consécutive à un recrutement massif des cellules planctoniques par le biofilm en formation. Nous avons visualisé, par microscopie à epi-fluorescence, l’intégration des bactéries planctoniques de la souche 407 dans son biofilm préformé. Les bactéries recrutées sont localisées dans des zones restreintes du biofilm, où la densité des cellules sessiles est faible, ce qui révèle une distribution spatiale hétérogène des cellules immigrantes au sein du biofilm. Pour identifier les mécanismes impliqués dans le recrutement des cellules planctoniques dans le biofilm, nous avons criblé une banque de mutants de la souche 407 obtenus par mutagenèse aléatoire, pour leur capacité à intégrer un biofilm pré-existant. L’un des mutants de la banque, fortement affecté dans sa capacité à intégrer un biofilm, est touché dans le gène Bthur62720. Celui-ci est porté par le plasmide BTB-9P et code pour une protéine de 21 kDa. Cette protéine est sans homologue, et présente un peptide signal, un domaine N-terminal de fonction inconnue et un domaine C-terminal membranaire. En utilisant deux méthodes, l’immunocytochimie et la fusion traductionnelle avec la GFP, nous avons montré que cette protéine est pariétale, polaire et que son domaine N-terminal est cytoplasmique. A l’aide d’un marqueur des phospholipides chargés, le 10-N-nonyl acridine orange, nous avons montré que la délétion de Bthur62720 désorganise la distribution des radeaux lipidiques, qui apparaissent essentiellement polaires chez la souche 407 sauvage. De plus, cette délétion affecte fortement la nage linéaire, mais pas la culbute, ni la présence de flagelles. Ces résultats nous permettent de formuler l’hypothèse que Bthur62720 stabilise les radeaux lipidiques aux pôles. La localisation polaire de ces radeaux, en permettant la formation des complexes de chémorécepteurs, serait nécessaire pour assurer la nage linéaire
Bacillus thuringiensis is able to produce a pellicle at the air-liquid interface in glass tubes under static conditions. During biofilm formation, two populations coexist: a sessile floating population and a planktonic population, located in the culture medium beneath the pellicle. Using spectrophotometric measurements, we followed the growth of both populations during the B. thuringiensis 407 pellicle formation. Our results show that while the biofilm biomass increases rapidly, the planktonic population growth drops sharply. This decrease is not observed with the 407 spoOA mutant or for strains unable to form a biofilm, and cannot be attributed to cell lysis or cell sedimentation. Therefore, it is the result of a massive integration of planktonic cells in the preformed pellicle. We also visualized, using epi-fluorescence microscopy, the integration of planktonic bacteria of the 407 strain in its preformed biofilm. The recruited cells are located in restricted areas of the biofilm, where the density of sessile cells is low, revealing a heterogeneous spatial distribution of the immigrant cells within the biofilm. To identify the mechanisms involved in the recruitment of planktonic cells in the biofilm, we screened a bank of mutants of the 407 strain, obtained by random mutagenesis, for their ability to integrate a pre-existing biofilm. One of the mutants in the library is strongly affected in its ability to integrate a biofilm. This deficiency is caused by the disruption of the Bthur62720 gene, which is carried by the BTB-9p plasmid and encodes a 21 kDa protein. This protein has no homolog and in silico analysis predict a signal peptide, a N-terminal domain of unknown function and a C-terminal membrane domain. Using immunocytochemistry and translational fusion assays with GFP, we showed that this protein is parietal, polar and that its N-terminal domain is cytoplasmic. With a specific dye of charged membrane phospholipids, 10-N-nonyl acridine orange, we showed that the deletion of Bthur002_62720 disorganizes the lipid rafts distribution, which appear essentially polar in wild type strain 407. Moreover, this deletion strongly affects linear swimming, but not bacterial tumbling or the presence of flagella. These results allow us to hypothesis that Bthur62720 stabilizes the lipid rafts located at the cell poles. The polar localization of these rafts, required for the clustering of chemoreceptors, would be necessary to ensure a normal chemotaxis function and thus, bacterial swimming

In the European Union (EU) the authorization of new active substances based on microbial agents is strictly regulated by several Regulations of the European Commission. A large amount of information is needed to achieve the authorization as: a) phylogenetic characterization, b) virulence traits; c) efficacy; d) mechanism of action; e) secondary metabolites production; f) toxic effects on the environment, human and animal. In the last years, there is an increasing interest in biopesticides based on microbial strains, including the bacterial genus Lysobacter, because of its biocontrol features. Specifically, L. capsici AZ78 is can effectively control phytopathogenic oomycetes, such as Plasmopara viticola e Phytophthora infestans. Although, some information about the biocontrol activity of the Lysobacter genus is available, the data that can be used to complete the registration dossier of L. capsici AZ78 are few. Therefore, the aim of the study was to characterize L. capsici AZ78. The genome of L. capsici AZ78 was sequenced and compared with two close-related pathogenic bacteria to confirm the lack of virulence factors to plant and human. The genome of L. capsici AZ78 includes a broad range of specific genes encoding lytic enzyme and in vitro trials confirmed the lytic activity on different substrates. The genome annotation showed the presence of genes involved in the biosynthesis of antibiotics and the antimicrobial activity of L. capsici AZ78 was tested in vitro against bacteria, oomycetes and fungi. The genome mining allowed the identification of genes encoding Type 4 Pilus (T4P), an interestingly motility mechanism that is involved in the host colonization, such as fungal hyphae. Moreover, the identification of a compound that positively modulates the expression of T4P genes and increases the bacterial efficacy against P. viticola, could be an important information for the final formulation of the bacterium. Genes involved in the resistance to environmental stressors and in the environmental fitness of L. capsici AZ78 under field conditions, were identified. Finally, the analysis of the L. capsici AZ78 transcriptome provided a complete overview of the mechanism of action displayed by the bacterium in the interaction with P. infestans. In conclusion the scientific literature produced during this work includes information that can speed up the preparation of the registration dossier of L. capsici AZ78.

In this thesis, I present a detailed analysis of the role cell morphology, solution rheology, and swimming mechanism has on the motility of Helicobacter Pylori. H. Pylori, the bacterium that causes gastric ulcers, has a helical cell shape that has long been believed to provide an advantage in penetrating the viscous mucus layer protecting the stomach lining, its niche environment. I present results obtained by performing optical microscopic live cell bacteria tracking of wild-type H. Pylori and cell shape and flagella mutants of H. Pylori. Bacteria tracking experiments show that helical shaped bacteria swim faster than straight rod-shaped bacteria, and bacteria with larger number of flagella swim faster. Altering cell shape is found to have a smaller effect on swimming speed than altering the number of flagella a bacterium has. These experimental observations are then compared to resistive force theory predictions. Resistive force theory shows qualitative agreement to our experimental observations, but overestimates the increase in swimming speed for a helical cell when compared to straight rod cell. In addition to effect of cell morphology on motility, I explore how motility is altered in different polymer environments by tracking bacteria in pig gastric mucin, methylcellulose, and gelatin solutions and gels. Bacteria are found to increase their swimming speed non-monotonically with increasing polymer concentration, while the number of mobile bacteria is found to decrease with increased polymer concentration. I also present an analysis of the swimming mechanism used by H. Pylori. H. Pylori is found to use a run-reverse swimming mechanism which I model as a random walk. This random walk model fits well to the experimental data and provides a theoretical tool for interpreting H. Pylori’s swimming mechanism. Taken together these results provide a detailed description of the motility of H. Pylori in different media and are applicable to the broad question of how H. pylori infects and colonizes the mucus layer of the stomach.

This work explores the effects of body shape and configuration of flagella on motility of Helicobacter pylori, a helical-shaped bacterium that inhabits the viscoelastic gastric mucosa and causes gastritis, ulcers and gastric cancer. Although it is well known that different shapes produce different hydrodynamic drag thus altering the speed and that helical shapes generate additional thrust this has not been quantitatively established for flagellated bacteria. Using fast time-resolution and high-magnification two-dimensional phase-contrast microscopy to simultaneously image and track individual H. pylori and its rod-shaped isogenic mutant in broth and mucin solutions, the shape as well as rotational and translational speed was determined. In collaboration with Professor Henry Fu and Mehdi Jabbarzadeh the experimental data was used to validate the method of regularized Stokeslets by directly comparing the observed speeds to numerical calculations. The results show that due to relatively slow body rotation rates, the helical shape makes at most a 15% contribution to speeds. In order to explore the effects of arrangement of flagella on motility three different Helicobacter spp. were examined: H. suis (bipolar, multiple flagella), H. cetorum (bipolar, single flagellum) and H. pylori (unipolar, multiple flagella) swimming in broth and mucin. Results show that regardless of media, the flagella bundles of bipolar bacteria can assume one of two configurations interchangeably: extended away from the body or wrapped around it. H. suis predominantly swims with the lagging flagella extended behind the body and the leading flagella wrapped around it, but cases where both bundles are extended or both are wrapped have also been observed. In addition the effects of varying pH on motility of H. suis in broth and mucin were investigated. In broth the rotational speed is not significantly affected by varying pH and the peak of the speed distribution shifts to lower values as the pH decreases. However in mucin the rotational speed decreases by a factor of 20 from pH5 to 4 and the motion is completely hindered below pH4. This indicates that H. suis is unable to move below pH4, in agreement with previous findings on H. pylori, due to gelation of mucin below pH4.

The basic structures of a bacterial and a eukaryotic cell are shown in Fig. 8.1. The differences whose origins call for an explanation are as follows: • The bacterial cell has a rigid outer cell wall, usually made of the peptidoglycan, murein. In eukaryotes, the rigid cell wall is not universal, and cell shape is maintained primarily by an internal cytoskeleton of filaments and microtubules. • Eukaryotic cells have a complex system of internal membranes, including the nuclear envelope, endoplasmic reticulum and lysosomes. • Bacteria have a single circular chromosome, attached to the rigid outer cell wall. In eukaryotes, linear chromosomes are contained within a nuclear envelope, which separates transcription from translation: communication between nucleus and cytoplasm is via pores in the nuclear envelope. • Eukaryotes have a complex cytoskeleton. The actomyosin system powers cell division, phagocytosis, amoeboid motion and the overall contractility to resist osmotic swelling. Microtubules and the associated motor proteins (kinesin, dynein and dynamin) ensure the accurate segregation of chromosomes in mitosis, ciliary motility and the movement of transport vesicles. Intermediate filaments form the structural basis for the association of the endomembranes and nuclear-pore complexes with the chromatin to form the nuclear envelope, while other intermediate filaments help to anchor the nucleus in the cytoplasm. One crucial difference between prokaryotes and most eukaryotes has been omitted from Fig. 8.1: this is the presence of mitochondria, and, in plants and algae, of chloroplasts. The reason for the omission is that, on the scenario for eukaryote origins that seems to us most plausible, these intracellular organelles originated later in time than the structures shown in the figure. The differences between these cell types justifies the recognition of two empires of life (above the kingdom level): Bacteria and Eukaryota (Cavalier-Smith, 199la; Table 8.1). (It is interesting that this taxonomic rank was recognized by Linnaeus.) Within each of the empires, there are two major categories: Bacteria consist of the kingdoms Eubacteria and Archaebacteria, and Eukaryota are divided into the superkingdoms Archaezoa and Metakaryota. The justification for these divisions is as follows. The Archaebacteria, in contrast to the Eubacteria, never have murein cell walls, and their single cell membrane contains isoprenoidal ether rather than acyl ester lipids.

Many pathogens have evolved common mechanisms for effecting the early stages of infecting target organisms, be they plants or animals. These include secretion systems, iron-chelating systems (siderophores), and motility. Secretion systems are the protein complexes used by bacteria to transport substances, particularly proteins, across cell membranes. They are the cellular devices whereby pathogenic bacteria secrete virulence factors that they use either to invade host cells or attack cells biochemically. Pathogens require iron for virulence and have evolved chelation systems to ensure that they can absorb enough. Pathogenic microbes need to attach to epithelial cells and use motility and chemotaxis to penetrate the protective hydrogels that overlay the epithelium. The target organisms have evolved common mechanisms for recognizing when they are being attacked, such as the detection of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors. The pathogens fight back by mutating in various ways including using phagocytosis as an infection strategy.

Many bacteria use motility described as swarming to colonize surfaces and form biofilm. Swarming motility has been shown important to biofilm formation [1], where cells act not as individuals but as coordinated groups to move across surfaces, often within a thin-liquid film [2]. Production of a surfactant during swarm improves bacterial motility by lowering surface tension of the liquid film [2]. The mechanism of cell motion during swarming are currently best described for Escherichia coli and Paenibacillus spp., which spread as monolayers of motile cells [3,4]. For Pseudomonas aeruginosa (P. aeruginosa), which does not swarm as a monolayer, the cell and fluid patterns are difficult to discern using current experimental methods. It is not yet known if swarming P. aeruginosa cells behave solely as swimming cells [5] or if twitching, sliding, or walking motility [6] are also important to swarming.

Abstract Active fluids are often is known as the aqueous suspensions of self-propelled elements such as bacteria, algae, or sperm cells, which their properties fundamentally differ from conventional fluids. Active fluids exhibit remarkable physical manifestations over a wide range of scales, from time-dependent microscopic diffusion to the large-scale colonization of aqueous spaces. Properties of active fluids depend on the behavior of microbial suspensions, among which motility plays a crucial role. In this work, we focus on the effect of microbial growth and aging on microorganism motility. Hence, the motility behavior of cyanobacterium Synechocystis sp. CPCC 534, and its relationship with aging were investigated in a closed microfluidic chip. The growth of Synechocystis cultures was followed from the lag phase, through exponential and linear growth up to the stationary phase. Culture samples were periodically examined; cell populations were measured by spectroscopy technique and cell trajectories were tracked by video-microscopy. Cell trajectory length and average cell motility were extracted from the video recordings and were correlated with the age and growth phase of the bacterium.

PGPR bacteria include taxonomically diverse bacterial species that function for improving plant mineral nutrition, stress tolerance, and disease suppression. A number of PGPR are being developed and commercialized as soil and seed inoculants, but to date, their interactions with resident bacterial populations are still poorly understood, and-almost nothing is known about the effects of soil management practices on their population size and activities. To this end, the original objectives of this research project were: 1) To examine microbial community interactions with plant-growth-promoting rhizobacteria (PGPR) and their plant hosts. 2) To explore the factors that affect PGPR population size and activity on plant root surfaces. In our original proposal, we initially prqposed the use oflow-resolution methods mainly involving the use of PCR-DGGE and PLFA profiles of community structure. However, early in the project we recognized that the methods for studying soil microbial communities were undergoing an exponential leap forward to much more high resolution methods using high-throughput sequencing. The application of these methods for studies on rhizosphere ecology thus became a central theme in these research project. Other related research by the US team focused on identifying PGPR bacterial strains and examining their effective population si~es that are required to enhance plant growth and on developing a simulation model that examines the process of root colonization. As summarized in the following report, we characterized the rhizosphere microbiome of four host plant species to determine the impact of the host (host signature effect) on resident versus active communities. Results of our studies showed a distinct plant host specific signature among wheat, maize, tomato and cucumber, based on the following three parameters: (I) each plant promoted the activity of a unique suite of soil bacterial populations; (2) significant variations were observed in the number and the degree of dominance of active populations; and (3)the level of contribution of active (rRNA-based) populations to the resident (DNA-based) community profiles. In the rhizoplane of all four plants a significant reduction of diversity was observed, relative to the bulk soil. Moreover, an increase in DNA-RNA correspondence indicated higher representation of active bacterial populations in the residing rhizoplane community. This research demonstrates that the host plant determines the bacterial community composition in its immediate vicinity, especially with respect to the active populations. Based on the studies from the US team, we suggest that the effective population size PGPR should be maintained at approximately 105 cells per gram of rhizosphere soil in the zone of elongation to obtain plant growth promotion effects, but emphasize that it is critical to also consider differences in the activity based on DNA-RNA correspondence. The results ofthis research provide fundamental new insight into the composition ofthe bacterial communities associated with plant roots, and the factors that affect their abundance and activity on root surfaces. Virtually all PGPR are multifunctional and may be expected to have diverse levels of activity with respect to production of plant growth hormones (regulation of root growth and architecture), suppression of stress ethylene (increased tolerance to drought and salinity), production of siderophores and antibiotics (disease suppression), and solubilization of phosphorus. The application of transcriptome methods pioneered in our research will ultimately lead to better understanding of how management practices such as use of compost and soil inoculants can be used to improve plant yields, stress tolerance, and disease resistance. As we look to the future, the use of metagenomic techniques combined with quantitative methods including microarrays, and quantitative peR methods that target specific genes should allow us to better classify, monitor, and manage the plant rhizosphere to improve crop yields in agricultural ecosystems. In addition, expression of several genes in rhizospheres of both cucumber and whet roots were identified, including mostly housekeeping genes. Denitrification, chemotaxis and motility genes were preferentially expressed in wheat while in cucumber roots bacterial genes involved in catalase, a large set of polysaccharide degradation and assimilatory sulfate reduction genes were preferentially expressed.