Le système de sécrétion de type VI (SST6) est un complexe multi-protéique permettant l’export d’effecteurs. Ce mécanisme est impliqué à la fois dans la virulence envers les cellules eucaryotes, dans l’activité antibactérienne mais également dans l’acquisition d’ions présents dans ’environnement. Ainsi, le SST6 joue un rôle important dans l’adaptation et la compétition, éléments essentiels dans la colonisation et la persistance au sein d’une niche écologique. Actuellement, très peu d’études portent sur l’importance du SST6 chez des souches environnementales, contrairement aux nombreuses études portant sur des pathogènes tels que Pseudomonas aeruginosa, Burkholderia thailandensis, Vibrio cholerae ou Escherichia coli. Mon sujet de recherche avait pour objectif de caractériser le ou les rôles du SST6 de la souche environnementale Pseudomonas fluorescens MFE01. Ces travaux ont permis d’appréhender certaines fonctions du SST6 de cette souche. Le génome de MFE01 ne comporte qu’un seul cluster de gènes du SST6 où sont regroupés les gènes codant pour la machinerie du SST6 (le « core-component ») à l’exception des gènes hcp. Les protéines Hcp sont des éléments structuraux du SST6 dont elles forment le tube interne qui permet le transfert des effecteurs. Différents gènes hcp sont disséminés sur le chromosome et parmi ces « hcp » orphelins, hcp2 et hcp3 codent respectivement pour les protéines Hcp2 et Hcp3. Ces deux Hcp sécrétées par le SST6, sont associées à l’activité antibactérienne de MFE01 sur différentes souches pathogènes et environnementales, tels que P. aeruginosa, P. fluorescens MFN1032 et Pectobacterium atrosepticum. La protéine Hcp1, codée par le gène orphelin hcp1, est impliquée dans l’inhibition de mobilité de souche compétitrice. Hcp1 permettrait la sécrétion d’au moins deux toxines qui perturberaient l’assemblage du flagelle. Chez MFE01Δhcp1 et MFE01ΔtssC (TssC est un élément de la gaine contractile du SST6), ces toxines seraient accumulées dans le cytoplasme, inhibant ainsi ’assemblage de leur propre flagelle. La surproduction du régulateur FliA, qui contrôle notamment l’assemblage du filament flagellaire, restaure la mobilité chez ces deux mutants. En parallèle, le SST6 de la souche MFE01 est essentiel à la formation et la maturation de biofilm mais également à la compétition bactérienne en biofilm mixte. Ce système interviendrait dans la communication bactérienne indispensable au comportement social, requis lors de l’élaboration des biofilms. / Type VI secretion system (T6SS) is a multiproteic apparatus that secreted proteinaceous effectors. T6SS participate in a variety of functions, whose eukaryote virulence, antibacterial activity or metal ion uptake. These capacities conferring an advantage in adaptation and competition, crucial to colonization or persistence within ecological niche. As well, only a few studies have focused on the T6SS functions of environmental strains, contrary to numerous studies dealing with pathogens as Pseudomonas aeruginosa, Burkholderia thailandensis, Vibrio cholerae or Escherichia coli. The purpose of my research project was to characterize the T6SS function(s) of the environmental strain Pseudomonas fluorescens MFE01. This work had led to understand the various functions of T6SS of MFE01 strain. This strain has a single T6SS cluster where all the core component proteins were gathered, except hcp genes. Three orphan hcp genes where found and are scattered in genome. Hcp proteins form the inner tube allowing effectors secretion. Both Hcp2 and Hcp3 proteins were involved in antibacterial activity on pathogens or environmental strains like P. aeruginosa, P. fluorescens or Pectobacterium atrosepticum. Characterization of Hcp1 proteins role constituted a major focus of this project. Hcp1 proteins participate to motility inhibition of competitive strains through T6SS. Hcp1 may be associated with secretion of at least two toxins perturbing the flagellar filament assembly. In MFE01Δhcp1 and MFE01ΔtssC mutants (Tss is a contractile sheath constituent), these toxins may be accumulated into cytoplasm and perturb assembly of their own flagella. Interestingly, overproduction of FliA flagellar regulator, which controls assembly of flagellar filament, restores motility of both mutants. Simultaneously, T6SS of MFE01 strain contributes to maturation and biofilm formation but also in bacterial competition within mixed biofilm. T6SS may be a mean of bacterial communication and thus coordinate a social behavior, primordial for biofilm formation.

What is flagellar swimming? Cilia and flagella are whip-like cell appendages that can exhibit regular bending waves. This active process emerges from the non-equilibrium dynamics of molecular motors distributed along the length of cilia and flagella. Eukaryotic cells can possess many cilia and flagella that beat in a coordinated fashion, thus transporting fluids, as in mammalian airways or the ventricular system inside the brain. Many unicellular organisms posses just one or two flagella, rendering them microswimmers that are propelled through fluids by the flagellar beat including sperm cells and the biflagellate green alga Chlamydomonas. Objectives. In this thesis in theoretical biological physics, we seek to understand the nonlinear dynamics of flagellar swimming and synchronization. We investigate the flow fields induced by beating flagella and how in turn external hydrodynamic flows change speed and shape of the flagellar beat. This flagellar load-response is a prerequisite for flagellar synchronization. We want to find the physical principals underlying stable synchronization of the two flagella of Chlamydomonas cells. Results. First, we employed realistic hydrodynamic simulations of flagellar swimming based on experimentally measured beat patterns. For this, we developed analysis tools to extract flagellar shapes from high-speed videoscopy data. Flow-signatures of flagellated swimmers are analysed and their effect on a neighboring swimmer is compared to the effect of active noise of the flagellar beat. We were able to estimate a chemomechanical energy efficiency of the flagellar beat and determine its waveform compliance by comparing findings from experiments, in which a clamped Chlamydomonas is exposed to external flow, to predictions from an effective theory that we designed. These mechanical properties have interesting consequences for the synchronization dynamics of Chlamydomonas, which are revealed by computer simulations. We propose that direct elastic coupling between the two flagella of Chlamydomonas, as suggested by recent experiments, in combination with waveform compliance is crucial to facilitate in-phase synchronization of the two flagella of Chlamydomonas.

No description available.

Für die E. coli K12 Biofilmbildung ist die Expression des Masterregulators CsgD essentiell. Dies erfordert das Signalmolekül c-di-GMP, dessen Auf- und Abbau durch 12 Diguanylatzyklasen (DGCs mit GGDEF-Domänen) und 13 Phosphodiesterasen (PDEs mit EAL-Domänen) erfolgt. DgcE ist mit einer MASE1-umfassenden Transmembranregion (TM), drei PAS-, einer GGDEF- und einer degenerierten EAL-Domäne die strukturell komplexeste DGC und notwendig für die Biofilmbildung. Diese Arbeit zeigt, dass die Aktivität von DgcE einer hoch komplexen Regulation unterliegt. Einzelnen DgcE-Domänen konnten aktivierende bzw. inhibierende Rollen hinsichtlich der Biofilmmatrixsynthese zugeordnet werden. Die Biofilmbildung hängt von DgcE-produziertem c-di-GMP ab, wobei die DgcE-Dimerisierung v.a. durch die PAS-Region vermittelt wird. Die EAL-Domäne wirkt einer aktiven DgcE-Form entgegen. Für die DgcE-vermittelte Matrixproduktion sind die GTPase YjdA und sein Partnerprotein YjcZ nötig. Über Interaktionen mit YjcZ und der TM von DgcE vermittelt YjdA eine Komplexbildung. Die Interaktion von YjdA und DgcE sowie die Matrixproduktion hängen von der GTPase-Aktivität von YjdA ab. GTP wird daher als intrazelluläres Signal vorgeschlagen, das die Aktivierung von DgcE durch YjdA/YjcZ reguliert. Die MASE1-umfassende TM agiert als Zentrale der Signalintegration. Einerseits ist sie nötig für die DgcE-Aktivität und andererseits ist sie an einem massiven Abbau von DgcE beteiligt. Zudem wurden neu identifizierte Curli-regulierende Gene (rbsK, rbsR, ydcI, yieP, puuR) untersucht, wobei keines über das PdeR/DgcM/MlrA-Modul in die c-di-GMP-vermittelte CsgD-Expression eingreift. Flagellare Verknotungen in der unteren Schicht von E. coli Makrokolonien tragen zur Morphogenese dieser Makrokolonien bei. Diese Arbeit zeigt, dass Flagellenverknotungen zu einer verminderten Expression der Master-PDE PdeH beitragen, wodurch vermutlich die zelluläre c-di-GMP-Konzentration steigt und somit die Biofilmbildung begünstigt wird. / Biofilm formation of E. coli K12 requires the expression of the biofilm master regulator CsgD. This process depends on the signaling molecule c-di-GMP, which is synthesized by 12 diguanylate cyclases (DGCs with GGDEF domains) and degraded by 13 phosphodiesterases (PDEs with EAL domains). DgcE is the most complex DGC with a MASE1-containing transmembrane region (TM), three PAS, a GGDEF and a degenerate EAL domain, and it is essential for biofilm formation. This work shows that the regulation of the DgcE activity is highly complex. It was possible to assign activating and inhibitory roles to single domains of DgcE with regard to the expression of biofilm matrix components. C-di-GMP produced by DgcE is necessary for biofilm matrix production. The dimerization of DgcE is mainly mediated by the PAS region, whereas the EAL domain counteracts an active form of DgcE. DgcE-mediated matrix synthesis requires the activating signal input of the GTPase YjdA and its partner protein YjcZ. DgcE, YjdA and YjcZ form a protein complex in which YjdA directly interacts with YjcZ and the TM of DgcE. The interaction between DgcE and YjdA as well as the matrix expression depend on the GTPase activity of YjdA. Thus, it is proposed that GTP serves as an intracellular signal regulating the activation of DgcE by YjdA/YjcZ. The MASE1-containing TM proved to be a central hub for signal integration. It is both required for DgcE activity and for a massive degradation of DgcE. Furthermore, newly discovered curli-regulating genes (rbsK, rbsR, ydcI, yieP, puuR) have been analyzed. None of those gene products act on CsgD expression via the PdeR/DgcM/MlrA module. Flagellar entangling within the bottom layer of E. coli macrocolonies determines morphogenesis of macrocolonies. The data presented here suggest that the master PDE PdeH is somehow down-regulated by flagellar entangling, which probably results in a higher cellular c-di-GMP concentration, thereby promoting biofilm formation.

What is flagellar swimming? Cilia and flagella are whip-like cell appendages that can exhibit regular bending waves. This active process emerges from the non-equilibrium dynamics of molecular motors distributed along the length of cilia and flagella. Eukaryotic cells can possess many cilia and flagella that beat in a coordinated fashion, thus transporting fluids, as in mammalian airways or the ventricular system inside the brain. Many unicellular organisms posses just one or two flagella, rendering them microswimmers that are propelled through fluids by the flagellar beat including sperm cells and the biflagellate green alga Chlamydomonas. Objectives. In this thesis in theoretical biological physics, we seek to understand the nonlinear dynamics of flagellar swimming and synchronization. We investigate the flow fields induced by beating flagella and how in turn external hydrodynamic flows change speed and shape of the flagellar beat. This flagellar load-response is a prerequisite for flagellar synchronization. We want to find the physical principals underlying stable synchronization of the two flagella of Chlamydomonas cells. Results. First, we employed realistic hydrodynamic simulations of flagellar swimming based on experimentally measured beat patterns. For this, we developed analysis tools to extract flagellar shapes from high-speed videoscopy data. Flow-signatures of flagellated swimmers are analysed and their effect on a neighboring swimmer is compared to the effect of active noise of the flagellar beat. We were able to estimate a chemomechanical energy efficiency of the flagellar beat and determine its waveform compliance by comparing findings from experiments, in which a clamped Chlamydomonas is exposed to external flow, to predictions from an effective theory that we designed. These mechanical properties have interesting consequences for the synchronization dynamics of Chlamydomonas, which are revealed by computer simulations. We propose that direct elastic coupling between the two flagella of Chlamydomonas, as suggested by recent experiments, in combination with waveform compliance is crucial to facilitate in-phase synchronization of the two flagella of Chlamydomonas.:1 Introduction 1.1 Physics of cell motility: flagellated swimmers as model system 2 1.1.1 Tissue cells and unicellular eukaryotic organisms have cilia and flagella 2 1.1.2 The conserved architecture of flagella 3 1.1.3 Synchronization in collections of flagella 5 1.2 Hydrodynamics at the microscale 9 1.2.1 Navier-Stokes equation 10 1.2.2 The limit of low Reynolds number 10 1.2.3 Multipole expansion of flow fields 11 1.3 Self-propulsion by viscous forces 13 1.3.1 Self propulsion requires broken symmetries 13 1.3.2 Signatures of flowfields: pusher & puller 15 1.4 Overview of the thesis 16 2 Flow signatures of flagellar swimming 2.1 Self-propulsion of flagellated swimmers 20 2.1.1 Representation of flagellar shapes 20 2.1.2 Computation of hydrodynamic friction forces 21 2.1.3 Material frame and rigid-body transformations 22 2.1.4 The grand friction matrix 23 2.1.5 Dynamics of swimming 23 2.2 The hydrodynamic far field: pusher and puller 26 2.2.1 The flow generated by a swimmer 26 2.2.2 Force dipole characterization 27 2.2.3 Flagellated swimmers alternate between pusher and puller 29 2.2.4 Implications for two interacting Chlamydomonas cells 31 2.3 Inertial screening of oscillatory flows 32 2.3.1 Convection and oscillatory acceleration 33 2.3.2 The oscilet: fundamental solution of unsteady flow 35 2.3.3 Screening length of oscillatory flows 35 2.4 Energetics of flagellar self-propulsion 36 2.4.1 Impact of inertial screening on hydrodynamic dissipation 37 2.4.2 Case study: the green alga Chlamydomonas 38 2.4.3 Discussion: evolutionary optimization and the number of molecular motors 38 2.5 Summary 39 3 The load-response of the flagellar beat 3.1 Experimental collaboration: flagellated swimmers exposed to flows 41 3.1.1 Description of the experimental setup 42 3.1.2 Computed flow profile in the micro-fluidic device 43 3.1.3 Image processing and flagellar tracking 43 3.1.4 Mode decomposition and limit-cycle reconstruction 47 3.1.5 Changes of limit-cycle dynamics: deformation, translation, acceleration 49 3.2 An effective theory of flagellar oscillations 50 3.2.1 A balance of generalized forces 50 3.2.2 Hydrodynamic friction in generalized coordinates 51 3.2.3 Intra-flagellar friction 52 3.2.4 Calibration of active flagellar driving forces 52 3.2.5 Stability of the limit cycle of the flagellar beat 53 3.2.6 Equations of motion 55 3.3 Comparison of theory and experiment 56 3.3.1 Flagellar mean curvature 57 3.3.2 Susceptibilities of phase speed and amplitude 57 3.3.3 Higher modes and stalling of the flagellar beat at high external load 59 3.3.4 Non-isochrony of flagellar oscillations 63 3.4 Summary 63 4 Flagellar load-response facilitates synchronization 4.1 Synchronization to external driving 65 4.2 Inter-flagellar synchronization in the green alga Chlamydomonas 67 4.2.1 Equations of motion for inter-flagellar synchronization 68 4.2.2 Synchronization strength for free-swimming and clamped cells 70 4.2.3 The synchronization strength depends on energy efficiency and waveform compliance 73 4.2.4 The case of an elastically clamped cell 74 4.2.5 Basal body coupling facilitates in-phase synchronization 75 4.2.6 Predictions for experiments 78 4.3 Summary 80 5 Active flagellar fluctuations 5.1 Effective description of flagellar oscillations 84 5.2 Measuring flagellar noise 84 5.2.1 Active phase fluctuations are much larger than thermal noise 84 5.2.2 Amplitude fluctuations are correlated 85 5.3 Active flagellar fluctuations result in noisy swimming paths 86 5.3.1 Effective diffusion of swimming circles of sperm cell 86 5.3.2 Comparison of the effect of noise and hydrodynamic interactions 87 5.4 Summary 88 6 Summary and outlook 6.1 Summary of our results 89 6.2 Outlook on future work 90 A Solving the Stokes equation A.1 Multipole expansion 95 A.2 Resistive-force theory 96 A.3 Fast multipole boundary element method 97 B Linearized Navier-Stokes equation B.1 Linearized Navier-Stokes equation 101 B.2 The case of an oscillating sphere 102 B.3 The small radius limit 103 B.4 Greens function 104 C Hydrodynamic friction C.1 A passive particle 107 C.2 Multiple Particles 107 C.3 Generalized coordinates 108 D Data analysis methods D.1 Nematic filter 111 D.1.1 Nemat 111 D.1.2 Nematic correlation 111 D.2 Principal-component analysis 112 D.3 The quality of the limit-cycle projections of experimental data 113 E Adler equation F Sensitivity analysis for computational results F.1 The distance function of basal coupling 117 F.2 Computed synchronization strength for alternative waveform 118 F.3 Insensitivity of computed load-response to amplitude correlation time 118 List of Symbols List of Figures Bibliography

Subject of study: Cilia and flagella are slender appendages of eukaryotic cells. They are actively bending structures and display regular bending waves. These active flagellar bending waves drive fluid flows on cell surfaces like in the case of the ciliated trachea or propels single cell micro-swimmers like sperm or alga. Objective: The axoneme is the evolutionarily conserved mechanical apparatus within cilia and flagella. It is comprised of a cylindrical arrangement of microtubule doublets, which are the elastic elements and dyneins, which are the force generating elements in the axonemal structure. Dyneins collectively bend the axoneme and must be specifically regulated to generate symmetric and highly asymmetric waveforms. In this thesis, I address the question of the molecular origin of the asymmetric waveform and test different theoretical descriptions for motor regulation. Approach: I introduce the isolated and reactivated Chlamydomonas axoneme as an experimental model for the symmetric and asymmetric flagellar beat. This system allows to study the beat in a controlled and cell free environment. I use high-speed microscopy to record shapes with high spatial and temporal resolution. Through image analysis and shape parameterization I extract a minimal set of parameters that describe the flagellar waveform. Using Chlamydomonas, I make use of different structural and motor mutants to study their effect on the shape in different reactivation conditions. Although the isolated axoneme system has many advantages compared to the cell-bound flagellum, to my knowledge, it has not been characterized yet. Results: I present a shape parameterization of the asymmetric beat using Fourier decomposition methods and find, that the asymmetric waveform can be understood as a sinusoidal beat around a circular arc. This reveals the similarities of the two different beat types: the symmetric and the asymmetric beat. I investigate the origin of the beat-asymmetry and find evidence for a specific dynein motor to be responsible for the asymmetry. I furthermore find experimental evidence for a strong sliding restriction at the basal end of the axoneme, which is important to establish a static bend. In collaboration with P. Sartori and F. Jülicher, I compare theoretical descriptions of different motor control mechanisms and find that a curvature controlled motor-regulation mechanism describes the experimental data best. We furthermore find, that in the dynamic case an additional sliding restriction at the base is unnecessary. By comparing the waveforms of intact cells and isolated reactivated axonemes, I reveal the effect of hydrodynamic loading, and the influence of boundary conditions on the shape of the beating flagella.

Subject of study: Cilia and flagella are slender appendages of eukaryotic cells. They are actively bending structures and display regular bending waves. These active flagellar bending waves drive fluid flows on cell surfaces like in the case of the ciliated trachea or propels single cell micro-swimmers like sperm or alga. Objective: The axoneme is the evolutionarily conserved mechanical apparatus within cilia and flagella. It is comprised of a cylindrical arrangement of microtubule doublets, which are the elastic elements and dyneins, which are the force generating elements in the axonemal structure. Dyneins collectively bend the axoneme and must be specifically regulated to generate symmetric and highly asymmetric waveforms. In this thesis, I address the question of the molecular origin of the asymmetric waveform and test different theoretical descriptions for motor regulation. Approach: I introduce the isolated and reactivated Chlamydomonas axoneme as an experimental model for the symmetric and asymmetric flagellar beat. This system allows to study the beat in a controlled and cell free environment. I use high-speed microscopy to record shapes with high spatial and temporal resolution. Through image analysis and shape parameterization I extract a minimal set of parameters that describe the flagellar waveform. Using Chlamydomonas, I make use of different structural and motor mutants to study their effect on the shape in different reactivation conditions. Although the isolated axoneme system has many advantages compared to the cell-bound flagellum, to my knowledge, it has not been characterized yet. Results: I present a shape parameterization of the asymmetric beat using Fourier decomposition methods and find, that the asymmetric waveform can be understood as a sinusoidal beat around a circular arc. This reveals the similarities of the two different beat types: the symmetric and the asymmetric beat. I investigate the origin of the beat-asymmetry and find evidence for a specific dynein motor to be responsible for the asymmetry. I furthermore find experimental evidence for a strong sliding restriction at the basal end of the axoneme, which is important to establish a static bend. In collaboration with P. Sartori and F. Jülicher, I compare theoretical descriptions of different motor control mechanisms and find that a curvature controlled motor-regulation mechanism describes the experimental data best. We furthermore find, that in the dynamic case an additional sliding restriction at the base is unnecessary. By comparing the waveforms of intact cells and isolated reactivated axonemes, I reveal the effect of hydrodynamic loading, and the influence of boundary conditions on the shape of the beating flagella.:Contents 1 Introduction. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.1 Biology of Cilia and Flagella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.1.1 The dimensions of flagellated micro-swimmers . . . . . . . . . . . . . . . . . 4 1.1.2 The symmetric and the asymmetric beat . . . . . . . . .. . . . . . . . . . . . 5 1.1.3 Chlamydomonas reinhardtii as a flagella model . . . . . . . . . . 5 1.2 The axoneme is the internal structure in eukaryotic cilia and flagella . . 6 1.3 Structure and function of microtubules and dyneins . . . . . . . . . . . 9 1.3.1 Microtubules: The structural elements in the axoneme . . . . . . 9 1.3.2 Dyneins: The force generators that drive the axonemal beat . . . 10 1.3.3 The asymmetries in the axoneme and consequences for the beat 17 1.4 Axonemal waveform models and mechanisms: from sliding to bending to beating . . . . . . . . . . . . . . 20 1.5 Geometrical representation and parameterization of the axonemal beat . . . . . . . . . . . . . . . 23 2 Questions addressed in this thesis . . . . . . . . . . . . . . 27 3 Material and Methods . . . . . . . . . . . . . . 29 3.1 Chlamydomonas cells: Axoneme preparation and motility assays . . . . 29 3.1.1 Culturing of Chlamydomonas reinhardtii cells . . . . . . . . . . . 29 3.1.2 Isolation, demembranation and storage of axonemes . . . . . . . 33 3.1.3 Reactivation of axonemes in controlled conditions . . . . . . . . . 35 3.1.4 Axoneme gliding assay using kinesin 1 . . . . . . . . . . . . . . . 36 3.2 Imaging and image processing . . . . . . . . . . . . . . . . . . . . . . . . 38 3.2.1 High-speed imaging of the flagella and axonemes . . . . . . . . . 38 3.2.2 Precise tracking of isolated axonemes and the flagella of cells . . 42 3.2.3 High throughput frequency evaluation of isolated axonemes . . . 47 3.2.4 Beat frequency characterization of the reactivated WT axoneme . . . . . . . . . . . . . . 49 4 Results and Discussion . . . . . . . . . . . . . . 53 4.1 The beat of the axoneme propagates from base to tip . . . . . . . . . . . 53 4.1.1 TEM study reveals no sliding at the base of a bend axoneme . . 53 4.1.2 The direction of wave propagation is directly determined from the reactivation of polarity marked axonemes . . . . . . . . . . 57 4.1.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4.2 The asymmetric beat is the superposition of a static semi-circular arc and a sinusoidal beat . . . . . . .. . . . . . . . . . . . . . . . . 61 4.2.1 The waveform is parameterized by Fourier decomposition in time . . . . . . . . . . . . . . 61 4.2.2 The 0th and 1st Fourier modes describe the axonemal waveform . . . . . . . . . . . . . . 65 4.2.3 General dependence of shape parameters on axoneme length and beat frequency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 4.2.4 The isolated axoneme is a good model for the intact flagellum . .. . . . . . . . . . . . . . 71 4.2.5 Summary: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 4.3 The circular motion is a consequence of the axonemal waveform . . . . . . . . . . . . . . . . . . . 79 4.3.1 Hydrodynamic relations for small amplitude waves explain the relation between swimming and shape of axonemes . . . . 79 4.3.2 The swimming path can be reconstructed using shape information and a hydrodynamic model . . . . . . . . . . . . . . . . 83 4.3.3 Motor mutations alter the direction of rotation of reactivated axonemes. . . . . . . . . . . . . . . . . . . . . . . . 84 4.3.4 Summary: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 4.4 The molecular origin of the circular mean shape. . . . . . . . . . . . . . 89 4.4.1 Motor Mutations do not abolish the mean shape, a structural mutation does . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 4.4.2 The axoneme is straight in absence of ATP but bend at low ATP concentrations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 4.4.3 Viscous load decreases the mean curvature . . . . . . . . . . . . 99 4.4.4 Summary: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 4.5 Curvature-dependent dynein activation accounts for the shape of the beat of the isolated axoneme . . . . . . . . . . . . . . . . 103 5 Conclusions and Outlook . . . . . . . . . . . . . . . . 109 5.1 Summary of the results . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 5.2 Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Abbreviations . . . . . . . . . . . . . . . . 111 List of figures . . . . . . . . . . . . . . . . 116 List of tables . . . . . . . . . . . . . . . . 118 Bibliography

Durch Parasiten stellen eine starke Gefährdung für die Aquakultur dar. Die durch diplomonaden Flagellaten bei der Regenbogenforelle Oncorhynchus mykiss verursachte Morbidität und Mortalität wurde bisher in Deutschland noch nicht gründlich untersucht. Ich habe diese Parasiten mittels SEM & TEM charakterisiert und wurde die Art Spironucleus salmonis bestimmt. Erstmals konnten die caudale Projektion, sich entleerende Vakuolen und verformbare Kernloben nachgewiesen werden. Ich untersuchte Mikrohabitatpräferenz von des Parasiten sowie pH-Profile in vier Darmabschnitten der Fische. Das Vorkommen und die Dichte von S. salmonis war in der Pylorusregion wesentlich höher als in anderen Bereichen. Das pH-Profil war bei infizierten und nicht infizierten Fischen gleich. Der optimale pH-Wert für S. salmonis war 7,1-7,5. Ich habe die Lebenszyklus der Diplomonaden mittels LM & SEM unter Kulturbedingungen untersucht. Die Enzystierung begann mit der Anheftung von Trophozoiten mit der Spitze der adhäsiven hinteren Flagellen aneinander oder an Fremdkörper. Die pyriformen Trophozoiten wurden kugelförmig, und die vorderen Flagellen inaktiv. Oberflächenbläschen produzierten eine lichtbrechende Zystenwand. Dies ist der erste Beschreibung der Multifunktionalität von Flagellen bei Diplomonaden. Ich untersuchte pathogene Mechanismen und sezierte die Pylorusregion sowie die Leber mittels H&E, PAS/AB. Bei infizierten Fischen trat eine signifikante Hypertrophie der Becherzellen auf. Zu erkennen war eine Hyperaktivität der Becherzellen, jedoch keine Hyperplasie. Ich entwickelte einen in vitro  Plasma-Inkubationstest zur Bestimmung der Suszeptibilität von Regenbogenforelle, Karpfen und Störe. Die unterschiedliche Resistenz von Stör, Karpfen und Regenbogenforelle gegen S. salmonis entsprechend zu den epizootiologischen Daten. Meine Untersuchungen führten zu einem neuen diagnostischen Hilfsmittel, Vorschlägen für neue Behandlungsmethoden, zu verbesserten in vitro-Kulturbedingungen und einem Modellsystem für die Multifunktionalität von Flagellen und flagellaren Signaltransduktion. / Parasitic diseases pose a significant threat to aquaculture. Diplomonad flagellates in rainbow trout Oncorhynchus mykiss are associated with morbidity and mortality; but in Germany has not been thoroughly studied. I characterised the species by SEM & TEM, which revealed Spironucleus salmonis, allowed its complete description including newly showing the caudal projection, discharging vacuoles, and deformable nuclear lobes; diagnostic keys were improved. The microhabitat preference of diplomonads was tested by recording occurrence and density of infection, and pH profile in 4 intestinal regions in fish. Occurrence and density of S. salmonis were significant higher in the pyloric region than elsewhere. The pH profile in uninfected and infected fish was similar; a causal relationship between microhabitat preference and pH was unlikely, and the optimal pH was between 7.1 – 7.5. I described life cycle and encystment using light and SEM. Encystment in culture began by trophozoites attaching at tip of adhesive posterior flagella to each other/debris. Pyriform trophozoites became sub-spherical, anterior flagella inactive, surface blebs produced a refractile cyst wall. Cysts clusters may exceed minimum infective dose for new infection; suggesting new treatment target. This is the first report of multi-functionality of flagella in diplomonads. I investigated pathogenic mechanism of diplomonads by sectioning and staining the pyloric region of the intestine and liver with H&E, and PAS/AB. There was significant hypertrophy of goblet cells in infected fish. The hyperactivity of goblet cells was seen, but no hyperplasia. This hyper-production of mucus may decrease nutrient absorption, underlying impaired growth in S. salmonis infected fish. I developed an in vitro plasma incubation test to predict host susceptibility of rainbow trout, carp, and sturgeon. The test showed the hierarchy of resistance of S. salmonis in sturgeon > carp > rainbow trout; this parallels epizootiological data. My research yielded new diagnostic tool, suggested new treatment target, improved in vitro conditions, and new model system for multi-functionality of flagella and flagellar signalling.

Les composés organiques hydrophobes (HOC), une grande famille de molécules naturelles ou d’origine anthropique incluant les lipides et les hydrocarbures, constituent une part significative de la matière organique dans les écosystèmes marins. Du fait de leur faible solubilité dans l’eau, les bactéries qui les dégradent requièrent la mise en place de fonctions cellulaires spécifiques permettant d’augmenter la fraction assimilable de ces HOC. La formation de biofilms à l’interface eau-HOC est une de ces stratégies adaptatives. C’est le cas pour Marinobacter hydrocarbonoclasticus SP17, modèle d’étude utilisé au laboratoire, qui est capable de former des biofilms sur un large spectre de HOC métabolisables tels que les alcanes, les triglycérides et les alcools gras. Le but de mes recherches consistait à améliorer la compréhension du processus d’adhésion et de développement des biofilms sur les HOC, à travers la caractérisation fonctionnelle de 10 gènes candidats mis en évidence lors d’analyses d’expression en protéomique et en transcriptomique. Pour mener à bien ce projet, des outils génétiques et une caractérisation fonctionnelle propre à chaque gène ont dû être développés. L’étude fonctionnelle du gène MARHY2686 a relevé son implication dans la formation de biofilm sur les alcanes. La co-expression de MARHY2686 et des gènes adjacents MARHY2687 et MARHY2685 en transcriptomique, leur distribution phylogénétique et leur conservation de la synthénie suggèreraient que ces trois gènes soient impliqués dans le même processus biologique. D’après l’identité forte de 36 % qui existe entre la protéine MARHY2686 et une protéine périplasmique AdeT d’un système de pompe d’efflux tripartite d’Acinetobacter baumanii, cette protéine, en association avec MARHY2687 et MARHY2685, pourrait faire partie d’un système de ce type. Par ailleurs, des observations ont permis d’envisager une implication potentielle de ce gène dans l’assimilation des HOC ou dans l’accumulation des réserves lipidiques intracellulaires. M. hydrocarbonoclasticus SP17 utilise les pili de type IV lors de la formation de biofilm sur les HOC. Ces appendices interviennent lors de l’adhésion de cette souche à des HOC ainsi que dans un processus de détachement d’un support hydrophobe. Les pili pourraient soit intervenir directement pour permettre à la bactérie de se détacher de la surface à laquelle elle s’est adhérée, soit indirectement par l’action de bactériophages. La présence d’une mobilité de type twitching sur les HOC a pu être également envisagée. Enfin, le rôle du système de sécrétion de type VI (T6SS), connu pour permettre à la bactérie d’interagir avec une cellule hôte, lors de la formation de biofilm mono-spécifique sur HOC, où aucun autre microorganisme que M. hydrocarbonoclasticus SP17 n’est présent, a été étudié. / Hydrophobic organic compounds (HOC), a large family of naturally-produced or anthropogenic molecules including lipids and hydrocarbons, represent a significant part of organic matter in marine ecosystems. Because of their low solubility in water, bacteria that degrade those compounds require the establishment of specific cell functions to increase their biodisponibility. Biofilm formation in water-HOC interface is one of these adaptations. The model of bacteria used in our laboratory, Marinobacter hydrocarbonoclasticus SP17, is able to form a biofilm on a wide range of HOC, such as alkanes, fatty alcohols and triglycerides, in order to use them as a carbon and energy source. The main purpose of my work was to broaden the knowledge of how bacteria adhere to and from biofilms on HOC, through the functional characterization of 10 candidate genes highlighted during proteomic and transcriptomic studies. Genetic tools and a gene-specific functional characterization have been developed in order to carry out this project. Functional study conducted on MARHY2686 revealed its involvement in the formation of biofilm on alkanes. Co-expression of MARHY2686 and the adjacent genes MARHY2687 and MARHY2685 durnig transcriptomic analysis together with their phylogenetic distribution and synteny conservation suggest that these three genes are involved in the same biological process. According to the high peptide sequence identity between MARHY2686 and AdeT, a periplasmic protein of a tripartite efflux pump system of Acinetobacter baumanii, MARHY2686 in combination with MARHY2687 and MARHY2685 could be the components of such a system. Other phenotypic observations would consider the involvement of MARHY2686 either in the assimilation of HOC or in the accumulation of intracellular lipid reserves. M. hydrocarbonoclasticus SP17 uses type IV pili during biofilm formation on HOC. These appendages are involved in the adhesion of this strain to and in a detachment process from HOC. Type IV pili could either act directly to allow bacteria to detach from the surface to which it is adhered, or indirectly through the action of bacteriophages. The presence of twitching motility on HOC has also been suggested. Finally, the role of the type VI secretion system (T6SS), a well-known protein system which allows interactions between bacteria and host cells, during the formation of a mono-species biofilm on HOC where no other microorganism than M. hydrocarbonoclasticus SP17 is present, has been studied.

Cilia and flagella are hair-like appendages of eukaryotic cells. They are actively bending structures that exhibit regular beat patterns and thereby play an important role in many different circumstances where motion on a cellular level is required. Most dramatic is the effect of nodal cilia whose vortical motion leads to a fluid flow that is directly responsible for establishing the left-right axis during embryological development in many vertebrate species, but examples range from the propulsion of single cells, such as the swimming of sperm, to the transport of mucus along epithelial cells, e.g. in the ciliated trachea. Cilia and flagella contain an evolutionary highly conserved structure called the axoneme, whose characteristic architecture is based on a cylindrical arrangement of elastic filaments (microtubules). In the presence of a chemical fuel (ATP), molecular motors (dynein) exert shear forces between neighbouring microtubules, leading to a bending of the axoneme through structural constraints. We address the following two questions: How can these organelles generate regular oscillatory beat patterns in the absence of a biochemical signal regulating the activity of the force generating elements? And how can the beat patterns be so different for apparently very similar structures? We present a theoretical description of the axonemal structure as an actively bending elastic cylinder, and show that in such a system bending waves emerge from a non-oscillatory state via a dynamic instability. The corresponding beat patterns are solutions to a set of coupled partial differential equations presented herein.