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Studies on the role of CheS in Sinorhizobium meliloti chemotaxisDogra, Gaurav 08 September 2011 (has links)
Chemotaxis is the ability of an organism to sense its environment and move towards attractants and away from repellents. The two-component system controlling chemotaxis in bacteria contains a histidine kinase CheA, which is autophosphorylated in response to a signal from a ligand-bound transmembrane methyl-accepting chemotaxis protein. CheA transfers the phosphate group to its cognate response regulator which modulates flagellar rotation. Signal termination by dephosphorylation of the response regulator is necessary for the organism to react rapidly to changes in the environment. The phosphorylated response regulator CheY in <i>Escherichia coli</i> is dephosphorylated by CheZ, a phosphatase; certain organisms, such as <i>Sinorhizobium meliloti</i>, that lack a CheZ homolog have developed alternate methods of signal termination. The signaling chain of S. meliloti contains two response regulators, CheY1 and CheY2, in which CheY2 modulates flagellar rotation and CheY1 causes signal termination by acting as a phosphate sink. In addition to known chemotaxis components, the second gene in the chemotaxis operon of <i>S. meliloti</i> codes a 97 amino acid protein, called CheS. The phenotype of a cheS deletion strain is similar to that of a cheY1 deletion strain. Therefore, the possibility that CheS causes signal termination was explored in this work. The derived amino acid sequence of CheS showed similarities with its orthologs from other °-proteobacteria. Sequence conservation was highest at the centrally located °4 and °5 helices. Earlier observations that CheS localizes at the polar chemotaxis cluster in a CheA-dependent manner were confirmed, and the co-localization of CheS with CheA was demonstrated by fluorescence microscopy. The stable expression of CheS in the presence of CheA was confirmed by immunoblot. The same approach was used to establish the stable expression of CheS only in the presence of the P2 domain of CheA, but not with the P1 or P345 domains. Limited proteolysis followed by mass spectrometry defined CheA<sub>163-256</sub> as the CheS binding domain, and this domain overlapped the previously defined CheY2-binding domain, CheA<sub>174-316</sub>. The role of CheS in the phosphate flux in S. meliloti chemotaxis was analyzed by assays using radio-labeled [?-?°P]ATP. CheS does not play a role in the autophosphorylation of CheA. However, CheS accelerated the rate of CheY1~P dephosphorylation by almost two-fold, but did not affect the rate of CheY2~P dephosphorylation. CheS also does not seem to affect phosphate flow in the retrophosphorylation from CheY2~P to CheA using acetyl [?°P]phosphate as phosphodonor. Since CheS increases the rate of CheY1 dephosphorylation, it can be envisioned that it either increases the association of CheY1 to CheA, increasing the flow of phosphate from CheA to CheY1, or directly accelerates the dephosphorylation of CheY1~P. The presence of a STAS domain and a conserved serine residue in CheS also raises the possibility that CheS may be phosphorylated by a yet unknown kinase, in a mechanism similar to the phosphorylation of <i>Bacillus subtilis</i> SpoIIAA by its cognate kinase SpoIIAB. Phosphorylated CheS may then switch CheA between a kinase or phosphotransferase ON/OFF state or activated CheS may directly interact with CheY1. Further studies are needed to determine the association of CheY1 with CheS to elucidate the mechanism of CheY1 dephosphorylation. This work has confirmed the <i>in vitro</i> association of CheS with CheA, determined the CheS binding domain on CheA, and indicated that CheS accelerates the dephosphorylation of CheY1~P. This has advanced our understanding of the role of CheS in the chemotaxis signaling chain of <i>S. meliloti</i>. / Master of Science
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The relationship between flagellar motor dynamics and the proton motive forceTipping, Murray January 2011 (has links)
The bacterial flagellar motor is one of the few rotary motors found in nature, and an excellent example of a complex molecular machine. Flagellar motors in the model organism Escherichia coli are products of the coordinated expression of ∼50 different genes. The E. coli flagellar motor is powered by the proton-motive force (pmf), an electrochemical gradient across the cell membrane. Motor torque is gen- erated by proton flow through membrane-embedded stator units which bind to the basal body of the motor. This thesis aimed to investigate the relationship between the pmf and the flag- ellar motor. A novel pmf control system was developed, based on the light-driven proton pump proteorhodopsin (pR). This system enabled pmf -dependent changes in motor behaviour to be precisely monitored in vivo. Expression of pR in E. coli was shown to be sufficient to drive the flagellar motor at wild-type speeds. Using the pR-based pmf control system, the motor was shown to respond to changes in pmf on a timescale of milliseconds. Surprisingly, motor speed increase was observed when pmf was increased above the physiological norm. Reduction of pmf to low levels enabled individual steps in motor rotation to be observed. Motor response to loss of pmf was investigated. Motors were shown to exhibit a two-stage speed decrease after disruption of pmf , with motor speed falling to ∼20 % of its initial value within milliseconds, reaching a complete stop after 1 s. Extended periods of pmf loss was shown to lead to disengagement of stators from the motor, with motor speed increasing in a stepwise fashion after pmf restoration. The integrity of the motor at different pmf levels was investigated by using TIRF microscopy to directly image positioning of fluorescently tagged motor components. The stator protein MotB was shown to physically leave and rejoin the motor after pmf disruption and restoration, with MotB dispersal following motor stop.
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Control of the unidirectional motor in Rhodobacter sphaeroidesBrown, Mostyn T. January 2009 (has links)
The control of the flagellar motor in Rhodobacter sphaeroides was investigated. Unlike most flagellar motors which are controlled by reversing the direction of rotation, the R. sphaeroides motor is controlled via a stop-start mechanism. Advanced optical microscopy was employed alongside genetic, biochemical, and behavioural techniques. High-resolution measurements of rotating beads on flagellar stubs revealed that the R. sphaeroides motor is similar to its E. coli counterpart, rotating counterclockwise at comparable torques/speeds (1,300 pNnm/rad at stall torque), and exhibiting transient step changes in speed. The mean stop duration, mean stop frequency (number of stops per s), and run bias (fraction of time spent rotating) of wild-type at steady-state were 0.66 ± 1.01 s, 0.31 ± 0.19 s-1, and 0.80 ± 0.20, respectively. Manipulating signal inputs to the motor genetically, or by exposing cells to chemotactic stimuli revealed that (i) without chemotactic stimulation the motor rotates continuously, (ii) phosphorylated CheYs are required to stop the motor, and (iii) the chemotaxis system cannot control the speed of rotation of the motor (termed chemokinesis) as previously reported. Complementation studies revealed that CheY3, CheY4, and CheY5 are functionally equivalent. The copy numbers per cell of important CheYs were found to vary greatly under the conditions tested (<1,000, ~3,000, ~60,000 for CheY3, CheY4, and CheY6 respectively). To determine how CheY-P binding causes the motor to stop, external force (viscous flow or optical tweezers) was applied to chemotactically stopped motors. CheY-P binding might either cause the torque-generating units to disengage from the rotor, analogous to a clutch, or trigger the rotor to jam, analogous to a brake. The rotor resisted re-orientation during a chemotactic stop implying that the motor was held in a locked state. The value of torque resisting forward motion (keeping it locked) was estimated to be 2-3 x stall torque (2,500-4,000 pNnm/rad). Furthermore beads attached to flagellar stubs stop at fixed angles for several seconds, showing no large-scale Brownian motion. Step analysis revealed that these stop events occur at 27-28 discrete angles around the motor, which most likely reflect the periodicity of the rotor (i.e. copies of FliG). This represents the first experimental resolution of steps in the rotation of a wild-type bacterial flagellar motor with a full complement of torque-generating units.
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Stepping dynamics of the bacterial flagellar motor and F₁-ATPaseNord, Ashley January 2014 (has links)
Rotary molecular motors are protein complexes which convert chemical or electrochemical energy from the environment into mechanical work in the form of rotary motion. The work in this thesis examines two of these motors: the F<sub>1</sub> portion of F<sub>1</sub>F<sub>O-</sub> ATP synthase, which is responsible for ATP production in bacteria and eukaryotes, and the bacterial flagellar motor (BFM), which rotates the flagella of a bacterium, enabling locomotion. The aim of these investigations was to measure the stepping dynamics of these motors, in order to further elucidate details of the stepping mechanism, the mechanism of rotation, and the mechanochemical cycle. A back-scattering laser dark field microscope of unprecedented resolution was designed and constructed to observe the rotation of gold nanoparticles attached to fixed motors. This micro- scope is capable of sub-nanometer and 20μs resolution. The protocols and algorithms to collect and analyze high resolution rotational data developed for these experiments have yielded novel discoveries for both F<sub>1</sub> and the BFM. While most of the previous single-molecule work has been done on F<sub>1</sub> from the thermophilic Bacilus PS3 (TF<sub>1</sub>), only mitochondrial F<sub>1</sub> has been well characterized by high-resolution crystal structures, and single-molecule studies of mesophilic F<sub>1</sub> are lacking. This thesis presents evidence that mesophilic F<sub>1</sub> from E. coli and wild type yeast F<sub>1</sub> from S. cerevisiae are governed by the same mechanism as TF<sub>1</sub> under laboratory conditions. Experiments with yeast F<sub>1</sub> mutants allow a direct comparison between single-molecule rotation studies and high resolution crystal structures. A data set of unprecedented size and resolution was acquired of high speed, low load BFM rotation, enabling the first observation of steps in the BFM under physiological conditions. Preliminary results from this analysis question previously published results of the dependence of speed on stator number at low load and provide novel hypotheses necessitating new models of BFM rotation.
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Application of magnetic torque on the bacterial flagellar motorLim, Ren Chong January 2015 (has links)
There is a strong need to develop a mechanical method to apply external torque to the bacterial flagellar motor. Such a method will allow us to probe the behaviour of the motor at a range of different speeds under different external conditions. In this thesis, I explored various methods to deliver torque at the single-molecule level, in particular the use of angular optical trapping and magnetic tweezers. I have identified rutile particles as suitable handles for use in angular optical trapping due to their high birefringence. Further progress was not achieved using angular optical trapping due to the lack of a suitable method to attach birefringent particles to the bacterial flagellar motor. On the other hand, I was able to make further progress using magnetic tweezers. A highly-reproducible and high-yielding magnetic bead assay was developed along with electromagnets capable of generating fast-rotating magnetic fields at magnitudes on the order of tens of mT. Using the system of delivering magnetic torque developed, I was able to stall and rotate the motor forward at speeds up to 220 Hz and in the reverse direction. Stalling experiments carried out on the motor revealed the stator mechanosensing depends on torque and not rotation. Signatures of stators dropping out at low load experiments further confirm the load dependence of stators.
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Osmotaxis in Escherichia coliRosko, Jerko January 2017 (has links)
Bacterial motility, and in particular repulsion or attraction towards specific chemicals, has been a subject of investigation for over 100 years, resulting in detailed understanding of bacterial chemotaxis and the corresponding sensory network in many bacterial species including Escherichia coli. E. Coli swims by rotating a bundle of flagellar filaments, each powered by an individual rotary motor located in the cell membrane. When all motors rotate counter-clockwise (CCW), a stable bundle forms and propels the cell forward. When one or more motors switch to clock-wise (CW) rotation, their respective filaments fall out of the bundle, leading to the cell changing orientation. Upon switching back to CCW, the bundle reforms and propels the cell in a new direction. Chemotaxis is performed by the bacterium through prolonging runs by suppressing CW rotation when moving towards nutrients and facilitating reorientation by increasing CW bias when close to a source of a harmful substance. Chemicals are sensed through interaction with membrane bound chemosensors. These proteins can interact with a very specific set of chemicals and the concentrations they are able to sense are in the range between 10-⁶ and 10-² M. However, experiments have shown that the osmotic pressure exerted by large (> 10-¹ M) concentrations of solutes, which have no specificity for binding to chemosensors (e.g. sucrose), is able to send a signal down the chemotactic network. Additionally, clearing of bacterial density away from sources of high osmolarity has been previously observed in experiments with agar plates. This behaviour has been termed osmotaxis. The aim of this doctoral thesis work is to understand how different environmental cues influence the tactic response and ultimately, combine at the network output to direct bacterial swimming. As tactic responses to chemical stimuli have been extensively studied, I focus purely on the response to non-specific osmotic stimuli, using sucrose to elevate osmolarity. I monitor the chemotactic network output, the rotation of a single bacterial flagellar motor, using Back Focal Plane Interferometry over a variety of osmotic conditions. Additionally, in collaboration with Vincent Martinez, I studied the effect of elevated osmolality on swimming speed of large (104) bacterial populations, using differential dynamic microscopy (DDM). I have found that sudden increases in media osmolarity lead to changes of both motor speed and motor clockwise bias, which is the fraction of time it spends rotating clockwise. Changes in CW Bias proceed in two phases. Initially, after elevating the osmolarity, CW Bias drops to zero, indicating that the motor is exclusively in the ‘cell run’ mode. This phase lasts from 2-5 minutes depending on the magnitude of the change in solute concentration. What follows then is a distinct second phase where the CW Bias is elevated with respect to the initial levels and this phase lasts longer than 15-20 minutes. In comparison, for defined chemical stimuli, the motor output resets after several seconds, a behaviour termed perfect adaptation. For changes of 100 mOsm/kg and 200 mOsm/kg in magnitude the motors speed up, often by as much as a factor of two, before experiencing a gradual slow down. Despite the slow down, motors still rotate faster 15-20 minutes after the change in osmolarity, than they did before. For changes of 400 mOsm/Kg in magnitude the motors decrease sharply in speed, coming to a near halt, recovering after 5 minutes and eventually, on average, speeding up. DDM studies of free swimming bacteria have shown that elevated osmolality leads to higher swimming speeds, in agreement with single motor data. Using theoretical models of bacterial swimming from the literature, it is discussed how this motor output, although different to what is expected for chemotaxis, is able to drive bacteria away from regions of space with high osmolalities. Additionally, I have started extending the work done with sucrose, to another solute often used to elevate osmolality, sodium chloride. While sucrose is outer membrane impermeable, NaCl can cross the outer membrane into the periplasmic space. Another layer of complexity is that NaCl has some specificty for the chemoreceptors. The preliminary results are shown and qualitatively agree with those obtain with sucrose.
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Dynamique fonctionnelle du moteur flagellaire bactérien entraîné par des stators marqués par des protéines fluorescentes et par des stators étrangers modifiés par évolution / Functional dynamics of the bacterial flagellar motor driven by fluorescent protein tagged stators and by evolutionary modified foreign statorsHeo, Minyoung 25 November 2016 (has links)
Le moteur flagellaire bactérien (BFM) est un complexe moléculaire qui permet aux bactéries de nager dans un milieu liquide. La rotation du moteur est générée à l’interface entre deux éléments clés: les protéines formant le stator (MotA and MoB) et l’anneau C “switching complex” à la base du rotor. Les stators sont des modules du moteur structurellement et fonctionnellement différentiables du reste du moteur, et leurs association et dissociation dynamique autour du rotor contrôle la génération du couple. Quand une protéine fluorescente (PF) est fusionnée à MotB, le moteur est en état de marche mais une réduction générale de la mobilité de la cellule a été observée. La raison précise d’une telle réduction de mobilité n’a pas été étudiée.Le but de cette étude est de comprendre comment la fusion PF de la protéine du stator modifie la génération du couple et le sens de rotation du moteur. C’est particulièrement important car le tag FP se trouve à l’interface entre le stator et le rotor, là où le couple et le changement du sens de rotation sont produits. Trois différentes PFs (eGFP, YPet, Dendra2) ont été fusionnées à la protéine MotB. Malgré la haute similarité de leurs structures, notre analyse a montré que les trois stators fusionnés génèrent des couples différents. Les stators marqués avec YPet produisent un couple moyen similaire au WT (stators sans tag PF), alors que les stators marqués avec eGFP et Dendra2 produisent respectivement 70% et 40% du couple moyen du WT. De plus, les moteurs utilisant les stators fusionnés ont montré des capacités de changement de sens de rotation réduites. Lors d’un changement de sens de rotation, la valeur absolue de la vitesse des moteurs WT ne change pas. Cette “symétrie” de vitesse lors du changement n’apparaît pas avec les moteurs à stators fusionnés et le changement peut être accompagné d’une importante diminution (~30%) de la vitesse absolue.En observant par microcopie TIRF avec détection de molécules uniques, des stators marqués dans un moteur en état de marche, les signaux de fluorescence sont détectés à la membrane comme prévu pour ces protéines, montrant une population de stators diffusant dans celle-ci. Les clusters fluorescents étaient visibles au centre des cellules en rotation, attachés au couvre-glace par une seule flagelle, confirmant que le tag de fluorescence peut être visualisé dans des moteurs en état de marche. Dans un second projet développé dans le laboratoire Bertus Beaumont à TU Delft, en prenant le BFM en tant que système modèle d’évolution expérimentale, sa modularité et son « évolubilité » ont été explorés pour apprendre les détails au niveau moléculaire de l’évolution de ce type de machine. Les stators de E.coli ont été échangés par un set de 21 stators étrangers homologues. L’expérience a révélé que les protéines du stator peuvent être échangées entre espèces de bactéries distantes et certains stators non compatibles peuvent être modifiés positivement par un procédé d’évolution pour devenir fonctionnels. Au cours de cette évolution, les bactéries ont accumulé des mutations avantageuses dans leurs gènes MotA et MotB étrangers, tout particulièrement dans leur domaine fonctionnel. Des mutations identiques dans des stators différents ont été observées, indiquant que l’évolution peut se reproduire. L’analyse fonctionnelle au niveau d’un seul moteur a révélé que ces mutations avantageuses amélioraient la génération du couple et/ou la capacité du moteur à changer de sens. Les investigations détaillées du génotype et du phénotype du BFM modifié par évolution apportés par cette étude, pourraient donner une idée sur la façon dont des machines moléculaires comme le BFM ont évolué, et les effets fonctionnels des mutations bénéfiques qui facilitent l'intégration fonctionnelle. / The bacterial flagellar motor (BFM) is the macromolecular complex which allows bacteria to swim in liquid media. Located at the base of the flagellum, anchored in the cell membrane, this remarkably small (~45nm) yet powerful rotary motor rotates each flagellum of the cell switching between counterclockwise (CCW) and clockwise (CW) direction. The motor rotation is generated at the interface between the two key components of the motor: the stator protein complexes (each composed of 4 MotA and 2 MotB proteins) and the C- ring protein complex at the base of the rotor. The stator complexes are structurally and functionally discernible modules of the motor, and their dynamical association and dissociation around the rotor controls the generation of torque.The first project of this study aims to investigate how the FP tag on the stator protein modifies the torque generation and switching of the motor. This is particularly important because the fluorescent protein tag lies at the interface between stator and rotor, where torque and switching are produced. Three different FPs (eGFP, YPet, Dendra2) were fused to MotB. Interestingly, despite the high similarity of their structures, our analysis revealed that the three fusion stators generate different torque. Furthermore, in the presence of fusion stators, the motor showed significantly impaired switching abilities. When switching direction of the rotation, the absolute value of the speed of WT motors does not change, whereas this symmetry of speed upon switching is not observed in the fusion stator motors, and switching can be accompanied with a significant (~30%) decrease in absolute speed. Both the impaired torque generation and the switching ability were improved by introducing a rigid linker between the stator and the FP tag. Taken together, this study provides a further insight into the dynamics of the stator and rotor interaction at its interface.When the cells carrying the fluorescently labeled stators were observed in a custom made TIRF-fluorescence microscope with single molecule capability, the fluorescence signals were detected as concentrated clusters in the membrane as expected for these membrane proteins around the motors, together with a population of stators diffusing in the membrane. Fluorescent clusters were visible at the center of rotating cells tethered to the glass slide by a single flagellum, confirming that the fluorescent tags can be visualized in functioning motors.In a second project developed in Bertus Beaumont lab at TU Delft, taking BFM as an experimental evolutionary model system, its modularity and evolvability have been explored to learn the molecular details of the evolution of molecular machines. The stators of E.coli have been exchanged by a set of 21 homologue foreign stators. The experiments revealed that the stator proteins can be exchanged between distant bacteria species, and some of the non-compatible stators can be positively modified by evolution to become functional. Those evolved strains accumulated beneficial mutations in their foreign motA and motB genes, especially on their functional domains. Identical mutations in different stators were common, indicating that evolution is repeatable. The functional investigation at the single motor level revealed that those beneficial mutations improved the torque generation and/or the switching ability of the motor. The detailed genotype and phenotype investigations of the evolutionary modified BFM may bring an insight into how molecular machines such as BFM have evolved as well as the functional effects of the beneficial mutations that facilitate functional integration.
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