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Sample records for intact flagellar motor

  1. Molecular Architecture of the Bacterial Flagellar Motor in Cells

    PubMed Central

    2015-01-01

    The flagellum is one of the most sophisticated self-assembling molecular machines in bacteria. Powered by the proton-motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior. Escherichia coli and Salmonella enterica have served as important model systems for extensive genetic, biochemical, and structural analysis of the flagellum, providing unparalleled insights into its structure, function, and gene regulation. Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part because of the limited structural information available regarding the intact rotor–stator complex and secretion apparatus. Cryo-electron tomography (cryo-ET) has become a valuable imaging technique capable of visualizing the intact flagellar motor in cells at molecular resolution. Because the resolution that can be achieved by cryo-ET with large bacteria (such as E. coli and S. enterica) is limited, analysis of small-diameter bacteria (including Borrelia burgdorferi and Campylobacter jejuni) can provide additional insights into the in situ structure of the flagellar motor and other cellular components. This review is focused on the application of cryo-ET, in combination with genetic and biophysical approaches, to the study of flagellar structures and its potential for improving the understanding of rotor–stator interactions, the rotational switching mechanism, and the secretion and assembly of flagellar components. PMID:24697492

  2. Protein turbines. I: The bacterial flagellar motor.

    PubMed Central

    Elston, T C; Oster, G

    1997-01-01

    The bacterial flagellar motor is driven by a flux of ions between the cytoplasm and the periplasmic lumen. Here we show how an electrostatic mechanism can convert this ion flux into a rotary torque. We demonstrate that, with reasonable parameters, the model can reproduce many of the experimental measurements. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 11 FIGURE 14 FIGURE 15 p720-a PMID:9251788

  3. Structural diversity of bacterial flagellar motors

    PubMed Central

    Chen, Songye; Beeby, Morgan; Murphy, Gavin E; Leadbetter, Jared R; Hendrixson, David R; Briegel, Ariane; Li, Zhuo; Shi, Jian; Tocheva, Elitza I; Müller, Axel; Dobro, Megan J; Jensen, Grant J

    2011-01-01

    The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species. PMID:21673657

  4. Flagellar motor based micro hybrid devices.

    PubMed

    Tung, S; Kim, J-W

    2004-01-01

    We are in the process of developing a series of micro hybrid devices based on tethered flagellar motors. Examples of the devices include a microfluidic pump and a micro AC dynamo. The microfluidic pump is realized through the tethering of a harmless strain of Escherichia coli cells to a MEMS based micro channel. Each E. coli cell is about 3 mum long and 1 mum in diameter, with several flagella that are driven at the base by molecular rotary motors. The operational principle of the micro pump is based on the viscous pumping effect where continuous rotation of the tethered cells forms a fluidic conveyor belt that 'drags' fluid from one end of the channel to the other. We used hydrodynamic loading to synchronize cell rotation in order to maximize the fluid pumping capability. The micro dynamo is realized through the integration of tethered flagellar motors with micro ferromagnetic beads and micro copper coils. The micro dynamo generates AC power by using the tethered cells to create a rotating magnetic field around the copper coils. Preliminary result indicates a high power density when compared to other biologically based micro power generators. PMID:17270806

  5. Limiting Speed of the Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Nirody, Jasmine; Berry, Richard; Oster, George

    The bacterial flagellar motor (BFM) drives swimming in a wide variety of bacterial species, making it crucial for several fundamental biological processes including chemotaxis and community formation. Recent experiments have shown that the structure of this nanomachine is more dynamic than previously believed. Specifically, the number of active torque-generating units (stators) was shown to vary across applied loads. This finding invalidates the experimental evidence reporting that limiting (zero-torque) speed is independent of the number of active stators. Here, we put forward a model for the torque generation mechanism of this motor and propose that the maximum speed of the motor increases as additional torque-generators are recruited. This is contrary to the current widely-held belief that there is a universal upper limit to the speed of the BFM. Our result arises from the assumption that stators disengage from the motor for a significant portion of their mechanochemical cycles at low loads. We show that this assumption is consistent with current experimental evidence and consolidate our predictions with arguments that a processive motor must have a high duty ratio at high loads.

  6. Exchange of rotor components in functioning bacterial flagellar motor

    SciTech Connect

    Fukuoka, Hajime; Inoue, Yuichi; Terasawa, Shun; Takahashi, Hiroto; Ishijima, Akihiko

    2010-03-26

    The bacterial flagellar motor is a rotary motor driven by the electrochemical potential of a coupling ion. The interaction between a rotor and stator units is thought to generate torque. The overall structure of flagellar motor has been thought to be static, however, it was recently proved that stators are exchanged in a rotating motor. Understanding the dynamics of rotor components in functioning motor is important for the clarifying of working mechanism of bacterial flagellar motor. In this study, we focused on the dynamics and the turnover of rotor components in a functioning flagellar motor. Expression systems for GFP-FliN, FliM-GFP, and GFP-FliG were constructed, and each GFP-fusion was functionally incorporated into the flagellar motor. To investigate whether the rotor components are exchanged in a rotating motor, we performed fluorescence recovery after photobleaching experiments using total internal reflection fluorescence microscopy. After photobleaching, in a tethered cell producing GFP-FliN or FliM-GFP, the recovery of fluorescence at the rotational center was observed. However, in a cell producing GFP-FliG, no recovery of fluorescence was observed. The transition phase of fluorescence intensity after full or partially photobleaching allowed the turnover of FliN subunits to be calculated as 0.0007 s{sup -1}, meaning that FliN would be exchanged in tens of minutes. These novel findings indicate that a bacterial flagellar motor is not a static structure even in functioning state. This is the first report for the exchange of rotor components in a functioning bacterial flagellar motor.

  7. An Element of Determinism in a Stochastic Flagellar Motor Switch

    PubMed Central

    Xie, Li; Altindal, Tuba; Wu, Xiao-Lun

    2015-01-01

    Marine bacterium Vibrio alginolyticus uses a single polar flagellum to navigate in an aqueous environment. Similar to Escherichia coli cells, the polar flagellar motor has two states; when the motor is counter-clockwise, the cell swims forward and when the motor is clockwise, the cell swims backward. V. alginolyticus also incorporates a direction randomization step at the start of the forward swimming interval by flicking its flagellum. To gain an understanding on how the polar flagellar motor switch is regulated, distributions of the forward Δf and backward Δb intervals are investigated herein. We found that the steady-state probability density functions, P(Δf) and P(Δb), of freely swimming bacteria are strongly peaked at a finite time, suggesting that the motor switch is not Poissonian. The short-time inhibition is sufficiently strong and long lasting, i.e., several hundred milliseconds for both intervals, which is readily observed and characterized. Treating motor reversal dynamics as a first-passage problem, which results from conformation fluctuations of the motor switch, we calculated P(Δf) and P(Δb) and found good agreement with the measurements. PMID:26554590

  8. Multiple kinetic states for the flagellar motor switch.

    PubMed Central

    Kuo, S C; Koshland, D E

    1989-01-01

    By means of a computerized video processing system, the flagellar motors of Escherichia coli were shown to have multiple kinetic states for each rotational direction. High-resolution analysis of flagellar motors revealed new kinetic states both in wild-type cells and in a strain deleted of other signal-transducing genes to which CheY had been introduced. This strain, RP1091, retained residual kinase activity that could phosphorylate CheY, complicating the biochemical identification of certain kinetic states. The behavioral effect of CheY on single flagellar motors was ultrasensitive, with an apparent Hill coefficient of 5.5 +/- 1.9 (SD) and a half-maximal effect at 10.1 +/- 0.5 (SD) microM CheY. Based on the CheY concentration dependence, a two-state model is clearly excluded, even for the simpler system of CheY-induced rotational reversals in the deletion strain. The data are best described by a four-state model, with two clockwise and two counterclockwise states. Images PMID:2681161

  9. Motility protein interactions in the bacterial flagellar motor.

    PubMed Central

    Garza, A G; Harris-Haller, L W; Stoebner, R A; Manson, M D

    1995-01-01

    Five proteins (MotA, MotB, FliG, FliM, and FliN) have been implicated in energizing flagellar rotation in Escherichia coli and Salmonella typhimurium. One model for flagellar function envisions that MotA and MotB comprise the stator of a rotary motor and that FliG, FliM, and FliN are part of the rotor. MotA probably functions as a transmembrane proton channel, and MotB has been proposed to anchor MotA to the peptidoglycan of the cell wall. To study interactions between the Mot proteins themselves and between them and other components of the flagellar motor, we attempted to isolate extragenic suppressors of 13 dominant or partially dominant motB missense mutations. Four of these yielded suppressors, which exhibited widely varying efficiencies of suppression. The pattern of suppression was partially alleles-specific, but no suppressor seriously impaired motility in a motB+ strain. Of 20 suppressors from the original selection, 15 were characterized by DNA sequencing. Fourteen of these cause single amino acid changes in MotA. Thirteen alter residues in, or directly adjacent to, the putative periplasmic loops of MotA, and the remaining one alters a residue in the middle of the fourth predicted transmembrane helix of MotA. We conclude that the MotA and MotB proteins form a complex and that their interaction directly involves or is strongly influenced by the periplasmic loops of MotA. The 15th suppressor from the original selection and 2 motB suppressors identified during a subsequent search cause single amino acid substitutions in FliG. This finding suggests that the postulated Mot-protein complex may be in close proximity to FliG at the stator-rotor interface of the flagellar motor. Images Fig. 1 Fig. 2 PMID:7892209

  10. Hybrid-fuel bacterial flagellar motors in Escherichia coli.

    PubMed

    Sowa, Yoshiyuki; Homma, Michio; Ishijima, Akihiko; Berry, Richard M

    2014-03-01

    The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H(+) or Na(+) ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H(+)- and Na(+)-driven stator components and runs on both types of ion gradient. We controlled the number of each type of stator unit in the motor by protein expression levels and Na(+) concentration ([Na(+)]), using speed changes of single motors driving 1-μm polystyrene beads to determine stator unit numbers. De-energized motors changed from locked to freely rotating on a timescale similar to that of spontaneous stator unit exchange. Hybrid motor speed is simply the sum of speeds attributable to individual stator units of each type. With Na(+) and H(+) stator components expressed at high and medium levels, respectively, Na(+) stator units dominate at high [Na(+)] and are replaced by H(+) units when Na(+) is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H(+) and Na(+) stator components sharing a common rotor. PMID:24550452

  11. The Limiting Speed of the Bacterial Flagellar Motor.

    PubMed

    Nirody, Jasmine A; Berry, Richard M; Oster, George

    2016-08-01

    Recent experiments on the bacterial flagellar motor have shown that the structure of this nanomachine, which drives locomotion in a wide range of bacterial species, is more dynamic than previously believed. Specifically, the number of active torque-generating complexes (stators) was shown to vary across applied loads. This finding brings under scrutiny the experimental evidence reporting that limiting (zero-torque) speed is independent of the number of active stators. In this study, we propose that, contrary to previous assumptions, the maximum speed of the motor increases as additional stators are recruited. This result arises from our assumption that stators disengage from the motor for a significant portion of their mechanochemical cycles at low loads. We show that this assumption is consistent with current experimental evidence in chimeric motors, as well as with the requirement that a processive motor driving a large load via an elastic linkage must have a high duty ratio. PMID:27508439

  12. Resurrection of the flagellar rotary motor near zero load

    PubMed Central

    Yuan, Junhua; Berg, Howard C.

    2008-01-01

    Flagellated bacteria, such as Escherichia coli, are propelled by helical flagellar filaments, each driven at its base by a reversible rotary motor, powered by a transmembrane proton flux. Torque is generated by the interaction of stator proteins, MotA and MotB, with a rotor protein FliG. The physiology of the motor has been studied extensively in the regime of relatively high load and low speed, where it appears to operate close to thermodynamic equilibrium. Here, we describe an assay that allows systematic study of the motor near zero load, where proton translocation and movement of mechanical components are rate limiting. Sixty-nanometer-diameter gold spheres were attached to hooks of cells lacking flagellar filaments, and light scattered from a sphere was monitored at the image plane of a microscope through a small pinhole. Paralyzed motors of cells carrying a motA point mutation were resurrected at 23°C by expression of wild-type MotA, and speeds jumped from zero to a maximum value (≈300 Hz) in one step. Thus, near zero load, the speed of the motor is independent of the number of torque-generating units. Evidently, the units act independently (they do not interfere with one another), and there are no intervals during which a second unit can add to the speed generated by the first (the duty ratio is close to 1). PMID:18202173

  13. Coordinated Switching of Bacterial Flagellar Motors: Evidence for Direct Motor-Motor Coupling?

    NASA Astrophysics Data System (ADS)

    Hu, Bo; Tu, Yuhai

    2013-04-01

    The swimming of Escherichia coli is powered by its multiple flagellar motors. Each motor spins either clockwise or counterclockwise, under the control of an intracellular regulator, CheY-P. There can be two mechanisms (extrinsic and intrinsic) to coordinate the switching of bacterial motors. The extrinsic one arises from the fact that different motors in the same cell sense a common input (CheY-P) which fluctuates near the motors’ response threshold. An alternative, intrinsic mechanism is direct motor-motor coupling which makes synchronized switching energetically favorable. Here, we develop simple models for both mechanisms and uncover their different hallmarks. A quantitative comparison to the recent experiments suggests that the direct coupling mechanism may be accountable for the observed sharp correlation between motors in a single Escherichia coli. Possible origins of this coupling (e.g., hydrodynamic interaction) are discussed.

  14. The Limiting Speed of the Bacterial Flagellar Motor

    NASA Astrophysics Data System (ADS)

    Nirody, Jasmine A.; Berry, Richard M.; Oster, George

    2016-08-01

    Recent experiments on the bacterial flagellar motor have shown that the structure of this nanomachine, which drives locomotion in a wide range of bacterial species, is more dynamic than previously believed. Specifically, the number of active torque-generating units (stators) was shown to vary across applied loads. This finding invalidates the experimental evidence reporting that limiting (zero-torque) speed is independent of the number of active stators. Here, we propose that, contrary to previous assumptions, the maximum speed of the motor is not universal, but rather increases as additional torque-generators are recruited. This result arises from our assumption that stators disengage from the motor for a significant portion of their mechanochemical cycles at low loads. We show that this assumption is consistent with current experimental evidence and consolidate our predictions with arguments that a processive motor must have a high duty ratio at high loads.

  15. Constraints on models for the flagellar rotary motor.

    PubMed

    Berg, H C

    2000-04-29

    Most bacteria that swim are propelled by flagellar filaments, each driven at its base by a rotary motor embedded in the cell wall and cytoplasmic membrane. A motor is about 45 nm in diameter and made up of about 20 different kinds of parts. It is assembled from the inside out. It is powered by a proton (or in some species, a sodium-ion) flux. It steps at least 400 times per revolution. At low speeds and high torques, about 1000 protons are required per revolution, speed is proportional to protonmotive force, and torque varies little with temperature or hydrogen isotope. At high speeds and low torques, torque increases with temperature and is sensitive to hydrogen isotope. At room temperature, torque varies remarkably little with speed from about -100 Hz (the present limit of measurement) to about 200 Hz, and then it declines rapidly reaching zero at about 300 Hz. These are facts that motor models should explain. None of the existing models for the flagellar rotary motor completely do so. PMID:10836502

  16. Mechanics of torque generation in the bacterial flagellar motor

    PubMed Central

    Mandadapu, Kranthi K.; Nirody, Jasmine A.; Berry, Richard M.; Oster, George

    2015-01-01

    The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual “power stroke.” Specifically, we propose that ion-induced conformational changes about a proline “hinge” residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque–speed and speed–ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator. PMID:26216959

  17. Model Studies of the Dynamics of Bacterial Flagellar Motors

    SciTech Connect

    Bai, F; Lo, C; Berry, R; Xing, J

    2009-03-19

    The Bacterial Flagellar Motor is a rotary molecular machine that rotates the helical filaments which propel swimming bacteria. Extensive experimental and theoretical studies exist on the structure, assembly, energy input, power generation and switching mechanism of the motor. In our previous paper, we explained the general physics underneath the observed torque-speed curves with a simple two-state Fokker-Planck model. Here we further analyze this model. In this paper we show (1) the model predicts that the two components of the ion motive force can affect the motor dynamics differently, in agreement with the latest experiment by Lo et al.; (2) with explicit consideration of the stator spring, the model also explains the lack of dependence of the zero-load speed on stator number in the proton motor, recently observed by Yuan and Berg; (3) the model reproduces the stepping behavior of the motor even with the existence of the stator springs and predicts the dwelling time distribution. Predicted stepping behavior of motors with two stators is discussed, and we suggest future experimental verification.

  18. The Gearbox of the Bacterial Flagellar Motor Switch.

    PubMed

    Pandini, Alessandro; Morcos, Faruck; Khan, Shahid

    2016-07-01

    Switching of flagellar motor rotation sense dictates bacterial chemotaxis. Multi-subunit FliM-FliG rotor rings couple signal protein binding in FliM with reversal of a distant FliG C-terminal (FliGC) helix involved in stator contacts. Subunit dynamics were examined in conformer ensembles generated by molecular simulations from the X-ray structures. Principal component analysis extracted collective motions. Interfacial loop immobilization by complex formation coupled elastic fluctuations of the FliM middle (FliMM) and FliG middle (FliGM) domains. Coevolved mutations captured interfacial dynamics as well as contacts. FliGM rotation was amplified via two central hinges to the FliGC helix. Intrinsic flexibility, reported by the FliGMC ensembles, reconciled conformers with opposite FliGC helix orientations. FliG domain stacking deformed the inter-domain linker and reduced flexibility; but conformational changes were not triggered by engineered linker deletions that cause a rotation-locked phenotype. These facts suggest that binary rotation states arise from conformational selection by stacking interactions. PMID:27345932

  19. Torque-generating units of the bacterial flagellar motor step independently.

    PubMed Central

    Samuel, A D; Berg, H C

    1996-01-01

    Measurements of the variance in rotation period of tethered cells as a function of mean rotation rate have shown that the flagellar motor of Escherichia coli is a stepping motor. Here, by measurement of the variance in rotation period as a function of the number of active torque-generating units, it is shown that each unit steps independently. PMID:8842231

  20. Dual stator dynamics in the Shewanella oneidensis MR-1 flagellar motor.

    PubMed

    Paulick, Anja; Delalez, Nicolas J; Brenzinger, Susanne; Steel, Bradley C; Berry, Richard M; Armitage, Judith P; Thormann, Kai M

    2015-06-01

    The bacterial flagellar motor is an intricate nanomachine which converts ion gradients into rotational movement. Torque is created by ion-dependent stator complexes which surround the rotor in a ring. Shewanella oneidensis MR-1 expresses two distinct types of stator units: the Na(+)-dependent PomA4 B2 and the H(+)-dependent MotA4 B2. Here, we have explored the stator unit dynamics in the MR-1 flagellar system by using mCherry-labeled PomAB and MotAB units. We observed a total of between 7 and 11 stator units in each flagellar motor. Both types of stator units exchanged between motors and a pool of stator complexes in the membrane, and the exchange rate of MotAB, but not of PomAB, units was dependent on the environmental Na(+)-levels. In 200 mM Na(+), the numbers of PomAB and MotAB units in wild-type motors was determined to be about 7:2 (PomAB:MotAB), shifting to about 6:5 without Na(+). Significantly, the average swimming speed of MR-1 cells at low Na(+) conditions was increased in the presence of MotAB. These data strongly indicate that the S. oneidensis flagellar motors simultaneously use H(+) and Na(+) driven stators in a configuration governed by MotAB incorporation efficiency in response to environmental Na(+) levels. PMID:25727785

  1. Bio-Hybrid Micro/Nanodevices Powered by Flagellar Motor: Challenges and Strategies

    PubMed Central

    Kim, Jin-Woo; Tung, Steve

    2015-01-01

    Molecular motors, which are precision engineered by nature, offer exciting possibilities for bio-hybrid engineered systems. They could enable real applications ranging from micro/nano fluidics, to biosensing, to medical diagnoses. This review describes the fundamental biological insights and fascinating potentials of these remarkable sensing and actuation machines, in particular, bacterial flagellar motors, as well as their engineering perspectives with regard to applications in bio-engineered hybrid systems. PMID:26284237

  2. Monitoring bacterial chemotaxis by using bioluminescence resonance energy transfer: Absence of feedback from the flagellar motors

    PubMed Central

    Shimizu, Thomas S.; Delalez, Nicolas; Pichler, Klemens; Berg, Howard C.

    2006-01-01

    We looked for a feedback system in Escherichia coli that might sense the rotational bias of flagellar motors and regulate the activity of the chemotaxis receptor kinase. Our search was based on the assumption that any machinery that senses rotational bias will be perturbed if flagellar rotation stops. We monitored the activity of the kinase in swimming cells by bioluminescence resonance energy transfer (BRET) between Renilla luciferase fused to the phosphatase, CheZ, and yellow fluorescent protein fused to the response regulator, CheY. Then we jammed the flagellar motors by adding an antifilament antibody that crosslinks adjacent filaments in flagellar bundles. At steady state, the rate of phosphorylation of CheY is equal to the rate of dephosphorylation of CheY-P, which is proportional to the degree of association between CheZ and CheY-P, the quantity sensed by BRET. No changes were observed, even upon addition of an amount of antibody that stopped the swimming of >95% of cells within a few seconds. So, the kinase does not appear to be sensitive to motor output. The BRET technique is complementary to one based on FRET, described previously. Its reliability was confirmed by measurements of the response of cells to the addition of attractants. PMID:16452163

  3. A novel type bacterial flagellar motor that can use divalent cations as a coupling ion.

    PubMed

    Imazawa, Riku; Takahashi, Yuka; Aoki, Wataru; Sano, Motohiko; Ito, Masahiro

    2016-01-01

    The bacterial flagellar motor is a sophisticated nanomachine embedded in the cell envelope and powered by an electrochemical gradient of H(+), Na(+), or K(+)across the cytoplasmic membrane. Here we describe a new member of the bacterial flagellar stator channel family (MotAB1 of Paenibacillus sp. TCA20 (TCA-MotAB1)) that is coupled to divalent cations (Ca(2+)and Mg(2+)). In the absence of divalent cations of alkaline earth metals, no swimming was observed in Paenibacillus sp. TCA20, which grows optimally in Ca(2+)-rich environments. This pattern was confirmed by swimming assays of a stator-free Bacillus subtilis mutant expressing TCA-MotAB1. Both a stator-free and major Mg(2+)uptake system-deleted B. subtilis mutant expressing TCA-MotAB1 complemented both growth and motility deficiency under low Mg(2+)conditions and exhibited [Mg(2+)]in identical to that of the wild-type. This is the first report of a flagellar motor that can use Ca(2+)and Mg(2+)as coupling ions. These findings will promote the understanding of the operating principles of flagellar motors and molecular mechanisms of ion selectivity. PMID:26794857

  4. A novel type bacterial flagellar motor that can use divalent cations as a coupling ion

    PubMed Central

    Imazawa, Riku; Takahashi, Yuka; Aoki, Wataru; Sano, Motohiko; Ito, Masahiro

    2016-01-01

    The bacterial flagellar motor is a sophisticated nanomachine embedded in the cell envelope and powered by an electrochemical gradient of H+, Na+, or K+across the cytoplasmic membrane. Here we describe a new member of the bacterial flagellar stator channel family (MotAB1 of Paenibacillus sp. TCA20 (TCA-MotAB1)) that is coupled to divalent cations (Ca2+and Mg2+). In the absence of divalent cations of alkaline earth metals, no swimming was observed in Paenibacillus sp. TCA20, which grows optimally in Ca2+-rich environments. This pattern was confirmed by swimming assays of a stator-free Bacillus subtilis mutant expressing TCA-MotAB1. Both a stator-free and major Mg2+uptake system-deleted B. subtilis mutant expressing TCA-MotAB1 complemented both growth and motility deficiency under low Mg2+conditions and exhibited [Mg2+]in identical to that of the wild-type. This is the first report of a flagellar motor that can use Ca2+and Mg2+as coupling ions. These findings will promote the understanding of the operating principles of flagellar motors and molecular mechanisms of ion selectivity. PMID:26794857

  5. Coordinated Switching of Bacterial Flagellar Motors in a Single E. Coli

    NASA Astrophysics Data System (ADS)

    Hu, Bo; Tu, Yuhai

    2013-03-01

    The swimming of Escherichia coli is propelled by its multiple flagellar motors. Each motor spins either clockwise or counterclockwise, under the control of an intracellular regulator, CheY-P. A long standing question is whether these motors work independently or not. There can be two mechanisms (extrinsic and intrinsic) to coordinate the switching of bacterial motors. The extrinsic one arises from the fact that different motors in the same cell sense a common biochemical signal (CheY-P) which fluctuates near the motors' response threshold. An alternative, intrinsic mechanism is direct motor-motor coupling which makes synchronized switching energetically favorable. Here, we develop simple models for both mechanisms and uncover their different hallmarks. A quantitative comparison to the recent experiments suggest that the direct coupling mechanism may be accountable for the observed sharp correlation between motors in a single E. coli. Possible origins of this coupling are discussed. This research is supported by the NIH Grant GM081747

  6. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.

    PubMed

    Beeby, Morgan; Ribardo, Deborah A; Brennan, Caitlin A; Ruby, Edward G; Jensen, Grant J; Hendrixson, David R

    2016-03-29

    Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes. PMID:26976588

  7. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

    PubMed Central

    Ribardo, Deborah A.; Brennan, Caitlin A.; Ruby, Edward G.; Jensen, Grant J.; Hendrixson, David R.

    2016-01-01

    Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes. PMID:26976588

  8. Structure and Function of the Bi-Directional Bacterial Flagellar Motor

    PubMed Central

    Morimoto, Yusuke V.; Minamino, Tohru

    2014-01-01

    The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane. The flagellar motor consists of a rotor made of FliF, FliG, FliM and FliN and a dozen stators consisting of MotA and MotB. FliG, FliM and FliN also act as a molecular switch, enabling the motor to spin in both counterclockwise and clockwise directions. Each stator is anchored to the peptidoglycan layer through the C-terminal periplasmic domain of MotB and acts as a proton channel to couple the proton flow through the channel with torque generation. Highly conserved charged residues at the rotor–stator interface are required not only for torque generation but also for stator assembly around the rotor. In this review, we will summarize our current understanding of the structure and function of the proton-driven bacterial flagellar motor. PMID:24970213

  9. Rotational speed control of Na +-driven flagellar motor by dual pipettes.

    PubMed

    Nogawa, Kousuke; Kojima, Masaru; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Fukuda, Toshio

    2009-12-01

    Single cell analysis has attracted much attention to reveal the detailed and localized biological information. Local environmental control technique is desired to analyze the detailed and localized properties of single cells. In this paper, we propose the local environmental control system with nano/micro dual pipettes to control the local reagent concentration dynamically and arbitrarily. Local environmental control by dual pipettes is applied to the rotational speed control of bacterial flagellar motor, which is a rotary molecular machine. We demonstrate quick response and iterative rotational speed control of Na (+)-driven flagellar motor in both accelerating and relaxing directions by switching the local spout between Na (+)-containing and Na (+) -free solutions with dual pipettes. It is shown that the rotational speed might be controllable by changing the spouting velocity of Na (+)-containing solution with multiplying the applied dc voltage. PMID:19887330

  10. Torque generated by the flagellar motor of Escherichia coli.

    PubMed Central

    Berg, H C; Turner, L

    1993-01-01

    Cells of the bacterium Escherichia coli were tethered and spun in a high-frequency rotating electric field at a series of discrete field strengths. This was done first at low field strengths, then at field strengths generating speeds high enough to disrupt motor function, and finally at low field strengths. Comparison of the initial and final speed versus applied-torque plots yielded relative motor torque. For backward rotation, motor torque rose steeply at speeds close to zero, peaking, on average, at about 2.2 times the stall torque. For forward rotation, motor torque remained approximately constant up to speeds of about 60% of the zero-torque speed. Then the torque dropped linearly with speed, crossed zero, and reached a minimum, on average, at about -1.7 times the stall torque. The zero-torque speed increased with temperature (about 90 Hz at 11 degrees C, 140 Hz at 16 degrees C, and 290 Hz at 23 degrees C), while other parameters remained approximately constant. Sometimes the motor slipped at either extreme (delivered constant torque over a range of speeds), but eventually it broke. Similar results were obtained whether motors broke catastrophically (suddenly and completely) or progressively or were de-energized by brief treatment with an uncoupler. These results are consistent with a tightly coupled ratchet mechanism, provided that elastic deformation of force-generating elements is limited by a stop and that mechanical components yield at high applied torques. PMID:8298044

  11. High hydrostatic pressure induces counterclockwise to clockwise reversals of the Escherichia coli flagellar motor.

    PubMed

    Nishiyama, Masayoshi; Sowa, Yoshiyuki; Kimura, Yoshifumi; Homma, Michio; Ishijima, Akihiko; Terazima, Masahide

    2013-04-01

    The bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to clockwise (CW), and vice versa, in response to input from the chemotaxis signaling circuit. CW rotation is normally caused by binding of the phosphorylated response regulator CheY (CheY-P), and strains lacking CheY are typically locked in CCW rotation. The detailed mechanism of switching remains unresolved because it is technically difficult to regulate the level of CheY-P within the concentration range that produces flagellar reversals. Here, we demonstrate that high hydrostatic pressure can induce CW rotation even in the absence of CheY-P. The rotation of single flagellar motors in Escherichia coli cells with the cheY gene deleted was monitored at various pressures and temperatures. Application of >120 MPa pressure induced a reversal from CCW to CW at 20°C, although at that temperature, no motor rotated CW at ambient pressure (0.1 MPa). At lower temperatures, pressure-induced changes in direction were observed at pressures of <120 MPa. CW rotation increased with pressure in a sigmoidal fashion, as it does in response to increasing concentrations of CheY-P. Application of pressure generally promotes the formation of clusters of ordered water molecules on the surfaces of proteins. It is possible that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY-P. PMID:23417485

  12. A Non-Poissonian Flagellar Motor Switch Increases Bacterial Chemotactic Potential.

    PubMed

    Yang, Yang; He, Jing; Altindal, Tuba; Xie, Li; Wu, Xiao-Lun

    2015-09-01

    We investigate bacterial chemotactic strategies using run-tumble and run-reverse-flick motility patterns. The former is typically observed in enteric bacteria such as Escherichia coli and Salmonella and the latter was recently observed in the marine bacteria Vibrio alginolyticus and is possibly exhibited by other polar flagellated species. It is shown that although the three-step motility pattern helps the bacterium to localize near hot spots, an exploitative behavior, its exploratory potential in short times can be significantly enhanced by employing a non-Poissonian regulation scheme for its flagellar motor switches. PMID:26331263

  13. The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backward

    PubMed Central

    Lele, Pushkar P.; Roland, Thibault; Shrivastava, Abhishek; Chen, Yihao; Berg, Howard C.

    2016-01-01

    Caulobacter crescentus, a monotrichous bacterium, swims by rotating a single right-handed helical filament. CW motor rotation thrusts the cell forward 1, a mode of motility known as the pusher mode; CCW motor rotation pulls the cell backward, a mode of motility referred to as the puller mode 2. The situation is opposite in E. coli, a peritrichous bacterium, where CCW rotation of multiple left-handed filaments drives the cell forward. The flagellar motor in E. coli generates more torque in the CCW direction than the CW direction in swimming cells 3,4. However, monotrichous bacteria including C. crescentus swim forward and backward at similar speeds, prompting the assumption that motor torques in the two modes are the same 5,6. Here, we present evidence that motors in C. crescentus develop higher torques in the puller mode than in the pusher mode, and suggest that the anisotropy in torque-generation is similar in two species, despite the differences in filament handedness and motor bias (probability of CW rotation). PMID:27499800

  14. Chemoreceptors and Flagellar Motors Are Subterminally Located in Close Proximity at the Two Cell Poles in Spirochetes ▿ ‡

    PubMed Central

    Xu, Hongbin; Raddi, Gianmarco; Liu, Jun; Charon, Nyles W.; Li, Chunhao

    2011-01-01

    Green fluorescent protein (GFP) fusions, immunofluorescence microscopy, and cryo-electron tomography revealed that the chemoreceptors of the Lyme disease spirochete Borrelia burgdorferi form long, thin arrays near both cell poles. These arrays are in close proximity to the flagellar motors. This information provides a basis for further understanding motility, chemotaxis, and protein localization in spirochetes. PMID:21441520

  15. Temperature dependences of torque generation and membrane voltage in the bacterial flagellar motor.

    PubMed

    Inoue, Yuichi; Baker, Matthew A B; Fukuoka, Hajime; Takahashi, Hiroto; Berry, Richard M; Ishijima, Akihiko

    2013-12-17

    In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na(+)-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H(+)-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1-2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors. PMID:24359752

  16. Switching of Bacterial Flagellar Motors Is Triggered by Mutant FliG

    PubMed Central

    Lele, Pushkar P.; Berg, Howard C.

    2015-01-01

    Binding of the chemotaxis response regulator CheY-P promotes switching between rotational states in flagellar motors of the bacterium Escherichia coli. Here, we induced switching in the absence of CheY-P by introducing copies of a mutant FliG locked in the clockwise (CW) conformation (FliGCW). The composition of the mixed FliG ring was estimated via fluorescence imaging, and the probability of CW rotation (CWbias) was determined from the rotation of tethered cells. The results were interpreted in the framework of a 1D Ising model. The data could be fit by assuming that mutant subunits are more stable in the CW conformation than in the counterclockwise conformation. We found that CWbias varies depending on the spatial arrangement of the assembled subunits in the FliG ring. This offers a possible explanation for a previous observation of hysteresis in the switch function in analogous mixed FliM motors—in motors containing identical fractions of mutant FliMCW in otherwise wild-type motors, the CWbias differed depending on whether mutant subunits were expressed in strains with native motors or native subunits were expressed in strains with mutant motors. PMID:25762339

  17. Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

    PubMed Central

    Inoue, Yuichi; Baker, Matthew A.B.; Fukuoka, Hajime; Takahashi, Hiroto; Berry, Richard M.; Ishijima, Akihiko

    2013-01-01

    In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na+-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H+-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors. PMID:24359752

  18. The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backwards

    NASA Astrophysics Data System (ADS)

    Lele, Pushkar P.; Roland, Thibault; Shrivastava, Abhishek; Chen, Yihao; Berg, Howard C.

    2016-02-01

    The bacterium Caulobacter crescentus swims by rotating a single right-handed helical filament. These cells have two swimming modes: a pusher mode, in which clockwise (CW) rotation of the filament thrusts the cell body forwards, and a puller mode, in which counterclockwise (CCW) rotation pulls it backwards. The situation is reversed in Escherichia coli, a bacterium that rotates several left-handed filaments CCW to drive the cell body forwards. The flagellar motor in E. coli generates more torque in the CCW direction than the CW direction in swimming cells. However, C. crescentus and other bacteria with single filaments swim forwards and backwards at similar speeds, prompting the assumption that motor torques in the two modes are the same. Here, we present evidence that motors in C. crescentus develop higher torques in the puller mode than in the pusher mode, and suggest that the anisotropy in torque generation is similar in the two species, despite the differences in filament handedness and motor bias.

  19. MotX, the channel component of the sodium-type flagellar motor.

    PubMed Central

    McCarter, L L

    1994-01-01

    Thrust for propulsion of flagellated bacteria is generated by rotation of a propeller, the flagellum. The power to drive the polar flagellar rotary motor of Vibrio parahaemolyticus is derived from the transmembrane potential of sodium ions. Force is generated by the motor on coupling of the movement of ions across the membrane to rotation of the flagellum. A gene, motX, encoding one component of the torque generator has been cloned and sequenced. The deduced protein sequence is 212 amino acids in length. MotX was localized to the membrane and shown to interact with MotY, which is the presumed stationary component of the motor. Overproduction of MotX, but not that of a nonfunctional mutant MotX, was lethal to Escherichia coli. The rate of lysis caused by induction of motX was proportional to the sodium ion concentration. Li+ and K+ substituted for Na+ to promote lysis, while Ca2+ did not enhance lysis. Protection from the lethal effects of induction of motX was afforded by the sodium channel blocker amiloride. The data suggest that MotX forms a sodium channel. The deduced protein sequence for MotX shows no homology to its ion-conducting counterpart in the proton-driven motor; however, in possessing only one hydrophobic domain, it resembles other channels formed by small proteins with single membrane-spanning domains. Images PMID:7928960

  20. Physiological characterization of motor unit properties in intact cats.

    PubMed

    O'Donovan, M J; Hoffer, J A; Loeb, G E

    1983-02-01

    Single motor units were isolated in intact cats, by microstimulation through chronically implanted microwires in the L5 ventral roots. Motor unit axonal and mechanical properties were obtained by stimulus-triggered averaging the signals from an implanted femoral nerve recording cuff and patellar tendon force transducer. All unit types were sampled with this technique, and it was also possible to stimulate in isolation an axon whose ventral root spike was recorded during treadmill locomotion. A new technique was described, spike-triggered microstimulation, for verifying the identity of a stimulated and a recorded axon. PMID:6300565

  1. Fluctuations in rotation rate of the flagellar motor of Escherichia coli.

    PubMed Central

    Kara-Ivanov, M; Eisenbach, M; Caplan, S R

    1995-01-01

    The purpose of this work was to study the changes in rotation rate of the bacterial motor and to try to discriminate between various sources of these changes with the aim of understanding the mechanism of force generation better. To this end Escherichia coli cells were tethered and videotaped with brief stroboscopic light flashes. The records were scanned by means of a computerized motion analysis system, yielding cell size, radius of rotation, and accumulated angle of rotation as functions of time for each cell selected. In conformity with previous studies, fluctuations in the rotation rate of the flagellar motor were invariably found. Employing an exclusively counterclockwise rotating mutant ("gutted" RP1091 strain) and using power spectral density, autocorrelation and residual mean square angle analysis, we found that a simple superposition of rotational diffusion on a steady rotary motion is insufficient to describe the observed rotation. We observed two additional rotational components, one fluctuating (0.04-0.6 s) and one oscillating (0.8-7 s). However, the effective rotational diffusion coefficient obtained after taking these two components into account generally exceeded that calculated from external friction by two orders of magnitude. This is consistent with a model incorporating association and dissociation of force-generating units. PMID:7669902

  2. Intracellular Na+ kinetically interferes with the rotation of the Na(+)-driven flagellar motors of Vibrio alginolyticus.

    PubMed

    Yoshida, S; Sugiyama, S; Hojo, Y; Tokuda, H; Imae, Y

    1990-11-25

    To understand the mechanism of Na+ movement through the force-generating units of the Na(+)-driven flagellar motors of Vibrio alginolyticus, the effect of intracellular Na+ concentration on motor rotation was investigated. Control cells containing about 50 mM Na+ showed good motility even at 10 mM Na+ in the medium, i.e. in the absence of an inwardly directed Na+ gradient. In contrast, Na(+)-loaded cells containing about 400 mM Na+ showed very poor motility at 500 mM Na+ in the medium, i.e. even in the presence of an inwardly directed Na+ gradient. The membrane potential of the cells, which is a major driving force for the motor under these conditions, was not detectably altered, and consistently with this, Na(+)-coupled sucrose transport was only partly reduced in the Na(+)-loaded cells. Motility of the Na(+)-loaded cells was restored by decreasing the intracellular Na+ concentration, and the rate of restoration of motility correlated with the rate of the Na+ decrease. These results indicate that the absolute concentration of the intracellular Na+ is a determinant of the rotation rate of the Na(+)-driven flagellar motors of V. alginolyticus. A simple explanation for this phenomenon is that the force-generating unit of the motor has an intracellular Na(+)-binding site, at which the intracellular Na+ kinetically interferes with the rate of Na+ influx for motor rotation. PMID:2243095

  3. Cell cycle-controlled proteolysis of a flagellar motor protein that is asymmetrically distributed in the Caulobacter predivisional cell.

    PubMed Central

    Jenal, U; Shapiro, L

    1996-01-01

    Flagellar biogenesis and release are developmental events tightly coupled to the cell cycle of Caulobacter crescentus. A single flagellum is assembled at the swarmer pole of the predivisional cell and is released later in the cell cycle. Here we show that the MS-ring monomer FliF, a central motor component that anchors the flagellum in the cell membrane, is synthesized only in the predivisional cell and is integrated into the membrane at the incipient swarmer cell pole, where it initiates flagellar assembly. FliF is proteolytically turned over during swarmer-to-stalked cell differentiation, coinciding with the loss of the flagellum, suggesting that its degradation is coupled to flagellar release. The membrane topology of FliF was determined and a region of the cytoplasmic C-terminal domain was shown to be required for the interaction with a component of the motor switch. The very C-terminal end of FliF contains a turnover determinant, required for the cell cycle-dependent degradation of the MS-ring. The cell cycle-dependent proteolysis of FliF and the targeting of FliF to the swarmer pole together contribute to the asymmetric localization of the MS-ring in the predivisional cell. Images PMID:8665847

  4. Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics*

    PubMed Central

    Subota, Ines; Julkowska, Daria; Vincensini, Laetitia; Reeg, Nele; Buisson, Johanna; Blisnick, Thierry; Huet, Diego; Perrot, Sylvie; Santi-Rocca, Julien; Duchateau, Magalie; Hourdel, Véronique; Rousselle, Jean-Claude; Cayet, Nadège; Namane, Abdelkader; Chamot-Rooke, Julia; Bastin, Philippe

    2014-01-01

    Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity. PMID:24741115

  5. Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium.

    PubMed Central

    Asai, Y; Kojima, S; Kato, H; Nishioka, N; Kawagishi, I; Homma, M

    1997-01-01

    The polar flagellum of Vibrio alginolyticus rotates remarkably fast (up to 1,700 revolutions per second) by using a motor driven by sodium ions. Two genes, motX and motY, for the sodium-driven flagellar motor have been identified in marine bacteria, Vibrio parahaemolyticus and V. alginolyticus. They have no similarity to the genes for proton-driven motors, motA and motB, whose products constitute a proton channel. MotX was proposed to be a component of a sodium channel. Here we identified additional sodium motor genes, pomA and pomB, in V. alginolyticus. Unexpectedly, PomA and PomB have similarities to MotA and MotB, respectively, especially in the predicted transmembrane regions. These results suggest that PomA and PomB may be sodium-conducting channel components of the sodium-driven motor and that the motor part consists of the products of at least four genes, pomA, pomB, motX, and motY. Furthermore, swimming speed was controlled by the expression level of the pomA gene, suggesting that newly synthesized PomA proteins, which are components of a force-generating unit, were successively integrated into the defective motor complexes. These findings imply that Na+-driven flagellar motors may have similar structure and function as proton-driven motors, but with some interesting differences as well, and it is possible to compare and study the coupling mechanisms of the sodium and proton ion flux for the force generation. PMID:9260952

  6. A New Player at the Flagellar Motor: FliL Controls both Motor Output and Bias

    PubMed Central

    Partridge, Jonathan D.; Nieto, Vincent

    2015-01-01

    ABSTRACT The bacterial flagellum is driven by a bidirectional rotary motor, which propels bacteria to swim through liquids or swarm over surfaces. While the functions of the major structural and regulatory components of the flagellum are known, the function of the well-conserved FliL protein is not. In Salmonella and Escherichia coli, the absence of FliL leads to a small defect in swimming but complete elimination of swarming. Here, we tracked single motors of these bacteria and found that absence of FliL decreases their speed as well as switching frequency. We demonstrate that FliL interacts strongly with itself, with the MS ring protein FliF, and with the stator proteins MotA and MotB and weakly with the rotor switch protein FliG. These and other experiments show that FliL increases motor output either by recruiting or stabilizing the stators or by increasing their efficiency and contributes additionally to torque generation at higher motor loads. The increased torque enabled by FliL explains why this protein is essential for swarming on an agar surface expected to offer increased resistance to bacterial movement. PMID:25714720

  7. Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor

    PubMed Central

    Pandini, Alessandro; Kleinjung, Jens; Rasool, Shafqat; Khan, Shahid

    2015-01-01

    Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) “torque” helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could

  8. Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor

    PubMed Central

    Nishihara, Yasutaka; Kitao, Akio

    2015-01-01

    The proton permeation process of the stator complex MotA/B in the flagellar motor of Escherichia coli was investigated. The atomic model structure of the transmembrane part of MotA/B was constructed based on the previously published disulfide cross-linking and tryptophan scanning mutations. The dynamic permeation of hydronium/sodium ions and water molecule through the channel formed in MotA/B was observed using a steered molecular dynamics simulation. During the simulation, Leu46 of MotB acts as the gate for hydronium ion permeation, which induced the formation of water wire that may mediate the proton transfer to Asp32 on MotB. Free energy profiles for permeation were calculated by umbrella sampling. The free energy barrier for H3O+ permeation was consistent with the proton transfer rate deduced from the flagellar rotational speed and number of protons per rotation, which suggests that the gating is the rate-limiting step. Structure and dynamics of the MotA/B with nonprotonated and protonated Asp32, Val43Met, and Val43Leu mutants in MotB were investigated using molecular dynamics simulation. A narrowing of the channel was observed in the mutants, which is consistent with the size-dependent ion selectivity. In MotA/B with the nonprotonated Asp32, the A3 segment in MotA maintained a kink whereas the protonation induced a straighter shape. Assuming that the cytoplasmic domain not included in the atomic model moves as a rigid body, the protonation/deprotonation of Asp32 is inferred to induce a ratchet motion of the cytoplasmic domain, which may be correlated to the motion of the flagellar rotor. PMID:26056313

  9. Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor.

    PubMed

    Nishihara, Yasutaka; Kitao, Akio

    2015-06-23

    The proton permeation process of the stator complex MotA/B in the flagellar motor of Escherichia coli was investigated. The atomic model structure of the transmembrane part of MotA/B was constructed based on the previously published disulfide cross-linking and tryptophan scanning mutations. The dynamic permeation of hydronium/sodium ions and water molecule through the channel formed in MotA/B was observed using a steered molecular dynamics simulation. During the simulation, Leu46 of MotB acts as the gate for hydronium ion permeation, which induced the formation of water wire that may mediate the proton transfer to Asp32 on MotB. Free energy profiles for permeation were calculated by umbrella sampling. The free energy barrier for H3O(+) permeation was consistent with the proton transfer rate deduced from the flagellar rotational speed and number of protons per rotation, which suggests that the gating is the rate-limiting step. Structure and dynamics of the MotA/B with nonprotonated and protonated Asp32, Val43Met, and Val43Leu mutants in MotB were investigated using molecular dynamics simulation. A narrowing of the channel was observed in the mutants, which is consistent with the size-dependent ion selectivity. In MotA/B with the nonprotonated Asp32, the A3 segment in MotA maintained a kink whereas the protonation induced a straighter shape. Assuming that the cytoplasmic domain not included in the atomic model moves as a rigid body, the protonation/deprotonation of Asp32 is inferred to induce a ratchet motion of the cytoplasmic domain, which may be correlated to the motion of the flagellar rotor. PMID:26056313

  10. Effect of the MotB(D33N) mutation on stator assembly and rotation of the proton-driven bacterial flagellar motor

    PubMed Central

    Nakamura, Shuichi; Minamino, Tohru; Kami-ike, Nobunori; Kudo, Seishi; Namba, Keiichi

    2014-01-01

    The bacterial flagellar motor generates torque by converting the energy of proton translocation through the transmembrane proton channel of the stator complex formed by MotA and MotB. The MotA/B complex is thought to be anchored to the peptidoglycan (PG) layer through the PG-binding domain of MotB to act as the stator. The stator units dynamically associate with and dissociate from the motor during flagellar motor rotation, and an electrostatic interaction between MotA and a rotor protein FliG is required for efficient stator assembly. However, the association and dissociation mechanism of the stator units still remains unclear. In this study, we analyzed the speed fluctuation of the flagellar motor of Salmonella enterica wild-type cells carrying a plasmid encoding a nonfunctional stator complex, MotA/B(D33N), which lost the proton conductivity. The wild-type motor rotated stably but the motor speed fluctuated considerably when the expression level of MotA/B(D33N) was relatively high compared to MotA/B. Rapid accelerations and decelerations were frequently observed. A quantitative analysis of the speed fluctuation and a model simulation suggested that the MotA/B(D33N) stator retains the ability to associate with the motor at a low affinity but dissociates more rapidly than the MotA/B stator. We propose that the stator dissociation process depends on proton translocation through the proton channel.

  11. Kinetically resolved states of the Halobacterium halobium flagellar motor switch and modulation of the switch by sensory rhodopsin I.

    PubMed Central

    McCain, D A; Amici, L A; Spudich, J L

    1987-01-01

    Spontaneous switching of the rotation sense of the flagellar motor of the archaebacterium Halobacterium halobium and modulation of the switch by attractant and repellent photostimuli were analyzed by using a computerized cell-tracking system with 67-ms resolution coupled to electronic shutters. The data fit a three-state model of the switch, in which a Poisson process governs the transition from state N (nonreversing) to state R (reversing). After a reversal, the switch returns to state N, passing through an intermediate state I (inactive), which produces a ca. 2-s period of low reversal frequency before the state N Poisson rate is restored. The stochastic nature of the H. halobium switch reveals a close similarity to Escherichia coli flagellar motor properties as elucidated previously. Sensory modulation of the switch by both photoattractant and photorepellent signals can be interpreted in terms of modulation of the single forward rate constant of the N to R transition. Insight into the mechanism of modulation by the phototaxis receptor sensory rhodopsin I (SR-I) was gained by increasing the lifetime of the principal photointermediate of the SR-I photochemical reaction cycle, S373, by replacing the native chromophore, all-trans-retinal, with the acyclic analog, 3,7,11-trimethyl-2,4,6,8-dodecapentaenal. Flash photolysis of analog-containing cells revealed an eightfold decrease in the rate of thermal decay of S373, and behavioral analysis showed longer periods of reversal suppression than that of cells with the native chromophore over similar ranges of illumination intensities. This indicates that attractant signaling is governed by the lifetime of the S373 intermediate rather than by the frequency of photocycling. In this sense, SR-I is similar to rhodopsin, whose function depends on an active photoproduct (Meta-II). PMID:3654583

  12. DNA sequence analysis, gene product identification, and localization of flagellar motor components of Escherichia coli.

    PubMed Central

    Malakooti, J; Komeda, Y; Matsumura, P

    1989-01-01

    The Escherichia coli operon designated flaA contains seven flagellar genes; among them are two switch protein genes whose products are believed to interface with the motility and chemotaxis machinery of the cell. Complementation analysis using several plasmids carrying different portions of the flaA operon and analysis of expression of these plasmids in minicells allowed the identification of two flagellar gene products. The MotD (now called FliN) protein, a flagellar switch protein, was determined to have an apparent molecular weight of 16,000, and the FlaAI (FliL) protein, encoded by a previously unidentified gene, had an apparent molecular weight of 17,000. DNA sequence analysis of the motD gene revealed an open reading frame of 414 base pairs. There were two possible initiation codons (ATG) for motD translation, the first of which overlapped with the termination codon of the upstream gene, flaAII (fliN). The wild-type flaAI gene on the chromosome was replaced with a flaAI gene mutated in vitro. Loss of the flaAI gene product resulted in a nonmotile and nonflagellated phenotype. The subcellular location for both the MotD and FlaAI proteins was determined; the FlaAI protein partitioned exclusively in the insoluble fraction of a whole minicell sonic extract, whereas the MotD protein remained in both the soluble and insoluble fractions. In addition, we subcloned a 2.2-kilobase-pair DNA fragment capable of complementing the remaining four genes of the flaA operon (flbD [fliO], flaR [fliP], flaQ [fliQ], and flaP [fliR]). Images PMID:2651416

  13. Defects in the Flagellar Motor Increase Synthesis of Poly-γ-Glutamate in Bacillus subtilis

    PubMed Central

    Chan, Jia Mun; Guttenplan, Sarah B.

    2014-01-01

    Bacillus subtilis swims in liquid media and swarms over solid surfaces, and it encodes two sets of flagellar stator homologs. Here, we show that B. subtilis requires only the MotA/MotB stator during swarming motility and that the residues required for stator force generation are highly conserved from the Proteobacteria to the Firmicutes. We further find that mutants that abolish stator function also result in an overproduction of the extracellular polymer poly-γ-glutamate (PGA) to confer a mucoid colony phenotype. PGA overproduction appeared to be the result of an increase in the expression of the pgs operon that encodes genes for PGA synthesis. Transposon mutagenesis was conducted to identify insertions that abolished colony mucoidy and disruptions in known transcriptional regulators of PGA synthesis (Com and Deg two-component systems) as well as mutants defective in transcription-coupled DNA repair (Mfd)-reduced expression of the pgs operon. A final class of insertions disrupted proteins involved in the assembly of the flagellar filament (FliD, FliT, and FlgL), and these mutants did not reduce expression of the pgs operon, suggesting a second mechanism of PGA control. PMID:24296669

  14. Structural insights into the interaction between the bacterial flagellar motor proteins FliF and FliG.

    PubMed

    Levenson, Robert; Zhou, Hongjun; Dahlquist, Frederick W

    2012-06-26

    The binding of the soluble cytoplasmic protein FliG to the transmembrane protein FliF is one of the first interactions in the assembly of the bacterial flagellum. Once established, this interaction is integral in keeping the flagellar cytoplasmic ring, responsible for both transmission of torque and control of the rotational direction of the flagellum, anchored to the central transmembrane ring on which the flagellum is assembled. Here we isolate and characterize the interaction between the N-terminal domain of Thermotoga maritima FliG (FliG(N)) and peptides corresponding to the conserved C-terminal portion of T. maritima FliF. Using nuclear magnetic resonance (NMR) and other techniques, we show that the last ~40 amino acids of FliF (FliF(C)) interact strongly (upper bound K(d) in the low nanomolar range) with FliG(N). The formation of this complex causes extensive conformational changes in FliG(N). We find that T. maritima FliG(N) is homodimeric in the absence of the FliF(C) peptide but forms a heterodimeric complex with the peptide, and we show that this same change in oligomeric state occurs in full-length T. maritima FliG, as well. We relate previously observed phenotypic effects of FliF(C) mutations to our direct observation of binding. Lastly, on the basis of NMR data, we propose that the primary interaction site for FliF(C) is located on a conserved hydrophobic patch centered along helix 1 of FliG(N). These results provide new detailed information about the bacterial flagellar motor and support efforts to understand the cytoplasmic ring's precise molecular structure and mechanism of rotational switching. PMID:22670715

  15. A Bacillus Flagellar Motor That Can Use Both Na+ and K+ as a Coupling Ion Is Converted by a Single Mutation to Use Only Na+

    PubMed Central

    Ito, Masahiro

    2012-01-01

    In bacteria, the sodium ion (Na+) cycle plays a critical role in negotiating the challenges of an extremely alkaline and sodium-rich environment. Alkaliphilic bacteria that grow optimally at high pH values use Na+ for solute uptake and flagellar rotation because the proton (H+) motive force is insufficient for use at extremely alkaline pH. Only three types of electrically driven rotary motors exist in nature: the F-type ATPase, the V-type ATPase, and the bacterial flagellar motor. Until now, only H+ and Na+ have been reported as coupling ions for these motors. Here, we report that the alkaliphilic bacterium Bacillus alcalophilus Vedder 1934 can grow not only under a Na+-rich and potassium ion (K+)-poor condition but also under the opposite condition in an extremely alkaline environment. In this organism, swimming performance depends on concentrations of Na+, K+ or Rb+. In the absence of Na+, swimming behavior is clearly K+- dependent. This pattern was confirmed in swimming assays of stator-less Bacillus subtilis and Escherichia coli mutants expressing MotPS from B. alcalophilus (BA-MotPS). Furthermore, a single mutation in BA-MotS was identified that converted the naturally bi-functional BA-MotPS to stators that cannot use K+ or Rb+. This is the first report that describes a flagellar motor that can use K+ and Rb+ as coupling ions. The finding will affect the understanding of the operating principles of flagellar motors and the molecular mechanisms of ion selectivity, the field of the evolution of environmental changes and stresses, and areas of nanotechnology. PMID:23049994

  16. Motor Rotation Is Essential for the Formation of the Periplasmic Flagellar Ribbon, Cellular Morphology, and Borrelia burgdorferi Persistence within Ixodes scapularis Tick and Murine Hosts

    PubMed Central

    Sultan, Syed Z.; Sekar, Padmapriya; Zhao, Xiaowei; Manne, Akarsh; Liu, Jun; Wooten, R. Mark

    2015-01-01

    Borrelia burgdorferi must migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotile flaB mutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized a motB mutant that is nonmotile but retains its periplasmic flagella. Even though ΔmotB bacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ΔmotB cells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ΔmotB cells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts. PMID:25690096

  17. Energy transduction in the bacterial flagellar motor. Effects of load and pH.

    PubMed Central

    Khan, S; Dapice, M; Humayun, I

    1990-01-01

    The effect of load and pH on the relation between proton potential and flagellar rotation has been studied in cells of a smooth-swimming Streptococcus strain. The driving potential, speeds of free-swimming bacteria, and rotation rates of bacteria tethered to glass by a single flagellum were measured. The relation between rotation rate of tethered bacteria and potential was remarkably linear up to nearly -200 mV. The relation between swimming speed and potential exhibited both saturation and threshold, as previously observed in other species. The form of these relations depended on pH. The equivalence of the electrical and chemical potential components of the proton potential in enabling swimming depended on the voltage. Our observations may be most simply accommodated by a kinetic scheme that links transmembrane proton transits to a tightly coupled work cycle. The properties of this scheme were elucidated by computer simulations of the experimental plots. These simulations indicated that the protonable groups that participate in the rate limiting reactions have a fractional electrical distance between three-fourths to all of the way toward the cytoplasm with a corresponding mean proton binding affinity of 10(-7.3)-10(-7.0) M, respectively. PMID:2160845

  18. The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium

    PubMed Central

    Takekawa, Norihiro; Terahara, Naoya; Kato, Takayuki; Gohara, Mizuki; Mayanagi, Kouta; Hijikata, Atsushi; Onoue, Yasuhiro; Kojima, Seiji; Shirai, Tsuyoshi; Namba, Keiichi; Homma, Michio

    2016-01-01

    Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor, and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA and MotB are four- and single-transmembrane proteins, respectively. To generate torque, the MotA/MotB stator unit changes its conformation in response to the ion influx, and interacts with the rotor protein FliG. Here, we overproduced and purified MotA of the hyperthermophilic bacterium Aquifex aeolicus. A chemical crosslinking experiment revealed that MotA formed a multimeric complex, most likely a tetramer. The three-dimensional structure of the purified MotA, reconstructed by electron microscopy single particle imaging, consisted of a slightly elongated globular domain and a pair of arch-like domains with spiky projections, likely to correspond to the transmembrane and cytoplasmic domains, respectively. We show that MotA molecules can form a stable tetrameric complex without MotB, and for the first time, demonstrate the cytoplasmic structure of the stator. PMID:27531865

  19. Serine 26 in the PomB Subunit of the Flagellar Motor Is Essential for Hypermotility of Vibrio cholerae

    PubMed Central

    Halang, Petra; Vorburger, Thomas; Steuber, Julia

    2015-01-01

    Vibrio cholerae is motile by means of its single polar flagellum which is driven by the sodium-motive force. In the motor driving rotation of the flagellar filament, a stator complex consisting of subunits PomA and PomB converts the electrochemical sodium ion gradient into torque. Charged or polar residues within the membrane part of PomB could act as ligands for Na+, or stabilize a hydrogen bond network by interacting with water within the putative channel between PomA and PomB. By analyzing a large data set of individual tracks of swimming cells, we show that S26 located within the transmembrane helix of PomB is required to promote very fast swimming of V. cholerae. Loss of hypermotility was observed with the S26T variant of PomB at pH 7.0, but fast swimming was restored by decreasing the H+ concentration of the external medium. Our study identifies S26 as a second important residue besides D23 in the PomB channel. It is proposed that S26, together with D23 located in close proximity, is important to perturb the hydration shell of Na+ before its passage through a constriction within the stator channel. PMID:25874792

  20. The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium.

    PubMed

    Takekawa, Norihiro; Terahara, Naoya; Kato, Takayuki; Gohara, Mizuki; Mayanagi, Kouta; Hijikata, Atsushi; Onoue, Yasuhiro; Kojima, Seiji; Shirai, Tsuyoshi; Namba, Keiichi; Homma, Michio

    2016-01-01

    Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor, and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA and MotB are four- and single-transmembrane proteins, respectively. To generate torque, the MotA/MotB stator unit changes its conformation in response to the ion influx, and interacts with the rotor protein FliG. Here, we overproduced and purified MotA of the hyperthermophilic bacterium Aquifex aeolicus. A chemical crosslinking experiment revealed that MotA formed a multimeric complex, most likely a tetramer. The three-dimensional structure of the purified MotA, reconstructed by electron microscopy single particle imaging, consisted of a slightly elongated globular domain and a pair of arch-like domains with spiky projections, likely to correspond to the transmembrane and cytoplasmic domains, respectively. We show that MotA molecules can form a stable tetrameric complex without MotB, and for the first time, demonstrate the cytoplasmic structure of the stator. PMID:27531865

  1. Flagellar Motor Switching in Caulobacter Crescentus Obeys First Passage Time Statistics

    NASA Astrophysics Data System (ADS)

    Morse, Michael; Bell, Jordan; Li, Guanglai; Tang, Jay X.

    2015-11-01

    A Caulobacter crescentus swarmer cell is propelled by a helical flagellum, which is rotated by a motor at its base. The motor alternates between rotating in clockwise and counterclockwise directions and spends variable intervals of time in each state. We measure the distributions of these intervals for cells either free swimming or tethered to a glass slide. A peak time of around one second is observed in the distributions for both motor directions with counterclockwise intervals more sharply peaked and clockwise intervals displaying a larger tail at long times. We show that distributions of rotation intervals fit first passage time statistics for a biased random walker and the dynamic binding of CheY-P to FliM motor subunits accounts for this behavior. Our results also suggest that the presence of multiple CheY proteins in C. crescentus may be responsible for differences between its switching behavior and that of the extensively studied E. coli.

  2. Reorganization of Intact Descending Motor Circuits to Replace Lost Connections After Injury.

    PubMed

    Fink, Kathren L; Cafferty, William B J

    2016-04-01

    Neurons have a limited capacity to regenerate in the adult central nervous system (CNS). The inability of damaged axons to re-establish original circuits results in permanent functional impairment after spinal cord injury (SCI). Despite abortive regeneration of axotomized CNS neurons, limited spontaneous recovery of motor function emerges after partial SCI in humans and experimental rodent models of SCI. It is hypothesized that this spontaneous functional recovery is the result of the reorganization of descending motor pathways spared by the injury, suggesting that plasticity of intact circuits is a potent alternative conduit to enhance functional recovery after SCI. In support of this hypothesis, several studies have shown that after unilateral corticospinal tract (CST) lesion (unilateral pyramidotomy), the intact CST functionally sprouts into the denervated side of the spinal cord. Furthermore, pharmacologic and genetic methods that enhance the intrinsic growth capacity of adult neurons or block extracellular growth inhibitors are effective at significantly enhancing intact CST reorganization and recovery of motor function. Owing to its importance in controlling fine motor behavior in primates, the CST is the most widely studied descending motor pathway; however, additional studies in rodents have shown that plasticity within other spared descending motor pathways, including the rubrospinal tract, raphespinal tract, and reticulospinal tract, can also result in restoration of function after incomplete SCI. Identifying the molecular mechanisms that drive plasticity within intact circuits is crucial in developing novel, potent, and specific therapeutics to restore function after SCI. In this review we discuss the evidence supporting a focus on exploring the capacity of intact motor circuits to functionally repair the damaged CNS after SCI. PMID:26846379

  3. Observed frequency-independent torque in flagellar bacterial motors optimizes space exploration.

    PubMed

    Di Salvo, Mario E; Condat, C A

    2012-12-01

    A surprising feature of many bacterial motors is the apparently conserved form of their torque-frequency relation. Experiments indicate that the torque provided by the bacterial rotary motor is approximately constant over a large range of angular speeds. This is observed in both monotrichous and peritrichous bacteria, independently of whether they are propelled by a proton flux or by a Na(+) ion flux. If the relation between angular speed ω and swimming speed is linear, a ω-independent torque implies that the power spent in active motion is proportional to the instantaneous bacterial speed. Using realistic values of the relevant parameters, we show that a constant torque maximizes the volume of the region explored by a bacterium in a resource-depleted medium. Given that nutrients in the ocean are often concentrated in separate, ephemeral patches, we propose that the observed constancy of the torque may be a trait evolved to maximize bacterial survival in the ocean. PMID:23367976

  4. Internal and external components of the bacterial flagellar motor rotate as a unit.

    PubMed

    Hosu, Basarab G; Nathan, Vedavalli S J; Berg, Howard C

    2016-04-26

    Most bacteria that swim, including Escherichia coli, are propelled by helical filaments, each driven at its base by a rotary motor powered by a proton or a sodium ion electrochemical gradient. Each motor contains a number of stator complexes, comprising 4MotA 2MotB or 4PomA 2PomB, proteins anchored to the rigid peptidoglycan layer of the cell wall. These proteins exert torque on a rotor that spans the inner membrane. A shaft connected to the rotor passes through the peptidoglycan and the outer membrane through bushings, the P and L rings, connecting to the filament by a flexible coupling known as the hook. Although the external components, the hook and the filament, are known to rotate, having been tethered to glass or marked by latex beads, the rotation of the internal components has remained only a reasonable assumption. Here, by using polarized light to bleach and probe an internal YFP-FliN fusion, we show that the innermost components of the cytoplasmic ring rotate at a rate similar to that of the hook. PMID:27071081

  5. Mutations alter the sodium versus proton use of a Bacillus clausii flagellar motor and confer dual ion use on Bacillus subtilis motors.

    PubMed

    Terahara, Naoya; Krulwich, Terry A; Ito, Masahiro

    2008-09-23

    Bacterial flagella contain membrane-embedded stators, Mot complexes, that harness the energy of either transmembrane proton or sodium ion gradients to power motility. Use of sodium ion gradients is associated with elevated pH and sodium concentrations. The Mot complexes studied to date contain channels that use either protons or sodium ions, with some bacteria having only one type and others having two distinct Mot types with different ion-coupling. Here, alkaliphilic Bacillus clausii KSM-K16 was shown to be motile in a pH range from 7 to 11 although its genome encodes only one Mot (BCl-MotAB). Assays of swimming as a function of pH, sodium concentration, and ion-selective motility inhibitors showed that BCl-MotAB couples motility to sodium at the high end of its pH range but uses protons at lower pH. This pattern was confirmed in swimming assays of a statorless Bacillus subtilis mutant expressing either BCl-MotAB or one of the two B. subtilis stators, sodium-coupled Bs-MotPS or proton-coupled Bs-MotAB. Pairs of mutations in BCl-MotB were identified that converted the naturally bifunctional BCl-MotAB to stators that preferentially use either protons or sodium ions across the full pH range. We then identified trios of mutations that added a capacity for dual-ion coupling on the distinct B. subtilis Bs-MotAB and Bs-MotPS motors. Determinants that alter the specificity of bifunctional and single-coupled flagellar stators add to insights from studies of other ion-translocating transporters that use both protons and sodium ions. PMID:18796609

  6. The C-terminal periplasmic domain of MotB is responsible for load-dependent control of the number of stators of the bacterial flagellar motor

    PubMed Central

    Castillo, David J.; Nakamura, Shuichi; Morimoto, Yusuke V.; Che, Yong-Suk; Kami-ike, Nobunori; Kudo, Seishi; Minamino, Tohru; Namba, Keiichi

    2013-01-01

    The bacterial flagellar motor is made of a rotor and stators. In Salmonella it is thought that about a dozen MotA/B complexes are anchored to the peptidoglycan layer around the motor through the C-terminal peptidoglycan-binding domain of MotB to become active stators as well as proton channels. MotB consists of 309 residues, forming a single transmembrane helix (30–50), a stalk (51–100) and a C-terminal peptidoglycan-binding domain (101–309). Although the stalk is dispensable for torque generation by the motor, it is required for efficient motor performance. Residues 51 to 72 prevent premature proton leakage through the proton channel prior to stator assembly into the motor. However, the role of residues 72–100 remains unknown. Here, we analyzed the torque-speed relationship of the MotB(Δ72–100) motor. At a low speed near stall, this mutant motor produced torque at the wild-type level. Unlike the wild-type motor, however, torque dropped off drastically by slight decrease in external load and then showed a slow exponential decay over a wide range of load by its further reduction. Since it is known that the stator is a mechano-sensor and that the number of active stators changes in a load-dependent manner, we interpreted this unusual torque-speed relationship as anomaly in load-dependent control of the number of active stators. The results suggest that residues 72–100 of MotB is required for proper load-dependent control of the number of active stators around the rotor.

  7. Normal motor adaptation in cervical dystonia: a fundamental cerebellar computation is intact.

    PubMed

    Sadnicka, Anna; Patani, Bansi; Saifee, Tabish A; Kassavetis, Panagiotis; Pareés, Isabel; Korlipara, Prasad; Bhatia, Kailash P; Rothwell, John C; Galea, Joseph M; Edwards, Mark J

    2014-10-01

    The potential role of the cerebellum in the pathophysiology of dystonia has become a focus of recent research. However, direct evidence for a cerebellar contribution in humans with dystonia is difficult to obtain. We examined motor adaptation, a test of cerebellar function, in 20 subjects with primary cervical dystonia and an equal number of aged matched controls. Adaptation to both visuomotor (distorting visual feedback by 30°) and forcefield (applying a velocity-dependent force) conditions were tested. Our hypothesis was that cerebellar abnormalities observed in dystonia research would translate into deficits of cerebellar adaptation. We also examined the relationship between adaptation and dystonic head tremor as many primary tremor models implicate the cerebellothalamocortical network which is specifically tested by this motor paradigm. Rates of adaptation (learning) in cervical dystonia were identical to healthy controls in both visuomotor and forcefield tasks. Furthermore, the ability to adapt was not clearly related to clinical features of dystonic head tremor. We have shown that a key motor control function of the cerebellum is intact in the most common form of primary dystonia. These results have important implications for current anatomical models of the pathophysiology of dystonia. It is important to attempt to progress from general statements that implicate the cerebellum to a more specific evidence-based model. The role of the cerebellum in this enigmatic disease perhaps remains to be proven. PMID:24872202

  8. Inactivation of a putative flagellar motor switch protein FliG1 prevents Borrelia burgdorferi from swimming in highly viscous media and blocks its infectivity

    PubMed Central

    Li, Chunhao; Xu, Hongbin; Zhang, Kai; Liang, Fang Ting

    2015-01-01

    Summary The flagellar motor switch complex protein FliG plays an essential role in flagella biosynthesis and motility. In most motile bacteria, only one fliG homologue is present in the genome. However, several spirochete species have two putative fliG genes (referred to as fliG1 and fliG2) and their roles in flagella assembly and motility remain unknown. In this report, the Lyme disease spirochete Borrelia burgdorferi was used as a genetic model to investigate the roles of these two fliG homologues. It was found that fliG2 encodes a typical motor switch complex protein that is required for the flagellation and motility of B. burgdorferi. In contrast, the function of fliG1 is quite unique. Disruption of fliG1 did not affect flagellation and the mutant was still motile but failed to translate in highly viscous media. GFP-fusion and motion tracking analyses revealed that FliG1 asymmetrically locates at one end of cells and the loss of fliG1 somehow impacted one bundle of flagella rotation. In addition, animal studies demonstrated that the fliG1− mutant was quickly cleared after inoculation into the murine host, which highlights the importance of the ability to swim in highly viscous media in the infectivity of B. burgdorferi and probably other pathogenic spirochetes. PMID:20180908

  9. Expression, purification and biochemical characterization of the cytoplasmic loop of PomA, a stator component of the Na+ driven flagellar motor

    PubMed Central

    Abe-Yoshizumi, Rei; Kobayashi, Shiori; Gohara, Mizuki; Hayashi, Kokoro; Kojima, Chojiro; Kojima, Seiji; Sudo, Yuki; Asami, Yasuo; Homma, Michio

    2013-01-01

    Flagellar motors embedded in bacterial membranes are molecular machines powered by specific ion flows. Each motor is composed of a stator and a rotor and the interactions of those components are believed to generate the torque. Na+ influx through the PomA/PomB stator complex of Vibrio alginolyticus is coupled to torque generation and is speculated to trigger structural changes in the cytoplasmic domain of PomA that interacts with a rotor protein in the C-ring, FliG, to drive the rotation. In this study, we tried to overproduce the cytoplasmic loop of PomA (PomA-Loop), but it was insoluble. Thus, we made a fusion protein with a small soluble tag (GB1) which allowed us to express and characterize the recombinant protein. The structure of the PomA-Loop seems to be very elongated or has a loose tertiary structure. When the PomA-Loop protein was produced in E. coli, a slight dominant effect was observed on motility. We conclude that the cytoplasmic loop alone retains a certain function. PMID:27493537

  10. Corticospinal activity evoked and modulated by non-invasive stimulation of the intact human motor cortex.

    PubMed

    Di Lazzaro, Vincenzo; Rothwell, John C

    2014-10-01

    A number of methods have been developed recently that stimulate the human brain non-invasively through the intact scalp. The most common are transcranial magnetic stimulation (TMS), transcranial electric stimulation (TES) and transcranial direct current stimulation (TDCS). They are widely used to probe function and connectivity of brain areas as well as therapeutically in a variety of conditions such as depression or stroke. They are much less focal than conventional invasive methods which use small electrodes placed on or in the brain and are often thought to activate all classes of neurones in the stimulated area. However, this is not true. A large body of evidence from experiments on the motor cortex shows that non-invasive methods of brain stimulation can be surprisingly selective and that adjusting the intensity and direction of stimulation can activate different classes of inhibitory and excitatory inputs to the corticospinal output cells. Here we review data that have elucidated the action of TMS and TES, concentrating mainly on the most direct evidence available from spinal epidural recordings of the descending corticospinal volleys. The results show that it is potentially possible to test and condition specific neural circuits in motor cortex that could be affected differentially by disease, or be used in different forms of natural behaviour. However, there is substantial interindividual variability in the specificity of these protocols. Perhaps in the future it will be possible, with the advances currently being made to model the electrical fields induced in individual brains, to develop forms of stimulation that can reliably target more specific populations of neurones, and open up the internal circuitry of the motor cortex for study in behaving humans. PMID:25172954

  11. Reconstitution of flagellar sliding.

    PubMed

    Alper, Joshua; Geyer, Veikko; Mukundan, Vikram; Howard, Jonathon

    2013-01-01

    The motile structure within eukaryotic cilia and flagella is the axoneme. This structure typically consists of nine doublet microtubules arranged around a pair of singlet microtubules. The axoneme contains more than 650 different proteins that have structural, force-generating, and regulatory functions. Early studies on sea urchin sperm identified the force-generating components, the dynein motors. It was shown that dynein can slide adjacent doublet microtubules in the presence of ATP. How this sliding gives rise to the beating of the axoneme is still unknown. Reconstitution assays provide a clean system, free from cellular effects, to elucidate the underlying beating mechanisms. These assays can be used to identify the components that are both necessary and sufficient for the generation of flagellar beating. PMID:23498749

  12. Hydrodynamic interaction of bacterial flagella - flagellar bundling

    NASA Astrophysics Data System (ADS)

    Lim, Sookkyung

    2013-11-01

    Flagellar bundling is an important aspect of locomotion in bacteria such as Escherichia coli. To study the hydrodynamic behavior of helical flagella, we present a computational model that is based on the geometry of the bacterial flagellar filament at the micrometer scale. We consider two model flagella, each of which has a rotary motor at its base with the rotation rate of the motor set at 100 Hz. Bundling occurs when both flagella are left-handed helices turning counterclockwise (when viewed from the nonmotor end of the flagellum looking back toward the motor) or when both flagella are right-handed helices turning clockwise. Helical flagella of the other combinations of handedness and rotation direction do not bundle. In this work we use the generalized immersed boundary method combined with the unconstrained Kirchhoff rod theory, which allows us to study the complicated hydrodynamics of flagellar behavior. This is a joint work with Charlie Peskin at NYU. NSF

  13. Mutations in the Borrelia burgdorferi Flagellar Type III Secretion System Genes fliH and fliI Profoundly Affect Spirochete Flagellar Assembly, Morphology, Motility, Structure, and Cell Division

    PubMed Central

    Gao, Lihui; Zhao, Xiaowei; Liu, Jun; Norris, Steven J.

    2015-01-01

    ABSTRACT The Lyme disease spirochete Borrelia burgdorferi migrates to distant sites in the tick vectors and mammalian hosts through robust motility and chemotaxis activities. FliH and FliI are two cytoplasmic proteins that play important roles in the type III secretion system (T3SS)-mediated export and assembly of flagellar structural proteins. However, detailed analyses of the roles of FliH and FliI in B. burgdorferi have not been reported. In this study, fliH and fliI transposon mutants were utilized to dissect the mechanism of the Borrelia type III secretion system. The fliH and fliI mutants exhibited rod-shaped or string-like morphology, greatly reduced motility, division defects (resulting in elongated organisms with incomplete division points), and noninfectivity in mice by needle inoculation. Mutants in fliH and fliI were incapable of translational motion in 1% methylcellulose or soft agar. Inactivation of either fliH or fliI resulted in the loss of the FliH-FliI complex from otherwise intact flagellar motors, as determined by cryo-electron tomography (cryo-ET). Flagellar assemblies were still present in the mutant cells, albeit in lower numbers than in wild-type cells and with truncated flagella. Genetic complementation of fliH and fliI mutants in trans restored their wild-type morphology, motility, and flagellar motor structure; however, full-length flagella and infectivity were not recovered in these complemented mutants. Based on these results, disruption of either fliH or fliI in B. burgdorferi results in a severe defect in flagellar structure and function and cell division but does not completely block the export and assembly of flagellar hook and filament proteins. PMID:25968649

  14. Incomplete flagellar structures in Escherichia coli mutants.

    PubMed Central

    Suzuki, T; Komeda, Y

    1981-01-01

    Escherichia coli mutants with defects in 29 flagellar genes identified so far were examined by electron microscopy for possession of incomplete flagellar structures in membrane-associated fractions. The results are discussed in consideration of the known transcriptional interaction of flagellar genes. Hook-basal body structures were detected in flaD, flaS, flaT, flbC, and hag mutants. The flaE mutant had a polyhook-basal body structure. An intact basal body appeared in flaK mutants. Putative precursors of the basal body were detected in mutants with defects in flaM, flaU, flaV, and flaY. No structures homologous to the flagellar basal body or its parts were detected in mutants with defects in flaA, flaB, flaC, flaG, flaH, flaI, flaL, flaN, flaO, flaP, flaQ, flaR, flaW, flaX, flbA, flbB, and flbD. One flaZ mutant had an incomplete flagellar basal body structure and another formed no significant structure, suggesting that flaZ is responsible for both basal body assembly and the transcription of the hag gene. Images PMID:7007337

  15. Flagellar waveform analysis of swimming algal cells

    NASA Astrophysics Data System (ADS)

    Kurtuldu, Huseyin; Johnson, Karl; Gollub, Jerry

    2011-11-01

    The twin flagella of the green alga Chlamydomas reinhardtii are driven by dynein molecular motors to oscillate at about 50-60 Hz in a breaststroke motion. For decades, Chlamydomas has been used as a model organism for studies of flagellar motility, and of genetic disorders of ciliary motion. However, little is known experimentally about the flagellar waveforms, and the resulting time-dependent force distribution along the 250 nm diameter flagella. Here, we study flagellar dynamics experimentally by confining cells in quasi-2D liquid films. From simultaneous measurements of the cell body velocity and the time-dependent velocities along the center lines of the two flagella, we determine the drag coefficients, and estimate the power expended by the body and the flagella, comparing our findings with measurements based on the induced fluid flow field. We contrast the results for the quite different beating patterns of synchronous and asynchronous flagella, respectively. Supported by NSF Grant DMR-0803153.

  16. Intact Acquisition and Short-Term Retention of Non-Motor Procedural Learning in Parkinson's Disease.

    PubMed

    Panouillères, Muriel T N; Tofaris, George K; Brown, Peter; Jenkinson, Ned

    2016-01-01

    Procedural learning is a form of memory where people implicitly acquire a skill through repeated practice. People with Parkinson's disease (PD) have been found to acquire motor adaptation, a form of motor procedural learning, similarly to healthy older adults but they have deficits in long-term retention. A similar pattern of normal learning on initial exposure with a deficit in retention seen on subsequent days has also been seen in mirror-reading, a form of non-motor procedural learning. It is a well-studied fact that disrupting sleep will impair the consolidation of procedural memories. Given the prevalence of sleep disturbances in PD, the lack of retention on following days seen in these studies could simply be a side effect of this well-known symptom of PD. Because of this, we wondered whether people with PD would present with deficits in the short-term retention of a non-motor procedural learning task, when the test of retention was done the same day as the initial exposure. The aim of the present study was then to investigate acquisition and retention in the immediate short term of cognitive procedural learning using the mirror-reading task in people with PD. This task involved two conditions: one where triads of mirror-inverted words were always new that allowed assessing the learning of mirror-reading skill and another one where some of the triads were presented repeatedly during the experiment that allowed assessing the word-specific learning. People with PD both ON and OFF their normal medication were compared to healthy older adults and young adults. Participants were re-tested 50 minutes break after initial exposure to probe for short-term retention. The results of this study show that all groups of participants acquired and retained the two skills (mirror-reading and word-specific) similarly. These results suggest that neither healthy ageing nor the degeneration within the basal ganglia that occurs in PD does affect the mechanisms that underpin the

  17. Studies on the mechanism of bacterial flagellar rotation and the flagellar number regulation.

    PubMed

    Kojima, Seiji

    2016-01-01

    Many motile bacteria have the motility organ, the flagellum. It rotates by the rotary motor driven by the ion-motive force and is embedded in the cell surface at the base of each flagellar filament. Many researchers have been studying its rotary mechanism for years, but most of the energy conversion processes have been remained in mystery. We focused on the flagellar stator, which works at the core process of energy conversion, and found that the periplasmic region of the stator changes its conformation to be activated only when the stator units are incorporated into the motor and anchored at the cell wall. Meanwhile, the physiologically important supramolecular complex is localized in the cell at the right place and the right time with a proper amount. How the cell achieves such a proper localization is the fundamental question for life science, and we undertake this problem by analyzing the mechanism for biogenesis of a single polar flagellum of Vibrio alginolyticus. Here I describe the molecular mechanism of how the flagellum is generated at the specific place with a proper number, and also how the flagellar stator is incorporated into the motor to complete the functional motor assembly, based on our studies. PMID:27581279

  18. Second-Chance Signal Transduction Explains Cooperative Flagellar Switching

    PubMed Central

    Zot, Henry G.; Hasbun, Javier E.; Van Minh, Nguyen

    2012-01-01

    The reversal of flagellar motion (switching) results from the interaction between a switch complex of the flagellar rotor and a torque-generating stationary unit, or stator (motor unit). To explain the steeply cooperative ligand-induced switching, present models propose allosteric interactions between subunits of the rotor, but do not address the possibility of a reaction that stimulates a bidirectional motor unit to reverse direction of torque. During flagellar motion, the binding of a ligand-bound switch complex at the dwell site could excite a motor unit. The probability that another switch complex of the rotor, moving according to steady-state rotation, will reach the same dwell site before that motor unit returns to ground state will be determined by the independent decay rate of the excited-state motor unit. Here, we derive an analytical expression for the energy coupling between a switch complex and a motor unit of the stator complex of a flagellum, and demonstrate that this model accounts for the cooperative switching response without the need for allosteric interactions. The analytical result can be reproduced by simulation when (1) the motion of the rotor delivers a subsequent ligand-bound switch to the excited motor unit, thereby providing the excited motor unit with a second chance to remain excited, and (2) the outputs from multiple independent motor units are constrained to a single all-or-none event. In this proposed model, a motor unit and switch complex represent the components of a mathematically defined signal transduction mechanism in which energy coupling is driven by steady-state and is regulated by stochastic ligand binding. Mathematical derivation of the model shows the analytical function to be a general form of the Hill equation (Hill AV (1910) The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J Physiol 40: iv–vii). PMID:22844429

  19. Novel Components of the Flagellar System in Epsilonproteobacteria

    PubMed Central

    Gao, Beile; Lara-Tejero, Maria; Lefebre, Matthew; Goodman, Andrew L.

    2014-01-01

    ABSTRACT Motility is essential for the pathogenesis of many bacterial species. Most bacteria move using flagella, which are multiprotein filaments that rotate propelled by a cell wall-anchored motor using chemical energy. Although some components of the flagellar apparatus are common to many bacterial species, recent studies have shown significant differences in the flagellar structures of different bacterial species. The molecular bases for these differences, however, are not understood. The flagella from epsilonproteobacteria, which include the bacterial pathogens Campylobacter jejuni and Helicobacter pylori, are among the most divergent. Using next-generation sequencing combined with transposon mutagenesis, we have conducted a comprehensive high-throughput genetic screen in Campylobacter jejuni, which identified several novel components of its flagellar system. Biochemical analyses detected interactions between the identified proteins and known components of the flagellar machinery, and in vivo imaging located them to the bacterial poles, where flagella assemble. Most of the identified new components are conserved within but restricted to epsilonproteobacteria. These studies provide insight into the divergent flagella of this group of bacteria and highlight the complexity of this remarkable structure, which has adapted to carry out its conserved functions in the context of widely diverse bacterial species. PMID:24961693

  20. Modulation of Training by Single-Session Transcranial Direct Current Stimulation to the Intact Motor Cortex Enhances Motor Skill Acquisition of the Paretic Hand

    PubMed Central

    Zimerman, Máximo; Heise, Kirstin F.; Hoppe, Julia; Cohen, Leonardo G.; Gerloff, Christian; Hummel, Friedhelm C.

    2016-01-01

    Background and Purpose Mechanisms of skill learning are paramount components for stroke recovery. Recent noninvasive brain stimulation studies demonstrated that decreasing activity in the contralesional motor cortex might be beneficial, providing transient functional improvements after stroke. The more crucial question, however, is whether this intervention can also enhance the acquisition of complex motor tasks, yielding longer-lasting functional improvements. In the present study, we tested the capacity of cathodal transcranial direct current stimulation (tDCS) applied over the contralesional motor cortex during training to enhance the acquisition and retention of complex sequential finger movements of the paretic hand. Method Twelve well-recovered chronic patients with subcortical stroke attended 2 training sessions during which either cathodal tDCS or a sham intervention were applied to the contralesional motor cortex in a double-blind, crossover design. Two different motor sequences, matched for their degree of complexity, were tested in a counterbalanced order during as well as 90 minutes and 24 hours after the intervention. Potential underlying mechanisms were evaluated with transcranial magnetic stimulation. Results tDCS facilitated the acquisition of a new motor skill compared with sham stimulation (P=0.04) yielding better task retention results. A significant correlation was observed between the tDCS-induced improvement during training and the tDCS-induced changes of intracortical inhibition (R2=0.63). Conclusions These results indicate that tDCS is a promising tool to improve not only motor behavior, but also procedural learning. They further underline the potential of noninvasive brain stimulation as an adjuvant treatment for long-term recovery, at least in patients with mild functional impairment after stroke. PMID:22618381

  1. Flagellar apparatus structure of choanoflagellates.

    PubMed

    Karpov, Sergey A

    2016-01-01

    Phylum choanoflagellata is the nearest unicellular neighbor of metazoa at the phylogenetic tree. They are single celled or form the colonies, can be presented by naked cells or live in theca or lorica, but in all cases they have a flagellum surrounded by microvilli of the collar. They have rather uniform and peculiar flagellar apparatus structure with flagellar basal body (FB) producing a flagellum, and non-flagellar basal body (NFB) lying orthogonal to the FB. Long flagellar transition zone contains a unique structure among eukaryotes, the central filament, which connects central microtubules to the transversal plate. Both basal bodies are composed of triplets and interconnected with fibrillar bridge. They also contain the internal arc-shaped connectives between the triplets. The FB has prominent transitional fibers similar to those of chytrid zoospores and choanocytes of sponges, and a radial microtubular root system. The ring-shaped microtubule organizing center (MTOC) produces radial root microtubules, but in some species a MTOC is represented by separate foci. The NFB has a narrow fibrillar root directed towards the Golgi apparatus in association with membrane-bounded sac. Prior to cell division, the basal bodies replicate and migrate to poles of elongated nucleus. The basal bodies serve as MTOCs for the spindle microtubules during nuclear division by semiopen orthomitosis. PMID:27148446

  2. Intact Acquisition and Short-Term Retention of Non-Motor Procedural Learning in Parkinson’s Disease

    PubMed Central

    Panouillères, Muriel T. N.; Tofaris, George K.; Brown, Peter; Jenkinson, Ned

    2016-01-01

    Procedural learning is a form of memory where people implicitly acquire a skill through repeated practice. People with Parkinson’s disease (PD) have been found to acquire motor adaptation, a form of motor procedural learning, similarly to healthy older adults but they have deficits in long-term retention. A similar pattern of normal learning on initial exposure with a deficit in retention seen on subsequent days has also been seen in mirror-reading, a form of non-motor procedural learning. It is a well-studied fact that disrupting sleep will impair the consolidation of procedural memories. Given the prevalence of sleep disturbances in PD, the lack of retention on following days seen in these studies could simply be a side effect of this well-known symptom of PD. Because of this, we wondered whether people with PD would present with deficits in the short-term retention of a non-motor procedural learning task, when the test of retention was done the same day as the initial exposure. The aim of the present study was then to investigate acquisition and retention in the immediate short term of cognitive procedural learning using the mirror-reading task in people with PD. This task involved two conditions: one where triads of mirror-inverted words were always new that allowed assessing the learning of mirror-reading skill and another one where some of the triads were presented repeatedly during the experiment that allowed assessing the word-specific learning. People with PD both ON and OFF their normal medication were compared to healthy older adults and young adults. Participants were re-tested 50 minutes break after initial exposure to probe for short-term retention. The results of this study show that all groups of participants acquired and retained the two skills (mirror-reading and word-specific) similarly. These results suggest that neither healthy ageing nor the degeneration within the basal ganglia that occurs in PD does affect the mechanisms that underpin the

  3. Dynamics of flagellar bundling

    NASA Astrophysics Data System (ADS)

    Janssen, Pieter; Graham, Michael

    2010-11-01

    Flagella are long thin appendages of microscopic organisms used for propulsion in low-Reynolds environments. For E. coli the flagella are driven by a molecular motor, which rotates the flagella in a counter-clockwise motion (CCM). When in a forward swimming motion, all flagella bundle up. If a motor reverses rotation direction, the flagella unbundle and the cell makes a tumbling motion. When all motors turn in the same CC direction again, the flagella bundle up, and forward swimming continues. To investigate the bundling, we consider two flexible helices next to each other, as well as several flagella attached to a spherical body. Each helix is modeled as several prolate spheroids connected at the tips by springs. For hydrodynamic interactions, we consider the flagella to made up of point forces, while the finite size of the body is incorporated via Fax'en's laws. We show that synchronization occurs quickly relative to the bundling process. For flagella next to each other, the initial deflection is generated by rotlet interactions generated by the rotating helices. At longer times, simulations show the flagella only wrap once around each other, but only for flagella that are closer than about 4 helix radii. Finally, we show a run-and-tumble motion of the body with attached flagella.

  4. Motility and Flagellar Glycosylation in Clostridium difficile▿ †

    PubMed Central

    Twine, Susan M.; Reid, Christopher W.; Aubry, Annie; McMullin, David R.; Fulton, Kelly M.; Austin, John; Logan, Susan M.

    2009-01-01

    In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In C. difficile strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein. PMID:19749038

  5. Nervous propagation along 'central' motor pathways in intact man: characteristics of motor responses to 'bifocal' and 'unifocal' spine and scalp non-invasive stimulation.

    PubMed

    Rossini, P M; Marciani, M G; Caramia, M; Roma, V; Zarola, F

    1985-10-01

    In 23 healthy adult volunteers motor action potentials (MAPs) were elicited in upper and lower limb muscles during stimulation of appropriate sites at spinal and scalp level, through skin electrodes. 'Bifocal' stimulation of scalp and spine motor tracts was performed with 2 plaques (3.5 cm2 each), delivering single pulses of 440-940 mA, less than 50 microseconds in duration, which elicited high voltage (up to 10 mV) MAPs in arm and leg muscles. 'Unifocal' stimulation of scalp was carried out through a cathode consisting in a belt or in a series of rectangular interconnected plaques secured around the head, 1-2 cm rostral to the nasion-inion plane, and in a circular anode placed on the appropriate scalp site. MAPs with similar amplitude-latency characteristics were recorded with both 'bifocal' and 'unifocal' stimulating methods. However, the 'unifocal' stimulation necessitated 5-10 times less current than the 'bifocal' one. The 'unifocal' device using the interconnected plaques (6-12 in number) provided the most tolerable stimuli with the lowest amount of current (60-106 mA, rectangular pulses of 100-150 microseconds). Conduction times and velocities of motor pathways in various 'central' and 'peripheral' districts were calculated. Voluntary contraction of target muscles remarkably enhanced MAP amplitudes during scalp, but not during spine stimulation. A nerve action potential was recorded from ulnar nerve during scalp stimulation. MAPs in hand muscles to scalp stimulation were obliterated by the simultaneous activation of the peripheral fibres innervating the target muscle, because of collision between ortho- and antidromically propagated motor impulses. Anodal stimuli showed liminal values significantly lower than the cathodal ones. Mapping studies have been carried out with 'unifocal' scalp stimulation by using different types of anode and of stimulus parameters. PMID:2411506

  6. Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium.

    PubMed

    Takekawa, Norihiro; Nishiyama, Masayoshi; Kaneseki, Tsuyoshi; Kanai, Tamotsu; Atomi, Haruyuki; Kojima, Seiji; Homma, Michio

    2015-01-01

    Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s(-1) at 85 °C. We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+). As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na(+) for energy coupling of the flagellar motor. The Na(+)-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution. PMID:26244427

  7. Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium

    PubMed Central

    Takekawa, Norihiro; Nishiyama, Masayoshi; Kaneseki, Tsuyoshi; Kanai, Tamotsu; Atomi, Haruyuki; Kojima, Seiji; Homma, Michio

    2015-01-01

    Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s−1 at 85 °C. We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na+. As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na+ for energy coupling of the flagellar motor. The Na+-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution. PMID:26244427

  8. Single-Molecule Studies of Rotary Molecular Motors

    NASA Astrophysics Data System (ADS)

    Pilizota, Teuta; Sowa, Yoshiyuki; Berry, Richard M.

    Rotary molecular motors are protein complexes that transform chemical or electrochemical energy into mechanical work. There are five known rotary molecular motors in nature; the bacterial flagellar motor, and two motors in each of ATP-synthase and V-ATPase. Rotation of the flagellar motor drives a helical propeller that powers bacterial swimming. The function of the other rotary motors is to couple electrochemical ion gradients to synthesis or hydrolysis of ATP, and rotation is a detail of the coupling mechanism rather than the ultimate purpose of the motors. Much has been learned about the mechanism of the F1 part of ATP-synthase and the flagellar motor by measuring the rotation of single motors with a variety of techniques under a wide range of conditions. This chapter will review the structures of ATP-synthase and the flagellar motor, and what has been learned about their mechanisms using single molecule techniques.

  9. Flagellar flows around bacterial swarms

    NASA Astrophysics Data System (ADS)

    Dauparas, Justas; Lauga, Eric

    2016-08-01

    Flagellated bacteria on nutrient-rich substrates can differentiate into a swarming state and move in dense swarms across surfaces. A recent experiment measured the flow in the fluid around an Escherichia coli swarm [Wu, Hosu, and Berg, Proc. Natl. Acad. Sci. USA 108, 4147 (2011)], 10.1073/pnas.1016693108. A systematic chiral flow was observed in the clockwise direction (when viewed from above) ahead of the swarm with flow speeds of about 10 μ m /s , about 3 times greater than the radial velocity at the edge of the swarm. The working hypothesis is that this flow is due to the action of cells stalled at the edge of a colony that extend their flagellar filaments outward, moving fluid over the virgin agar. In this work we quantitatively test this hypothesis. We first build an analytical model of the flow induced by a single flagellum in a thin film and then use the model, and its extension to multiple flagella, to compare with experimental measurements. The results we obtain are in agreement with the flagellar hypothesis. The model provides further quantitative insight into the flagella orientations and their spatial distributions as well as the tangential speed profile. In particular, the model suggests that flagella are on average pointing radially out of the swarm and are not wrapped tangentially.

  10. Successive incorporation of force-generating units in the bacterial rotary motor

    NASA Astrophysics Data System (ADS)

    Block, Steven M.; Berg, Howard C.

    1984-05-01

    Mot mutants of Escherichia coli are paralysed: their flagella appear to be intact but do not rotate1 . The motA and motB gene products are found in the cytoplasmic membrane2; they do not co-purify with flagellar basal bodies isolated in neutral detergents1. Silverman et al. found that mot mutants could be `resurrected' through protein synthesis directed by λ transducing phages carrying the wild-type genes2. Here, we have studied this activation at the level of a single flagellar motor. Cells of a motB strain carrying plasmids in which transcription of the wild-type motB gene was controlled by the lac promoter were tethered to a glass surface by a single flagellum. These cells began to spin within several minutes after the addition of a lac inducer, and their rotational speed changed in a series of equally spaced steps. As many as 7 steps were seen in individual cells and, from the final speeds attained, as many as 16 steps could be inferred. These experiments show that each flagellar motor contains several independent force-generating units comprised, at least in part, of motB protein.

  11. Bacterial flagellar microhydrodynamics: Laminar flow over complex flagellar filaments, analog archimedean screws and cylinders, and its perturbations.

    PubMed

    Trachtenberg, Shlomo; Fishelov, Dalia; Ben-Artzi, Matania

    2003-09-01

    The flagellar filament, the bacterial organelle of motility, is the smallest rotary propeller known. It consists of 1), a basal body (part of which is the proton driven rotary motor), 2), a hook (universal joint-allowing for off-axial transmission of rotary motion), and 3), a filament (propeller-a long, rigid, supercoiled helical assembly allowing for the conversion of rotary motion into linear thrust). Helically perturbed (so-called "complex") filaments have a coarse surface composed of deep grooves and ridges following the three-start helical lines. These surface structures, reminiscent of a turbine or Archimedean screw, originate from symmetry reduction along the six-start helical lines due to dimerization of the flagellin monomers from which the filament self assembles. Using high-resolution electron microscopy and helical image reconstruction methods, we calculated three-dimensional density maps of the complex filament of Rhizobium lupini H13-3 and determined its surface pattern and boundaries. The helical symmetry of the filament allows viewing it as a stack of identical slices spaced axially and rotated by constant increments. Here we use the closed outlines of these slices to explore, in two dimensions, the hydrodynamic effect of the turbine-like boundaries of the flagellar filament. In particular, we try to determine if, and under what conditions, transitions from laminar to turbulent flow (or perturbations of the laminar flow) may occur on or near the surface of the bacterial propeller. To address these questions, we apply the boundary element method in a manner allowing the handling of convoluted boundaries. We tested the method on several simple, well-characterized cylindrical structures before applying it to real, highly convoluted biological surfaces and to simplified mechanical analogs. Our results indicate that under extreme structural and functional conditions, and at low Reynolds numbers, a deviation from laminar flow might occur on the flagellar

  12. Arsenate arrests flagellar rotation in cytoplasm-free envelopes of bacteria.

    PubMed Central

    Margolin, Y; Barak, R; Eisenbach, M

    1994-01-01

    The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellular) prevented the inhibition by arsenate. In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function. PMID:8071237

  13. Energy source of flagellar type III secretion.

    PubMed

    Paul, Koushik; Erhardt, Marc; Hirano, Takanori; Blair, David F; Hughes, Kelly T

    2008-01-24

    Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane. This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion. The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export. Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the non-flagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a proton-driven protein exporter and that ATP hydrolysis is not essential for type III secretion. PMID:18216859

  14. Mesoscopic modeling of bacterial flagellar microhydrodynamics.

    PubMed

    Gebremichael, Yeshitila; Ayton, Gary S; Voth, Gregory A

    2006-11-15

    A particle-based hybrid method of elastic network model and smooth-particle hydrodynamics has been employed to describe the propulsion of bacterial flagella in a viscous hydrodynamic environment. The method explicitly models the two aspects of bacterial propulsion that involve flagellar flexibility and long-range hydrodynamic interaction of low-Reynolds-number flow. The model further incorporates the molecular organization of the flagellar filament at a coarse-grained level in terms of the 11 protofilaments. Each of these protofilaments is represented by a collection of material points that represent the flagellin proteins. A computational model of a single flexible helical segment representing the filament of a bacterial flagellum is presented. The propulsive dynamics and the flow fields generated by the motion of the model filament are examined. The nature of flagellar deformation and the influence of hydrodynamics in determining the shape of deformations are examined based on the helical filament. PMID:16935949

  15. Rhythmicity, recurrence, and recovery of flagellar beating

    NASA Astrophysics Data System (ADS)

    Wan, Kirsty; Goldstein, Raymond

    2015-03-01

    The eukaryotic flagellum beats with apparently unfailing periodicity, yet responds rapidly to stimuli. Like the human heartbeat, flagellar oscillations are now known to be noisy. Using the unicellular alga Chlamydomonas reinhardtii, we explore three aspects of nonuniform flagellar beating. We report the existence of rhythmicity, waveform noise peaking at transitions between power and recovery strokes, and fluctuations of interbeat intervals that are correlated and even recurrent, with memory extending to hundreds of beats. These features are altered qualitatively by physiological perturbations. Further, we quantify the recovery of periodic breaststroke beating from transient hydrodynamic forcing. These results will help constrain microscopic theories on the origins and regulation of flagellar beating. Financial support is acknowledged from the EPSRC, ERC Advanced Investigator Grant No. 247333, and a Senior Investigator Award from the Wellcome Trust.

  16. A Protein Methylation Pathway in Chlamydomonas Flagella Is Active during Flagellar Resorption

    PubMed Central

    Schneider, Mark J.; Ulland, Megan

    2008-01-01

    During intraflagellar transport (IFT), the regulation of motor proteins, the loading and unloading of cargo and the turnover of flagellar proteins all occur at the flagellar tip. To begin an analysis of the protein composition of the flagellar tip, we used difference gel electrophoresis to compare long versus short (i.e., regenerating) flagella. The concentration of tip proteins should be higher relative to that of tubulin (which is constant per unit length of the flagellum) in short compared with long flagella. One protein we have identified is the cobalamin-independent form of methionine synthase (MetE). Antibodies to MetE label flagella in a punctate pattern reminiscent of IFT particle staining, and immunoblot analysis reveals that the amount of MetE in flagella is low in full-length flagella, increased in regenerating flagella, and highest in resorbing flagella. Four methylated proteins have been identified in resorbing flagella, using antibodies specific for asymmetrically dimethylated arginine residues. These proteins are found almost exclusively in the axonemal fraction, and the methylated forms of these proteins are essentially absent in full-length and regenerating flagella. Because most cells resorb cilia/flagella before cell division, these data indicate a link between flagellar protein methylation and progression through the cell cycle. PMID:18701702

  17. DRC3 connects the N-DRC to dynein g to regulate flagellar waveform

    PubMed Central

    Awata, Junya; Song, Kangkang; Lin, Jianfeng; King, Stephen M.; Sanderson, Michael J.; Nicastro, Daniela; Witman, George B.

    2015-01-01

    The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo–electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC–dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform. PMID:26063732

  18. Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis.

    PubMed

    Dawson, Scott C; Sagolla, Meredith S; Mancuso, Joel J; Woessner, David J; House, Susan A; Fritz-Laylin, Lillian; Cande, W Zacheus

    2007-12-01

    Microtubule depolymerization dynamics in the spindle are regulated by kinesin-13, a nonprocessive kinesin motor protein that depolymerizes microtubules at the plus and minus ends. Here we show that a single kinesin-13 homolog regulates flagellar length dynamics, as well as other interphase and mitotic dynamics in Giardia intestinalis, a widespread parasitic diplomonad protist. Both green fluorescent protein-tagged kinesin-13 and EB1 (a plus-end tracking protein) localize to the plus ends of mitotic and interphase microtubules, including a novel localization to the eight flagellar tips, cytoplasmic anterior axonemes, and the median body. The ectopic expression of a kinesin-13 (S280N) rigor mutant construct caused significant elongation of the eight flagella with significant decreases in the median body volume and resulted in mitotic defects. Notably, drugs that disrupt normal interphase and mitotic microtubule dynamics also affected flagellar length in Giardia. Our study extends recent work on interphase and mitotic kinesin-13 functioning in metazoans to include a role in regulating flagellar length dynamics. We suggest that kinesin-13 universally regulates both mitotic and interphase microtubule dynamics in diverse microbial eukaryotes and propose that axonemal microtubules are subject to the same regulation of microtubule dynamics as other dynamic microtubule arrays. Finally, the present study represents the first use of a dominant-negative strategy to disrupt normal protein function in Giardia and provides important insights into giardial microtubule dynamics with relevance to the development of antigiardial compounds that target critical functions of kinesins in the giardial life cycle. PMID:17766466

  19. The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas.

    PubMed

    Reck, Jaimee; Schauer, Alexandria M; VanderWaal Mills, Kristyn; Bower, Raqual; Tritschler, Douglas; Perrone, Catherine A; Porter, Mary E

    2016-08-01

    The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms. PMID:27251063

  20. CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

    PubMed Central

    Craige, Branch; Tsao, Che-Chia; Diener, Dennis R.; Hou, Yuqing; Lechtreck, Karl-Ferdinand; Rosenbaum, Joel L.

    2010-01-01

    Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule–membrane links there. Ultrastructural analysis revealed defects in these microtubule–membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule–membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition. PMID:20819941

  1. Right lower limb apraxia in a patient with left supplementary motor area infarction: intactness of the corticospinal tract confirmed by transcranial magnetic stimulation

    PubMed Central

    Chang, Min Cheol; Chun, Min Ho

    2015-01-01

    We reported a 50-year-old female patient with left supplementary motor area infarction who presented right lower limb apraxia and investigated the possible causes using transcranial magnetic stimulation. The patient was able to walk and climb stairs spontaneously without any assistance at 3 weeks after onset. However, she was unable to intentionally move her right lower limb although she understood what she supposed to do. The motor evoked potential evoked by transcranial magnetic stimulation from the right lower limb was within the normal range, indicating that the corticospinal tract innervating the right lower limb was uninjured. Thus, we thought that her motor dysfunction was not induced by motor weakness, and confirmed her symptoms as apraxia. In addition, these results also suggest that transcranial magnetic stimulation is helpful for diagnosing apraxia. PMID:25883636

  2. A Single-Domain FlgJ Contributes to Flagellar Hook and Filament Formation in the Lyme Disease Spirochete Borrelia burgdorferi

    PubMed Central

    Zhang, Kai; Tong, Brian A.; Liu, Jun

    2012-01-01

    FlgJ plays a very important role in flagellar assembly. In the enteric bacteria, flgJ null mutants fail to produce the flagellar rods, hooks, and filaments but still assemble the integral membrane-supramembrane (MS) rings. These mutants are nonmotile. The FlgJ proteins consist of two functional domains. The N-terminal rod-capping domain acts as a scaffold for rod assembly, and the C-terminal domain acts as a peptidoglycan (PG) hydrolase (PGase), which allows the elongating flagellar rod to penetrate through the PG layer. However, the FlgJ homologs in several bacterial phyla (including spirochetes) often lack the PGase domain. The function of these single-domain FlgJ proteins remains elusive. Herein, a single-domain FlgJ homolog (FlgJBb) was studied in the Lyme disease spirochete Borrelia burgdorferi. Cryo-electron tomography analysis revealed that the flgJBb mutant still assembled intact flagellar basal bodies but had fewer and disoriented flagellar hooks and filaments. Consistently, Western blots showed that the levels of flagellar hook (FlgE) and filament (FlaB) proteins were substantially decreased in the flgJBb mutant. Further studies disclosed that the decreases of FlgE and FlaB in the mutant occurred at the posttranscriptional level. Microscopic observation and swarm plate assay showed that the motility of the flgJBb mutant was partially deficient. The altered phenotypes were completely restored when the mutant was complemented. Collectively, these results indicate that FlgJBb is involved in the assembly of the flagellar hook and filament but not the flagellar rod in B. burgdorferi. The observed phenotype is different from that of flgJ mutants in the enteric bacteria. PMID:22155773

  3. Flagellar Synchronization Independent of Hydrodynamic Interactions

    NASA Astrophysics Data System (ADS)

    Friedrich, Benjamin M.; Jülicher, Frank

    2012-09-01

    Inspired by the coordinated beating of the flagellar pair of the green algae Chlamydomonas, we study theoretically a simple, mirror-symmetric swimmer, which propels itself at low Reynolds number by a revolving motion of a pair of spheres. We show that perfect synchronization between these two driven spheres can occur due to the motion of the swimmer and local hydrodynamic friction forces. Hydrodynamic interactions, though crucial for net propulsion, contribute little to synchronization for this free-moving swimmer.

  4. Transplanting intact donor tissue enhances dopamine cell survival and the predictability of motor improvements in a rat model of Parkinson's disease.

    PubMed

    Fricker, Rosemary A; Kuiper, Jan Herman; Gates, Monte A

    2012-01-01

    Primary cell transplantation is currently the gold standard for cell replacement in Parkinson's disease. However, the number of donors needed to treat a single patient is high, and the functional outcome is sometimes variable. The present work explores the possibility of enhancing the viability and/or functionality of small amounts of ventral mesencephalic (VM) donor tissue by reducing its perturbation during preparation and implantation. Briefly, unilaterally lesioned rats received either: (1) an intact piece of half an embryonic day 13 (E13) rat VM; (2) dissociated cells from half an E13 rat VM; or (3) no transplant. D-amphetamine- induced rotations revealed that animals receiving pieces of VM tissue or dissociated cells showed significant improvement in ipsilateral rotation 4 weeks post transplantation. By 6 weeks post transplantation, animals receiving pieces of VM tissue showed a trend for further improvement, while those receiving dissociated cells remained at their 4 week scores. Postmortem cell counts showed that the number of dopaminergic neurons in dissociated cell transplants was significantly lower than that surviving in transplants of intact tissue. When assessing the correlation between the number of dopamine cells in each transplant, and the improvement in rotation bias in experimental animals, it was shown that transplants of whole pieces of VM tissue offered greater predictability of graft function based on their dopamine cell content. Such results suggest that maintaining the integrity of VM tissue during implantation improves dopamine cell content, and that the dopamine cell content of whole tissue grafts offers a more predictable outcome of graft function in an animal model of Parkinson's disease. PMID:23056602

  5. Studies on flagellar shortening in Chlamydomonas reinhardtii

    SciTech Connect

    Cherniack, J.

    1985-01-01

    Flagellar shortening of Chlamydomonas reinhardtii was promoted by sodium chloride, pyrophosphate (sodium, potassium and ammonium salts), EDTA and EGTA, succinate, citrate and oxalate (sodium salts), caffeine and aminophylline. Removal of calcium from the medium potentiated the effects of these agents in inducing shortening. Investigations of the release of phosphorylated compounds to the medium during pyrophosphate-induced flagellar shortening of cells pre-labelled with /sup 32/P, revealed an as yet unidentified /sup 32/P-labelled compound with distinct chromatographic properties. Chromatography and electrophoresis indicates that it is a small, highly polar molecule with a high charge to mass ratio, containing thermo- and acid-labile phosphate linkages. Investigations showed of the release of /sup 35/S-labelled protein to the medium from cells pre-labelled with /sup 35/S-sulfate showed that flagellated cells released two prominent polypeptides which comigrated with ..cap alpha..- and ..beta..-flagellar tubulin on SDS polyacrylamide gel electrophoresis, while deflagellated cells did not.

  6. Aeromonas hydrophila Lateral Flagellar Gene Transcriptional Hierarchy

    PubMed Central

    Wilhelms, Markus; Gonzalez, Victor; Merino, Susana

    2013-01-01

    Aeromonas hydrophila AH-3 lateral flagella are not assembled when bacteria grow in liquid media; however, lateral flagellar genes are transcribed. Our results indicate that A. hydrophila lateral flagellar genes are transcribed at three levels (class I to III genes) and share some similarities with, but have many important differences from, genes of Vibrio parahaemolyticus. A. hydrophila lateral flagellum class I gene transcription is σ70 dependent, which is consistent with the fact that lateral flagellum is constitutively transcribed, in contrast to the characteristics of V. parahaemolyticus. The fact that multiple genes are included in class I highlights that lateral flagellar genes are less hierarchically transcribed than polar flagellum genes. The A. hydrophila lafK-fliEJL gene cluster (where the subscript L distinguishes genes for lateral flagella from those for polar flagella) is exclusively from class I and is in V. parahaemolyticus class I and II. Furthermore, the A. hydrophila flgAMNL cluster is not transcribed from the σ54/LafK-dependent promoter and does not contain class II genes. Here, we propose a gene transcriptional hierarchy for the A. hydrophila lateral flagella. PMID:23335410

  7. Regulation of flagellar motility during biofilm formation

    PubMed Central

    Guttenplan, Sarah B.; Kearns, Daniel B.

    2013-01-01

    Many bacteria swim in liquid or swarm over solid surfaces by synthesizing rotary flagella. The same bacteria that are motile also commonly form non-motile multicellular aggregates held together by an extracellular matrix called biofilms. Biofilms are an important part of the lifestyle of pathogenic bacteria and it is assumed that there is a motility-to-biofilm transition wherein the inhibition of motility promotes biofilm formation. The transition is largely inferred from regulatory mutants that reveal the opposite regulation of the two phenotypes. Here we review the regulation of motility during biofilm formation in Bacillus, Pseudomonas, Vibrio, and Escherichia, and we conclude that the motility-to-biofilm transition, if necessary, likely involves two steps. In the short term, flagella are functionally regulated to either inhibit rotation or modulate the basal flagellar reversal frequency. Over the long term, flagellar gene transcription is inhibited and in the absence of de novo synthesis, flagella are likely diluted to extinction through growth. Both short term and long term control is likely important to the motility-to-biofilm transition to stabilize aggregates and optimize resource investment. We emphasize the newly discovered classes of flagellar functional regulators and speculate that others await discovery in the context of biofilm formation. PMID:23480406

  8. Isolation of the polar and lateral flagellum-defective mutants in Vibrio alginolyticus and identification of their flagellar driving energy sources.

    PubMed Central

    Kawagishi, I; Maekawa, Y; Atsumi, T; Homma, M; Imae, Y

    1995-01-01

    Vibrio alginolyticus has two types of flagella (polar and lateral) in one cell. We isolated mutants with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+). Using these mutants, we demonstrated that the energy sources of the lateral and polar flagellar motors in V. alginolyticus are H+ and Na+ motive forces, respectively, as in the related species V. parahaemolyticus. PMID:7665498

  9. The bacterial flagellar protein export apparatus processively transports flagellar proteins even with extremely infrequent ATP hydrolysis

    PubMed Central

    Minamino, Tohru; Morimoto, Yusuke V.; Kinoshita, Miki; Aldridge, Phillip D.; Namba, Keiichi

    2014-01-01

    For self-assembly of the bacterial flagellum, a specific protein export apparatus utilizes ATP and proton motive force (PMF) as the energy source to transport component proteins to the distal growing end. The export apparatus consists of a transmembrane PMF-driven export gate and a cytoplasmic ATPase complex composed of FliH, FliI and FliJ. The FliI6FliJ complex is structurally similar to the α3β3γ complex of FOF1-ATPase. FliJ allows the gate to efficiently utilize PMF to drive flagellar protein export but it remains unknown how. Here, we report the role of ATP hydrolysis by the FliI6FliJ complex. The export apparatus processively transported flagellar proteins to grow flagella even with extremely infrequent or no ATP hydrolysis by FliI mutation (E211D and E211Q, respectively). This indicates that the rate of ATP hydrolysis is not at all coupled with the export rate. Deletion of FliI residues 401 to 410 resulted in no flagellar formation although this FliI deletion mutant retained 40% of the ATPase activity, suggesting uncoupling between ATP hydrolysis and activation of the gate. We propose that infrequent ATP hydrolysis by the FliI6FliJ ring is sufficient for gate activation, allowing processive translocation of export substrates for efficient flagellar assembly. PMID:25531309

  10. Regulation of Flagellar Length in Chlamydomonas

    PubMed Central

    Wilson, Nedra F.; Iyer, Janaki Kannan; Buchheim, Julie A.; Meek, William

    2008-01-01

    Chlamydomonas reinhardtii has two apically localized flagella that are maintained at an equal and appropriate length. Assembly and maintenance of flagella requires a microtubule-based transport system known as intraflagellar transport (IFT). During IFT, proteins destined for incorporation into or removal from a flagellum are carried along doublet microtubules via IFT particles. Regulation of IFT activity therefore is pivotal in determining the length of a flagellum. Reviewed is our current understanding of the role of IFT and signal transduction pathways in the regulation of flagellar length. PMID:18692148

  11. Dynamics of a tightly coupled mechanism for flagellar rotation. Bacterial motility, chemiosmotic coupling, protonmotive force.

    PubMed

    Meister, M; Caplan, S R; Berg, H C

    1989-05-01

    The bacterial flagellar motor is a molecular engine that couples the flow of protons across the cytoplasmic membrane to rotation of the flagellar filament. We analyze the steady-state behavior of an explicit mechanical model in which a fixed number of protons carries the filament through one revolution. Predictions of this model are compared with experimentally determined relationships between protonmotive force, proton flux, torque, and speed. All such tightly coupled mechanisms produce the same torque when the motor is stalled but vary greatly in their behavior at high speed. The speed at zero load predicted by our model is limited by the rates of association and dissociation of protons at binding sites on the rotor and by the mobility of force generators containing transmembrane channels that interact with these sites. Our analysis suggests that more could be learned about the motor if it were driven by an externally applied torque backwards (at negative speed) or forwards at speeds greater than the zero-load speed. PMID:2720081

  12. A Complete Set of Flagellar Genes Acquired by Horizontal Transfer Coexists with the Endogenous Flagellar System in Rhodobacter sphaeroides▿ †

    PubMed Central

    Poggio, Sebastian; Abreu-Goodger, Cei; Fabela, Salvador; Osorio, Aurora; Dreyfus, Georges; Vinuesa, Pablo; Camarena, Laura

    2007-01-01

    Bacteria swim in liquid environments by means of a complex rotating structure known as the flagellum. Approximately 40 proteins are required for the assembly and functionality of this structure. Rhodobacter sphaeroides has two flagellar systems. One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum, while the other was found only after the genome sequence of this bacterium was completed. In this work we found that the second flagellar system of R. sphaeroides can be expressed and produces a functional flagellum. In many bacteria with two flagellar systems, one is required for swimming, while the other allows movement in denser environments by producing a large number of flagella over the entire cell surface. In contrast, the second flagellar system of R. sphaeroides produces polar flagella that are required for swimming. Expression of the second set of flagellar genes seems to be positively regulated under anaerobic growth conditions. Phylogenic analysis suggests that the flagellar system that was initially characterized was in fact acquired by horizontal transfer from a γ-proteobacterium, while the second flagellar system contains the native genes. Interestingly, other α-proteobacteria closely related to R. sphaeroides have also acquired a set of flagellar genes similar to the set found in R. sphaeroides, suggesting that a common ancestor received this gene cluster. PMID:17293429

  13. Polar features in the flagellar propulsion of E. coli bacteria

    NASA Astrophysics Data System (ADS)

    Bianchi, S.; Saglimbeni, F.; Lepore, A.; Di Leonardo, R.

    2015-06-01

    E. coli bacteria swim following a run and tumble pattern. In the run state all flagella join in a single helical bundle that propels the cell body along approximately straight paths. When one or more flagellar motors reverse direction the bundle unwinds and the cell randomizes its orientation. This basic picture represents an idealization of a much more complex dynamical problem. Although it has been shown that bundle formation can occur at either pole of the cell, it is still unclear whether these two run states correspond to asymmetric propulsion features. Using holographic microscopy we record the 3D motions of individual bacteria swimming in optical traps. We find that most cells possess two run states characterized by different propulsion forces, total torque, and bundle conformations. We analyze the statistical properties of bundle reversal and compare the hydrodynamic features of forward and backward running states. Our method is naturally multi-particle and opens up the way towards controlled hydrodynamic studies of interacting swimming cells.

  14. Flagellar synchronization through direct hydrodynamic interactions.

    PubMed

    Brumley, Douglas R; Wan, Kirsty Y; Polin, Marco; Goldstein, Raymond E

    2014-01-01

    Flows generated by ensembles of flagella are crucial to development, motility and sensing, but the mechanisms behind this striking coordination remain unclear. We present novel experiments in which two micropipette-held somatic cells of Volvox carteri, with distinct intrinsic beating frequencies, are studied by high-speed imaging as a function of their separation and orientation. Analysis of time series shows that the interflagellar coupling, constrained by lack of connections between cells to be hydrodynamical, exhibits a spatial dependence consistent with theory. At close spacings it produces robust synchrony for thousands of beats, while at increasing separations synchrony is degraded by stochastic processes. Manipulation of the relative flagellar orientation reveals in-phase and antiphase states, consistent with dynamical theories. Flagellar tracking with exquisite precision reveals waveform changes that result from hydrodynamic coupling. This study proves unequivocally that flagella coupled solely through a fluid can achieve robust synchrony despite differences in their intrinsic properties.DOI: http://dx.doi.org/10.7554/eLife.02750.001. PMID:25073925

  15. Flagellar synchronization through direct hydrodynamic interactions

    PubMed Central

    Brumley, Douglas R; Wan, Kirsty Y; Polin, Marco; Goldstein, Raymond E

    2014-01-01

    Flows generated by ensembles of flagella are crucial to development, motility and sensing, but the mechanisms behind this striking coordination remain unclear. We present novel experiments in which two micropipette-held somatic cells of Volvox carteri, with distinct intrinsic beating frequencies, are studied by high-speed imaging as a function of their separation and orientation. Analysis of time series shows that the interflagellar coupling, constrained by lack of connections between cells to be hydrodynamical, exhibits a spatial dependence consistent with theory. At close spacings it produces robust synchrony for thousands of beats, while at increasing separations synchrony is degraded by stochastic processes. Manipulation of the relative flagellar orientation reveals in-phase and antiphase states, consistent with dynamical theories. Flagellar tracking with exquisite precision reveals waveform changes that result from hydrodynamic coupling. This study proves unequivocally that flagella coupled solely through a fluid can achieve robust synchrony despite differences in their intrinsic properties. DOI: http://dx.doi.org/10.7554/eLife.02750.001 PMID:25073925

  16. Biochemical Characterization of the Flagellar Rod Components of Rhodobacter sphaeroides: Properties and Interactions

    PubMed Central

    Osorio-Valeriano, Manuel; de la Mora, Javier; Camarena, Laura

    2015-01-01

    ABSTRACT The flagellar basal body is a rotary motor that spans the cytoplasmic and outer membranes. The rod is a drive shaft that transmits torque generated by the motor through the hook to the filament that propels the bacterial cell. The assembly and structure of the rod are poorly understood. In a first attempt to characterize this structure in the alphaproteobacterium Rhodobacter sphaeroides, we overexpressed and purified FliE and the four related rod proteins (FlgB, FlgC, FlgF, and FlgG), and we analyzed their ability to form homo-oligomers. We found that highly purified preparations of these proteins formed high-molecular-mass oligomers that tended to dissociate in the presence of NaCl. As predicted by in silico modeling, the four rod proteins share architectural features. Using affinity blotting, we detected the heteromeric interactions between these proteins. In addition, we observed that deletion of the N- and C-terminal regions of FlgF and FlgG severely affected heteromeric but not homomeric interactions. On the basis of our findings, we propose a model of rod assembly in this bacterium. IMPORTANCE Despite the considerable amount of research on the structure and assembly of other flagellar axial structures that has been conducted, the rod has been barely studied. An analysis of the biochemical characteristics of the flagellar rod components of the Fla1 system of R. sphaeroides is presented in this work. We also analyze the interactions of these proteins with each other and with their neighbors, and we propose a model for the order in which they are assembled. PMID:26574514

  17. Identification of Escherichia coli region III flagellar gene products and description of two new flagellar genes.

    PubMed Central

    Bartlett, D H; Matsumura, P

    1984-01-01

    Region III flagellar genes in Escherichia coli are involved with the assembly and rotation of the flagella, as well as taxis. We subcloned the flaB operon from a lambda fla transducing phage onto plasmid pMK2004. Two additional genes were found at the flaB locus, and we subdivided the flaB gene into flaB1, flaBII, and flaBIII. The cheY suppressor mutations which have previously been mapped to flaB were further localized to flaB11 (Parkinson et al., J. Bacteriol. 155:265-274, 1983). Until now, gene product identification has not been possible for these genes because of their low levels of gene expression. Overexpression of the flagellar genes was accomplished by placing the flaB operon under the control of the lacUV5 or tac promoters. Plasmid-encoded proteins were examined in a minicell expression system. By correlating various deletions and insertions in the flaB operon with the ability to complement specific flagellar mutants and code for polypeptides, we made the following gene product assignments: flaB 1, 60 kilodaltons; flaB 11, 38 kilodaltons; flaB111, 28 kilodaltons; flaC, 56 kilodaltons; fla0, 16 kilodaltons; and flaE, 54 kilodaltons. Images PMID:6094477

  18. Characterization of Escherichia coli Flagellar Mutants That are Insensitive to Catabolite Repression

    PubMed Central

    Silverman, Michael; Simon, Melvin

    1974-01-01

    In Escherichia coli, the synthesis of the flagellar organelle is sensitive to catabolite repression. Synthesis requires the presence of the cyclic adenosine monophosphate receptor protein (Crp) and 3′,5′-cyclic adenosine monophosphate (cAMP); i.e., mutants that lack Crp or adenylcyclase (Cya) synthesize no flagella. We isolated and characterized a series of mutants (cfs) that restored flagella-forming ability in a Crp strain of E. coli. The mutations in these strains were transferred onto episomes and they were then introduced into a variety of other strains. The presence of the mutation resulted in flagella synthesis in Cya and Crp strains as well as in the wild type grown under conditions of catabolite repression. Deletion analysis and other genetic studies indicated that: (i) the cfs mutations had a dominant effect when they were in the transconfiguration in merodiploids: (ii) they occurred in or very close to the flaI gene: and (iii) their expression required the presence of an intact flaI gene adjacent to the cfs mutation. Biochemical studies showed that the synthesis of at least two flagellar polypeptides, the hook subunit and an amber fragment of flagellin, were absent in strains that carried a cya mutation. Their synthesis was depressed in strains grown under conditions of catabolite repression. The presence of the cfs mutation restored the specific synthesis of these two polypeptides. We suggest that the formation of the flaI gene product is the step in flagellar synthesis that is catabolite sensitive and requires cAMP. We propose a regulatory function for the product of the flaI gene. Images PMID:4373438

  19. Xylan-Degrading Catalytic Flagellar Nanorods.

    PubMed

    Klein, Ágnes; Szabó, Veronika; Kovács, Mátyás; Patkó, Dániel; Tóth, Balázs; Vonderviszt, Ferenc

    2015-09-01

    Flagellin, the main component of flagellar filaments, is a protein possessing polymerization ability. In this work, a novel fusion construct of xylanase A from B. subtilis and Salmonella flagellin was created which is applicable to build xylan-degrading catalytic nanorods of high stability. The FliC-XynA chimera when overexpressed in a flagellin deficient Salmonella host strain was secreted into the culture medium by the flagellum-specific export machinery allowing easy purification. Filamentous assemblies displaying high surface density of catalytic sites were produced by ammonium sulfate-induced polymerization. FliC-XynA nanorods were resistant to proteolytic degradation and preserved their enzymatic activity for a long period of time. Furnishing enzymes with self-assembling ability to build catalytic nanorods offers a promising alternative approach to enzyme immobilization onto nanostructured synthetic scaffolds. PMID:25966869

  20. Escherichia coli modulates its motor speed on sensing an attractant.

    PubMed

    Karmakar, Richa; Naaz, Farha; Tirumkudulu, Mahesh S; Venkatesh, K V

    2016-10-01

    It is well known that Escherichia coli achieves chemotaxis by modulating the bias of the flagellar motor. Recent experiments have shown that the bacteria vary their swimming speeds as well in presence of attractants. However, this increase in the swimming speed in response to the attractants has not been correlated with the increase in the flagellar motor speed. Using flickering dark-field microscopy, we measure the head-rotation speed of a large population of cells to correlate it with the flagellar motor speed. Experiments performed with wild-type and trg-deletion mutant strains suggest that the cells are capable of modulating the flagellar motor speed via mere sensing of a ligand. The motor speed can be further correlated with the swimming speed of the cells and was found to be linear. These results suggest the existence of a hitherto unknown intra-cellular pathway that modulates the flagellar motor speed in response to sensing of chemicals, thereby making chemotaxis more efficient than previously known. PMID:27318664

  1. Comprehensive Mapping of the Escherichia coli Flagellar Regulatory Network

    PubMed Central

    Fitzgerald, Devon M.; Bonocora, Richard P.; Wade, Joseph T.

    2014-01-01

    Flagellar synthesis is a highly regulated process in all motile bacteria. In Escherichia coli and related species, the transcription factor FlhDC is the master regulator of a multi-tiered transcription network. FlhDC activates transcription of a number of genes, including some flagellar genes and the gene encoding the alternative Sigma factor FliA. Genes whose expression is required late in flagellar assembly are primarily transcribed by FliA, imparting temporal regulation of transcription and coupling expression to flagellar assembly. In this study, we use ChIP-seq and RNA-seq to comprehensively map the E. coli FlhDC and FliA regulons. We define a surprisingly restricted FlhDC regulon, including two novel regulated targets and two binding sites not associated with detectable regulation of surrounding genes. In contrast, we greatly expand the known FliA regulon. Surprisingly, 30 of the 52 FliA binding sites are located inside genes. Two of these intragenic promoters are associated with detectable noncoding RNAs, while the others either produce highly unstable RNAs or are inactive under these conditions. Together, our data redefine the E. coli flagellar regulatory network, and provide new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process. PMID:25275371

  2. Stoichiometry and turnover of the bacterial flagellar switch protein FliN.

    PubMed

    Delalez, Nicolas J; Berry, Richard M; Armitage, Judith P

    2014-01-01

    Some proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, in Escherichia coli. Our results confirm that, in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments. Importance: The flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single

  3. Negative transcriptional regulation in the Caulobacter flagellar hierarchy.

    PubMed Central

    Xu, H; Dingwall, A; Shapiro, L

    1989-01-01

    The Caulobacter crescentus flagellum is formed at a specific time in the cell cycle and its assembly requires the ordered expression of a large number of genes. These genes are controlled in a positive trans-acting hierarchy that reflects the order of assembly of the flagellum. Using plasmids carrying transcriptional fusions of either a neo or a lux reporter gene to the promoters of three flagellar genes representing different ranks in the hierarchy (the hook operon, a basal body gene flbN, and the flaO gene), we have measured the level of chimeric gene expression in 13 flagellar mutant backgrounds. Mutants in the hook operon or in basal body genes caused overproduction of both hook operon and basal body gene chimeric mRNAs, suggesting that negative regulation is superimposed on the positive trans-acting control for these early events in the flagellar hierarchy. Mutants in the structural genes and in genes involved in flagellar assembly had no effect on flaO expression, placing the flaO gene near the top of the hierarchy. However, flaO expression appears to be under negative control by two regulatory genes flaS and flaW. Negative control, as a response to the completion of specific steps in the assembly process, may be an important mechanism used by the cell to turn off flagellar gene expression once the gene product is no longer needed. Images PMID:2771950

  4. Flagellar apparatus gene sequences of Aeromonas hydrophila AL09-73 isolate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flagellar apparatus genes of recent outbreak Aeromonas hydrophila AL09-73 isolate were sequenced and characterized. Total 28 flagellar genes were identified. The sizes of the genes range from 318 to 2001 nucleotides, which potentially encode different complex flagellar proteins. At nucleotide and...

  5. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles

    PubMed Central

    2016-01-01

    We show that DNA-based self-assembly can serve as a general and flexible tool to construct artificial flagella of several micrometers in length and only tens of nanometers in diameter. By attaching the DNA flagella to biocompatible magnetic microparticles, we provide a proof of concept demonstration of hybrid structures that, when rotated in an external magnetic field, propel by means of a flagellar bundle, similar to self-propelling peritrichous bacteria. Our theoretical analysis predicts that flagellar bundles that possess a length-dependent bending stiffness should exhibit a superior swimming speed compared to swimmers with a single appendage. The DNA self-assembly method permits the realization of these improved flagellar bundles in good agreement with our quantitative model. DNA flagella with well-controlled shape could fundamentally increase the functionality of fully biocompatible nanorobots and extend the scope and complexity of active materials. PMID:26821214

  6. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles.

    PubMed

    Maier, Alexander M; Weig, Cornelius; Oswald, Peter; Frey, Erwin; Fischer, Peer; Liedl, Tim

    2016-02-10

    We show that DNA-based self-assembly can serve as a general and flexible tool to construct artificial flagella of several micrometers in length and only tens of nanometers in diameter. By attaching the DNA flagella to biocompatible magnetic microparticles, we provide a proof of concept demonstration of hybrid structures that, when rotated in an external magnetic field, propel by means of a flagellar bundle, similar to self-propelling peritrichous bacteria. Our theoretical analysis predicts that flagellar bundles that possess a length-dependent bending stiffness should exhibit a superior swimming speed compared to swimmers with a single appendage. The DNA self-assembly method permits the realization of these improved flagellar bundles in good agreement with our quantitative model. DNA flagella with well-controlled shape could fundamentally increase the functionality of fully biocompatible nanorobots and extend the scope and complexity of active materials. PMID:26821214

  7. Flagellar force production during regeneration in Chlamydomonas reinhardtii

    NASA Astrophysics Data System (ADS)

    Yukich, John N.; Clodfelter, Catherine; Bernd, Karen K.

    2009-11-01

    Several respiratory, digestive, and reproductive disorders originate with motional dysfunction of cilia and flagella. The usefulness of cilia and flagella is understood, but the internal mechanism for creating their breast stroke-like motion is not. This study reports on standardization of calibration, trapping and cell movement recording methods. Our techniques permit us to measure the flagellar swimming force of Chlamydomonas during flagella regeneration. We find that as flagella length increases, the flagellar force is maximized after 50% of full length is achieved except for a significant dip at 75% of full length. These results raise many questions regarding the flagella infrastructure.

  8. Viscous Dynamics of Lyme Disease and Syphilis Spirochetes Reveal Flagellar Torque and Drag

    PubMed Central

    Harman, Michael; Vig, Dhruv K.; Radolf, Justin D.; Wolgemuth, Charles W.

    2013-01-01

    The spirochetes that cause Lyme disease (Borrelia burgdorferi) and syphilis (Treponema pallidum) swim through viscous fluids, such as blood and interstitial fluid, by undulating their bodies as traveling, planar waves. These undulations are driven by rotation of the flagella within the periplasmic space, the narrow (∼20–40 nm in width) compartment between the inner and outer membranes. We show here that the swimming speeds of B. burgdorferi and T. pallidum decrease with increases in viscosity of the external aqueous milieu, even though the flagella are entirely intracellular. We then use mathematical modeling to show that the measured changes in speed are consistent with the exertion of constant torque by the spirochetal flagellar motors. Comparison of simulations, experiments, and a simple model for power dissipation allows us to estimate the torque and resistive drag that act on the flagella of these major spirochetal pathogens. PMID:24268139

  9. Divalent Cation Control of Flagellar Motility in African Trypanosomes

    NASA Astrophysics Data System (ADS)

    Westergard, Anna M.; Hutchings, Nathan R.

    2005-03-01

    Changes in calcium concentration have been shown to dynamically affect flagellar motility in several eukaryotic systems. The African trypanosome is a monoflagellated protozoan parasite and the etiological agent of sleeping sickness. Although cell motility has been implicated in disease progression, very little is currently known about biochemical control of the trypanosome flagellum. In this study, we assess the effects of extracellular changes in calcium and nickel concentration on trypanosome flagellar movement. Using a flow through chamber, we determine the relative changes in motility in individual trypanosomes in response to various concentrations of calcium and nickel, respectively. Extracellular concentrations of calcium and nickel (as low as 100 micromolar) significantly inhibit trypanosome cell motility. The effects are reversible, as indicated by the recovery of motion after removal of the calcium or nickel from the chamber. We are currently investigating the specific changes in flagellar oscillation and coordination that result from calcium and nickel, respectively. These results verify the presence of a calcium-responsive signaling mechanism(s) that regulates flagellar beat in trypanosomes.

  10. Functional Activation of the Flagellar Type III Secretion Export Apparatus

    PubMed Central

    Phillips, Andrew M.; Calvo, Rebecca A.; Kearns, Daniel B.

    2015-01-01

    Flagella are assembled sequentially from the inside-out with morphogenetic checkpoints that enforce the temporal order of subunit addition. Here we show that flagellar basal bodies fail to proceed to hook assembly at high frequency in the absence of the monotopic protein SwrB of Bacillus subtilis. Genetic suppressor analysis indicates that SwrB activates the flagellar type III secretion export apparatus by the membrane protein FliP. Furthermore, mutants defective in the flagellar C-ring phenocopy the absence of SwrB for reduced hook frequency and C-ring defects may be bypassed either by SwrB overexpression or by a gain-of-function allele in the polymerization domain of FliG. We conclude that SwrB enhances the probability that the flagellar basal body adopts a conformation proficient for secretion to ensure that rod and hook subunits are not secreted in the absence of a suitable platform on which to polymerize. PMID:26244495

  11. Identification and characterization of flagellar biosynthetic genes in Yersinia ruckeri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using transposon mutagenesis we have identified a Yersinia ruckeri serovar I mutant defective in both motility and production of secreted lipase activity. Sequence analysis of this mutant revealed a single transposon insertion in an open reading frame (ORF) with homology to flhA, a flagellar biosynt...

  12. A distant homologue of the FlgT protein interacts with MotB and FliL and is essential for flagellar rotation in Rhodobacter sphaeroides.

    PubMed

    Fabela, Salvador; Domenzain, Clelia; De la Mora, Javier; Osorio, Aurora; Ramirez-Cabrera, Victor; Poggio, Sebastian; Dreyfus, Georges; Camarena, Laura

    2013-12-01

    In this work, we describe a periplasmic protein that is essential for flagellar rotation in Rhodobacter sphaeroides. This protein is encoded upstream of flgA, and its expression is dependent on the flagellar master regulator FleQ and on the class III flagellar activator FleT. Sequence comparisons suggest that this protein is a distant homologue of FlgT. We show evidence that in R. sphaeroides, FlgT interacts with the periplasmic regions of MotB and FliL and with the flagellar protein MotF, which was recently characterized as a membrane component of the flagellum in this bacterium. In addition, the localization of green fluorescent protein (GFP)-MotF is completely dependent on FlgT. The Mot(-) phenotype of flgT cells was weakly suppressed by point mutants of MotB that presumably keep the proton channel open and efficiently suppress the Mot(-) phenotype of motF and fliL cells, indicating that FlgT could play an additional role beyond the opening of the proton channel. The presence of FlgT in purified filament-hook-basal bodies of the wild-type strain was confirmed by Western blotting, and the observation of these structures under an electron microscope showed that the basal bodies from flgT cells had lost the ring that covers the LP ring in the wild-type structure. Moreover, MotF was detected by immunoblotting in the basal bodies obtained from the wild-type strain but not from flgT cells. From these results, we suggest that FlgT forms a ring around the LP ring, which anchors MotF and stabilizes the stator complex of the flagellar motor. PMID:24056105

  13. Flagellar motility is necessary for Aeromonas hydrophila adhesion.

    PubMed

    Qin, Yingxue; Lin, Guifang; Chen, Wenbo; Xu, Xiaojin; Yan, Qingpi

    2016-09-01

    Adhesion to host surface or cells is the initial step in bacterial pathogenesis, and the adhesion mechanisms of the fish pathogenic bacteria Aeromonas hydrophila were investigated in this study. First, a mutagenesis library of A. hydrophila that contained 332 random insertion mutants was constructed via mini-Tn10 Km mutagenesis. Four mutants displayed the most attenuated adhesion. Sequence analysis revealed that the mini-Tn10 insertion sites in the four mutant strains were flgC(GenBank accession numbers KX261880), cytb4(GenBank accession numbers JN133621), rbsR(GenBank accession numbers KX261881) and flgE(GenBank accession numbers JQ974982). To further study the roles of flgC and flgE in the adhesion of A. hydrophila, some biological characteristics of the wild-type strain B11, the mutants M121 and M240, and the complemented strains C121 and C240 were investigated. The results showed that the mutation in flgC or flgE led to the flagellar motility of A. hydrophila significant reduction or abolishment. flgC was not necessary for flagellar biosynthesis but was necessary for the full motility of A. hydrophila, flgE was involved in both flagellar biosynthesis and motility. The flagellar motility is necessary for A. hydrophila to adhere to the host mucus, which suggests flagellar motility plays crucial roles in the early infection process of this bacterium. PMID:27432325

  14. Effects of various agents on flagellar activity, flagellar autotomy and cell viability in four species of Chlamydomonas (chlorophyta: volvocales).

    PubMed

    Lewin, R A; Lee, T H; Fang, L S

    1982-01-01

    Over 200 strains of green algal flagellates, representing about 100 species, were examined for their suitability as experimental organisms for studies of flagellar activity. The cells of all species shed their flagella under unfavourable conditions of temperature or pH, or in the presence of alcohols, detergents or toxic agents of various kinds. For further studies of flagellar activity, motility and autotomy (biologically induced shedding) in particular, we selected four species of Chlamydomonas: C. dysosmos Moewus, C. moewusii Gerloff, C. monoica Strehlow and C. reinhardtii Dangeard. Agents found to inhibit motility without inducing death or flagellar autotomy included azide, arsenite, thiosulphate, cyanide, ferricyanide, hydroxylamine, chloral hydrate, malonate, p-chloro-mercury benzoate and cytochalasin-B, each in a limited range of concentrations which differed according to species and strain. Higher concentrations of these agents caused the flagella to be shed. Since flagellar autotomy is a means by which a cell can quickly reduce the area of its permeable surface, it may have a positive survival value for species liable to be subjected to unfavourable physicochemical conditions. PMID:6764045

  15. Mechanoregulation of molecular motors in flagella

    NASA Astrophysics Data System (ADS)

    Gadelha, Hermes

    2014-11-01

    Molecular motors are nano-biological machines responsible for exerting forces that drive movement in living organisms, from cargo transport to cell division and motility. Interestingly, despite the inherent complexity of many interacting motors, order and structure may arise naturally, as exemplified by the harmonic, self-organized undulatory motion of the flagellum. The real mechanisms behind this collective spontaneous oscillation are still unknown, and it is challenging task to measure experimentally the molecular motor dynamics within the flagellar structure in real time. In this talk we will explore different competing hypotheses that are capable of generating flagellar bending waves that ``resemble'' in-vitro observations, emphasizing the need for further mathematical analysis and model validation. It also highlight that this is a fertile and challenging area of inter-disciplinary research for applied mathematicians and demonstrates the importance of future observational and theoretical studies in understanding the underlying mechanics of these motile cell appendages.

  16. A mutation that uncouples flagellum assembly from transcription alters the temporal pattern of flagellar gene expression in Caulobacter crescentus.

    PubMed Central

    Mangan, E K; Bartamian, M; Gober, J W

    1995-01-01

    The transcription of flagellar genes in Caulobacter crescentus is regulated by cell cycle events that culminate in the synthesis of a new flagellum once every cell division. Early flagellar gene products regulate the expression of late flagellar genes at two distinct stages of the flagellar trans-acting hierarchy. Here we investigate the coupling of early flagellar biogenesis with middle and late flagellar gene expression. We have isolated mutants (bfa) that do not require early class II flagellar gene products for the transcription of middle or late flagellar genes. bfa mutant strains are apparently defective in a negative regulatory pathway that couples early flagellar biogenesis to late flagellar gene expression. The bfa regulatory pathway functions solely at the level of transcription. Although flagellin promoters are transcribed in class II/bfa double mutants, there is no detectable flagellin protein on immunoblots prepared from mutant cell extracts. This finding suggests that early flagellar biogenesis is coupled to gene expression by two distinct mechanisms: one that negatively regulates transcription, mediated by bfa, and another that functions posttranscriptionally. To determine whether bfa affects the temporal pattern of late flagellar gene expression, cell cycle experiments were performed in bfa mutant strains. In a bfa mutant strain, flagellin expression fails to shut off at its normal time in the cell division cycle. This experimental result indicates that bfa may function as a regulator of flagellar gene transcription late in the cell cycle, after early flagellar structures have been assembled. PMID:7768816

  17. Alternative flagellar filament types in the haloarchaeon Haloarcula marismortui.

    PubMed

    Pyatibratov, Michael G; Beznosov, Sergey N; Rachel, Reinhard; Tiktopulo, Elizabeth I; Surin, Alexei K; Syutkin, Alexei S; Fedorov, Oleg V

    2008-10-01

    Many Archaea use rotation of helical flagellar filaments for swimming motility. We isolated and characterized the flagellar filaments of Haloarcula marismortui, an archaeal species previously considered to be nonmotile. Two Haloarcula marismortui phenotypes were discriminated--their filaments are composed predominantly of either FlaB or FlaA2 flagellin, and the corresponding genes are located on different replicons. FlaB and FlaA2 filaments differ in antigenicity and thermostability. FlaA2 filaments are distinctly thicker (20-22 nm) than FlaB filaments (16-18 nm). The observed filaments are nearly twice as thick as those of other characterized euryarchaeal filaments. The results suggest that the helicity of Haloarcula marismortui filaments is provided by a mechanism different from that in the related haloarchaeon Halobacterium salinarum, where 2 different flagellin molecules present in comparable quantities are required to form a helical filament. PMID:18923552

  18. FlaA, a putative flagellar outer sheath protein, is not an immunodominant antigen associated with Lyme disease.

    PubMed Central

    Ge, Y; Charon, N W

    1997-01-01

    FlaA was recently found to be associated with flagellar filaments of Borrelia burgdorferi. We tested whether antibodies to this protein are a good indicator of infection, as antibodies to FlaA proteins in other spirochetal infections show an increase in titer. Although overproduction of intact FlaA was highly toxic to Escherichia coli, truncated proteins which lacked the N-terminal signal sequence could be successfully overexpressed. Immunoblotting with sera from mammalian hosts infected with B. burgdorferi indicated that FlaA is not an immunodominant antigen in Lyme disease. However, sera from two patients reacted with both recombinant and native FlaA protein, suggesting that B. burgdorferi FlaA was antigenic and expressed in vivo. PMID:9199479

  19. Incomplete flagellar structures in nonflagellate mutants of Salmonella typhimurium.

    PubMed Central

    Suzuki, T; Iino, T; Horiguchi, T; Yamaguchi, S

    1978-01-01

    Incomplete flagellar structures were detected in osmotically shocked cells or membrane-associated fraction of many nonflagellate mutants of Salmonella typhimurium by electron microscopy. The predominant types of these structures in the mutants were cistron specific. The incomplete basal bodies were detected in flaFI, flaFIV, flaFVIII, and flaFIX mutants, the structure homologous to a basal body in flaFV mutants, the polyhook-basal body complex in flaR mutants, and the hook-basal body complex in flaL and flaU mutants. No structures homologous to flagellar bases or their parts were detected in the early-fla group nonflagellate mutants of flaAI, flaAII, flaAIII, flaB, flaC, flaD, flaE, flaFII, flaFIII, flaFVI, flaFVII, flaFX, flaK, and flaM. From these observations, a process of flagellar morphogenesis was postulated. The functions of the early-fla group are essential to the formation of S ring-M ring-rod complexes bound to the membrane. The completion of basal bodies requires succeeding functions of flaFI, flaFIV, flaFVIII, and flaFIX. Next, the formation of hooks attached to basal bodies proceeds by the function of flaFV and by flaR, which controls the hook length. Flagellar filaments appear at the tips of hooks because of the functions of flaL, flaU, and flagellin genes. Images PMID:342514

  20. Direct evidence of flagellar synchronization through hydrodynamic interactions

    NASA Astrophysics Data System (ADS)

    Brumley, Douglas; Polin, Marco; Wan, Kirsty; Goldstein, Raymond

    2013-11-01

    Eukaryotic cilia and flagella exhibit striking coordination, from the synchronous beating of two flagella in Chlamydomonas to the metachronal waves and large-scale flows displayed by carpets of cilia. However, the precise mechanisms responsible for flagellar synchronization remain unclear. We perform a series of experiments involving two individual flagella in a quiescent fluid. Cells are isolated from the colonial alga Volvox carteri, held in place at a fixed distance d, and oriented so that their flagellar beating planes coincide. In this fashion, we are able to explicitly assess the role of hydrodynamics in achieving synchronization. For closely separated cells, the flagella are capable of exhibiting a phase-locked state for thousands of beats at a time, despite significant differences in their intrinsic frequencies. For intermediate values of d, synchronous periods are interrupted by brief phase slips, while for d >> 1 the flagellar phase difference drifts almost linearly with time. The coupling strength extracted through analysis of the synchronization statistics exhibits excellent agreement with hydrodynamic predictions. This study unambiguously reveals that flagella coupled only through hydrodynamics are capable of exhibiting robust synchrony.

  1. Approaches for functional analysis of flagellar proteins in African trypanosomes

    PubMed Central

    Oberholzer, Michael; Lopez, Miguel A.; Ralston, Katherine S.; Hill, Kent L.

    2013-01-01

    The eukaryotic flagellum is a highly conserved organelle serving motility, sensory and transport functions. Although genetic, genomic and proteomic studies have led to the identification of hundreds of flagellar and putative flagellar proteins, precisely how these proteins function individually and collectively to drive flagellum motility and other functions remains to be determined. In this chapter we provide an overview of tools and approaches available for studying flagellum protein function in the protozoan parasite Trypanosoma brucei. We begin by outlining techniques for in vitro cultivation of both T. brucei lifecycle stages, as well as transfection protocols for the delivery of DNA constructs. We then describe specific assays used to assess flagellum function including flagellum preparation and quantitative motility assays. We conclude the chapter with a description of molecular genetic approaches for manipulating gene function. In summary, the availability of potent molecular tools, as well as the health and economic relevance of T. brucei as a pathogen, combine to make the parasite an attractive and integral experimental system for the functional analysis of flagellar proteins. PMID:20409810

  2. Reduced Protein Synthesis Fidelity Inhibits Flagellar Biosynthesis and Motility

    PubMed Central

    Fan, Yongqiang; Evans, Christopher R.; Ling, Jiqiang

    2016-01-01

    Accurate translation of the genetic information from DNA to protein is maintained by multiple quality control steps from bacteria to mammals. Genetic and environmental alterations have been shown to compromise translational quality control and reduce fidelity during protein synthesis. The physiological impact of increased translational errors is not fully understood. While generally considered harmful, translational errors have recently been shown to benefit cells under certain stress conditions. In this work, we describe a novel regulatory pathway in which reduced translational fidelity downregulates expression of flagellar genes and suppresses bacterial motility. Electron microscopy imaging shows that the error-prone Escherichia coli strain lacks mature flagella. Further genetic analyses reveal that translational errors upregulate expression of a small RNA DsrA through enhancing its transcription, and deleting DsrA from the error-prone strain restores motility. DsrA regulates expression of H-NS and RpoS, both of which regulate flagellar genes. We demonstrate that an increased level of DsrA in the error-prone strain suppresses motility through the H-NS pathway. Our work suggests that bacteria are capable of switching on and off the flagellar system by altering translational fidelity, which may serve as a previously unknown mechanism to improve fitness in response to environmental cues. PMID:27468805

  3. (Photosynthesis in intact plants)

    SciTech Connect

    Not Available

    1990-01-01

    Progress in the two years since the last renewal application has been excellent. We have made substantial contributions on both main fronts of the projects, and are particularly happy with the progress of our research on intact plants. The approach of basing our field work on a sound foundation of laboratory studies has enabled is to use methods which provide unambiguous assays of well characterized reactions. We have also made excellent progress in several laboratory studies which will have direct applications in future field work, and have introduced to the laboratory a range of molecular genetics techniques which will allow us to explore new options in the attempt to understand function at the level of molecular structure.

  4. The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide.

    PubMed Central

    Albertini, A M; Caramori, T; Crabb, W D; Scoffone, F; Galizzi, A

    1991-01-01

    We cloned and sequenced 8.3 kb of Bacillus subtilis DNA corresponding to the flaA locus involved in flagellar biosynthesis, motility, and chemotaxis. The DNA sequence revealed the presence of 10 complete and 2 incomplete open reading frames. Comparison of the deduced amino acid sequences to data banks showed similarities of nine of the deduced products to a number of proteins of Escherichia coli and Salmonella typhimurium for which a role in flagellar functioning has been directly demonstrated. In particular, the sequence data suggest that the flaA operon codes for the M-ring protein, components of the motor switch, and the distal part of the basal-body rod. The gene order is remarkably similar to that described for region III of the enterobacterial flagellar regulon. One of the open reading frames was translated into a protein with 48% amino acid identity to S. typhimurium FliI and 29% identity to the beta subunit of E. coli ATP synthase. PMID:1828465

  5. The physics of intact capture

    NASA Technical Reports Server (NTRS)

    Tsou, Peter; Griffiths, D. J.; Albee, A. L.

    1994-01-01

    The ability to capture projectiles intact at hypervelocities in underdense media open a new area of study in physics. Underdense material behaves markedly different than solid, liquid, or gas upon hypervelocity impact. This new phenomenon enables applications in science that would either not be possible or would be very costly by other means. This phenomenon has been fully demonstrated in the laboratory and validated in space. Even more interesting is the fact that this hypervelocity intact capture was accomplished passively. A better understanding of the physics of intact capture will lead to improvements in intact capture. A collection of physical observations of this phenomenon is presented here.

  6. Involvement of the flagellar assembly pathway in Vibrio alginolyticus adhesion under environmental stresses

    PubMed Central

    Wang, Lu; Huang, Lixing; Su, Yongquan; Qin, Yingxue; Kong, Wendi; Ma, Ying; Xu, Xiaojin; Lin, Mao; Zheng, Jiang; Yan, Qingpi

    2015-01-01

    Adhesion is an important virulence factor of Vibrio alginolyticus. This factor may be affected by environmental conditions; however, its molecular mechanism remains unclear. In our previous research, adhesion deficient strains were obtained by culturing V. alginolyticus under stresses including Cu, Pb, Hg, and low pH. With RNA-seq and bioinformatics analysis, we found that all of these stress treatments significantly affected the flagellar assembly pathway, which may play an important role in V. alginolyticus adhesion. Therefore, we hypothesized that the environmental stresses of the flagellar assembly pathway may be one way in which environmental conditions affect adhesion. To verify our hypothesis, a bioinformatics analysis, QPCR, RNAi, in vitro adhesion assay and motility assay were performed. Our results indicated that (1) the flagellar assembly pathway was sensitive to environmental stresses, (2) the flagellar assembly pathway played an important role in V. alginolyticus adhesion, and (3) motility is not the only way in which the flagellar assembly pathway affects adhesion. PMID:26322276

  7. Individual Flagellar Waveform Affects Collective Behavior of Chlamydomonas reinhardtii.

    PubMed

    Kage, Azusa; Mogami, Yoshihiro

    2015-08-01

    Bioconvection is a form of collective motion that occurs spontaneously in the suspension of swimming microorganisms. In a previous study, we quantitatively described the "pattern transition," a phase transition phenomenon that so far has exclusively been observed in bioconvection of the unicellular green alga Chlamydomonas. We suggested that the transition could be induced by changes in the balance between the gravitational and shear-induced torques, both of which act to determine the orientation of the organism in the shear flow. As both of the torques should be affected by the geometry of the Chlamydomonas cell, alteration in the flagellar waveform might change the extent of torque generation by altering overall geometry of the cell. Based on this working hypothesis, we examined bioconvection behavior of two flagellar mutants of Chlamydomonas reinhardtii, ida1 and oda2, making reference to the wild type. Flagella of ida1 beat with an abnormal waveform, while flagella of oda2 show a normal waveform but lower beat frequency. As a result, both mutants had swimming speed of less than 50% of the wild type. ida1 formed bioconvection patterns with smaller spacing than those of wild type and oda2. Two-axis view revealed the periodic movement of the settling blobs of ida1, while oda2 showed qualitatively similar behavior to that of wild type. Unexpectedly, ida1 showed stronger negative gravitaxis than did wild type, while oda2 showed relatively weak gravitaxis. These findings suggest that flagellar waveform, not swimming speed or beat frequency, strongly affect bioconvection behavior in C. reinhardtii. PMID:26245228

  8. The Three-Dimensional Structure of the Flagellar Rotor from a Clockwise-Locked Mutant of Salmonella enterica Serovar Typhimurium

    PubMed Central

    Thomas, Dennis R.; Francis, Noreen R.; Xu, Chen; DeRosier, David J.

    2006-01-01

    Three-dimensional reconstructions from electron cryomicrographs of the rotor of the flagellar motor reveal that the symmetry of individual M rings varies from 24-fold to 26-fold while that of the C rings, containing the two motor/switch proteins FliM and FliN, varies from 32-fold to 36-fold, with no apparent correlation between the symmetries of the two rings. Results from other studies provided evidence that, in addition to the transmembrane protein FliF, at least some part of the third motor/switch protein, FliG, contributes to a thickening on the face of the M ring, but there was no evidence as to whether or not any portion of FliG also contributes to the C ring. Of the four morphological features in the cross section of the C ring, the feature closest to the M ring is not present with the rotational symmetry of the rest of the C ring, but instead it has the symmetry of the M ring. We suggest that this inner feature arises from a domain of FliG. We present a hypothetical docking in which the C-terminal motor domain of FliG lies in the C ring, where it can interact intimately with FliM. PMID:17015643

  9. Space research with intact organisms

    NASA Technical Reports Server (NTRS)

    Phillips, Robert W.; Haddy, Francis J.

    1992-01-01

    Effects of space exposure on intact organisms are briefly reviewed, and examples of future experiments that might provide new information on the role of gravity in the evolution of life are suggested. It is noted that long term experiments with intact plant and animals for studying gravitational thresholds will provide important new insights.

  10. Quorum sensing positively regulates flagellar motility in pathogenic Vibrio harveyi.

    PubMed

    Yang, Qian; Defoirdt, Tom

    2015-04-01

    Vibrios belonging to the Harveyi clade are among the major pathogens of aquatic organisms. Quorum sensing (QS) is essential for virulence of V. harveyi towards different hosts. However, most virulence factors reported to be controlled by QS to date are negatively regulated by QS, therefore suggesting that their impact on virulence is limited. In this study, we report that QS positively regulates flagellar motility. We found that autoinducer synthase mutants showed significantly lower swimming motility than the wild type, and the swimming motility could be restored by adding synthetic signal molecules. Further, motility of a luxO mutant with inactive QS (LuxO D47E) was significantly lower than that of the wild type and of a luxO mutant with constitutively maximal QS activity (LuxO D47A). Furthermore, we found that the expression of flagellar genes (both early, middle and late genes) was significantly lower in the luxO mutant with inactive QS when compared with wild type and the luxO mutant with maximal QS activity. Motility assays and gene expression also revealed the involvement of the quorum-sensing master regulator LuxR in the QS regulation of motility. Finally, the motility inhibitor phenamil significantly decreased the virulence of V. harveyi towards gnotobiotic brine shrimp larvae. PMID:24528485

  11. Interacting Protein Kinases Involved in the Regulation of Flagellar Length

    PubMed Central

    Erdmann, Maja; Scholz, Anne; Melzer, Inga M.; Schmetz, Christel; Wiese, Martin

    2006-01-01

    A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis. PMID:16467378

  12. Flagellar Glycosylation in Burkholderia pseudomallei and Burkholderia thailandensis▿

    PubMed Central

    Scott, Andrew E.; Twine, Susan M.; Fulton, Kelly M.; Titball, Richard W.; Essex-Lopresti, Angela E.; Atkins, Timothy P.; Prior, Joann L.

    2011-01-01

    Glycosylation of proteins is known to impart novel physical properties and biological roles to proteins from both eukaryotes and prokaryotes. In this study, gel-based glycoproteomics were used to identify glycoproteins of the potential biothreat agent Burkholderia pseudomallei and the closely related but nonpathogenic B. thailandensis. Top-down and bottom-up mass spectrometry (MS) analyses identified that the flagellin proteins of both species were posttranslationally modified by novel glycans. Analysis of proteins from two strains of each species demonstrated that B. pseudomallei flagellin proteins were modified with a glycan with a mass of 291 Da, while B. thailandensis flagellin protein was modified with related glycans with a mass of 300 or 342 Da. Structural characterization of the B. thailandensis carbohydrate moiety suggests that it is an acetylated hexuronic acid. In addition, we have identified through mutagenesis a gene from the lipopolysaccharide (LPS) O-antigen biosynthetic cluster which is involved in flagellar glycosylation, and inactivation of this gene eliminates flagellar glycosylation and motility in B. pseudomallei. This is the first report to conclusively demonstrate the presence of a carbohydrate covalently linked to a protein in B. pseudomallei and B. thailandensis, and it suggests new avenues to explore in order to examine the marked differences in virulence between these two species. PMID:21602339

  13. Step-Wise Loss of Bacterial Flagellar Torsion Confers Progressive Phagocytic Evasion

    PubMed Central

    Lovewell, Rustin R.; Collins, Ryan M.; Acker, Julie L.; O'Toole, George A.; Wargo, Matthew J.; Berwin, Brent

    2011-01-01

    Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria. PMID:21949654

  14. Flagellar kinematics and swimming of algal cells in viscoelastic fluids.

    PubMed

    Qin, B; Gopinath, A; Yang, J; Gollub, J P; Arratia, P E

    2015-01-01

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes. PMID:25778677

  15. Flagellar generated flow mediates attachment of Giardia lamblia

    NASA Astrophysics Data System (ADS)

    Urbach, Jeffrey; Luo, Haibei; Picou, Theodore; McAllister, Ryan; Elmendorf, Heidi

    2011-03-01

    Giardia lamblia is a protozoan parasite responsible for widespread diarrheal disease in humans and animals worldwide. Attachment to the host intestinal mucosa and resistance to peristalsis is necessary for establishing infection, but the physical basis for this attachment is poorly understood. We report results from TIRF and confocal fluorescence microscopy that demonstrate that the regular beating of the posterior flagella generate a flow through the ventral disk, a suction-cup shaped structure that is against the substrate during attachment. Finite element simulations are used to compare the negative pressure generated by the flow to the measured attachment force and the expected performance of the flagellar pump. NIH grant 1R21AI062934-0.

  16. Flagellar Kinematics and Swimming of Algal Cells in Viscoelastic Fluids

    PubMed Central

    Qin, B.; Gopinath, A.; Yang, J.; Gollub, J. P.; Arratia, P. E.

    2015-01-01

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes. PMID:25778677

  17. Composition and sensory function of the trypanosome flagellar membrane

    PubMed Central

    Maric, Danijela; Epting, Conrad L.; Engman, David M.

    2010-01-01

    Summary A cilium is an extension of the cell that contains an axonemal complex of microtubules and associated proteins bounded by a membrane which is contiguous with the cell body membrane. Cilia may be nonmotile or motile, the latter having additional specific roles in cell or fluid movement. The term flagellum refers to the motile cilium of free-living single cells (e.g., bacteria, archaea, spermatozoa and protozoa). In eukaryotes, both nonmotile and motile cilia possess sensory functions. The ciliary interior (cilioplasm) is separated from the cytoplasm by a selective barrier that prevents passive diffusion of molecules between the two domains. The sensory functions of cilia reside largely in the membrane and signals generated in the cilium are transduced into a variety of cellular responses. In this review we discuss the structure and biogenesis of the cilium, with special attention to the trypanosome flagellar membrane, its lipid and protein composition and its proposed roles in sensing and signaling. PMID:20580599

  18. Flagellar swimmers oscillate between pusher- and puller-type swimming

    NASA Astrophysics Data System (ADS)

    Klindt, Gary S.; Friedrich, Benjamin M.

    2015-12-01

    Self-propulsion of cellular microswimmers generates flow signatures, commonly classified as pusher and puller type, which characterize hydrodynamic interactions with other cells or boundaries. Using experimentally measured beat patterns, we compute that the flagellated green alga Chlamydomonas oscillates between pusher and puller, rendering it an approximately neutral swimmer, when averaging over its full beat cycle. Beyond a typical distance of 100 μ m from the cell, inertia attenuates oscillatory microflows. We show that hydrodynamic interactions between cells oscillate in time and are of similar magnitude as stochastic swimming fluctuations. From our analysis, we also find that the rate of hydrodynamic dissipation varies in time, which implies that flagellar beat patterns are not optimized with respect to this measure.

  19. Flagellar Kinematics and Swimming of Algal Cells in Viscoelastic Fluids

    NASA Astrophysics Data System (ADS)

    Qin, B.; Gopinath, A.; Yang, J.; Gollub, J. P.; Arratia, P. E.

    2015-03-01

    The motility of microorganisms is influenced greatly by their hydrodynamic interactions with the fluidic environment they inhabit. We show by direct experimental observation of the bi-flagellated alga Chlamydomonas reinhardtii that fluid elasticity and viscosity strongly influence the beating pattern - the gait - and thereby control the propulsion speed. The beating frequency and the wave speed characterizing the cyclical bending are both enhanced by fluid elasticity. Despite these enhancements, the net swimming speed of the alga is hindered for fluids that are sufficiently elastic. The origin of this complex response lies in the interplay between the elasticity-induced changes in the spatial and temporal aspects of the flagellar cycle and the buildup and subsequent relaxation of elastic stresses during the power and recovery strokes.

  20. Antiphase Synchronization in a Flagellar-Dominance Mutant of Chlamydomonas

    NASA Astrophysics Data System (ADS)

    Leptos, Kyriacos C.; Wan, Kirsty Y.; Polin, Marco; Tuval, Idan; Pesci, Adriana I.; Goldstein, Raymond E.

    2013-10-01

    Groups of beating flagella or cilia often synchronize so that neighboring filaments have identical frequencies and phases. A prime example is provided by the unicellular biflagellate Chlamydomonas reinhardtii, which typically displays synchronous in-phase beating in a low-Reynolds number version of breaststroke swimming. We report the discovery that ptx1, a flagellar-dominance mutant of C. reinhardtii, can exhibit synchronization in precise antiphase, as in the freestyle swimming stroke. High-speed imaging shows that ptx1 flagella switch stochastically between in-phase and antiphase states, and that the latter has a distinct waveform and significantly higher frequency, both of which are strikingly similar to those found during phase slips that stochastically interrupt in-phase beating of the wild-type. Possible mechanisms underlying these observations are discussed.

  1. Life with eight flagella: flagellar assembly and division in Giardia

    PubMed Central

    Dawson, Scott C.; House, Susan A.

    2016-01-01

    Summary of Recent Advances Flagellar movement in Giardia, a common intestinal parasitic protist, is critical to its survival in the host. Each axoneme is unique in possessing a long, cytoplasmic portion as well as a membrane-bound portion. Intraflagellar transport (IFT) is required for the assembly of membrane-bound regions, yet the cytoplasmic regions may be assembled by IFT-independent mechanisms. Steady-state axoneme length is maintained by IFT and by intrinsic and active microtubule dynamics. Following mitosis and prior to their segregation, giardial flagella undergo a multigenerational division cycle in which the parental eight flagella migrate and reposition to different cellular locations; eight new flagella are assembled de novo. Each daughter cell thus inherits four mature and four newly synthesized flagella. PMID:20580308

  2. Protein export through the bacterial flagellar type III export pathway.

    PubMed

    Minamino, Tohru

    2014-08-01

    For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. PMID:24064315

  3. Biochemical, immunological, metabolic, and molecular studies on flagellar development in Euglena gracilis

    SciTech Connect

    Levasseur, P.J.

    1989-01-01

    The emergent flagellum of Euglena gracilis arises from an anterior invagination of the organism and possesses, along with the typical eukaryotic axoneme, a glycoprotein surface layer, a complement of structurally complex mastigonemes and a paraxial rod. Nonionic detergent extraction of isolated flagella yielded a fraction containing 21% of the flagellar protein. This fraction contained at least 25 components. In vivo radiolabeling experiments indicated that Euglena possessed a pool of flagellar precursors. This was evidence by the observation that flagellar proteins radiolabeled during an initial regeneration could be mobilized to flagella of a subsequent regeneration. At least one component in the pool was present in sufficient quantity to support an entire regeneration. This protein was tentatively identified as a mastigonemal protein of M{sub r} {approximately} 220,000. A cDNA library was constructed to investigate flagellar gene expression in Euglena.

  4. Fusions of flagellar operons to lactose genes on a mu lac bacteriophage.

    PubMed Central

    Komeda, Y

    1982-01-01

    Previous studies have defined 29 genes necessary for synthesis of the Escherichia coli flagellar apparatus. This study analyzed the transcriptional control of flagellar genes, using Mu d (Apr lac) phage to generate flagellar mutants by insertion. These mutants contained operon fusions of flagellar genes to the lac genes of the Mu d phage and allowed the measurement of flagellar operon expression by detection of beta-galactosidase activity. These fusion mutants expressed the enzyme activity constitutively, and an autogenous regulation mechanism was not revealed. Lambda transducing phages carrying these chromosomal fla-lac fusions were also isolated and used to examine the effect of different fla mutations on expression of each flagellar operon. The results showed that flagellar operons are divided into six classes; (class 1) the flbB operon, which controls all of the other flagellar operons; (class 2) the flaU and flbC operons, which are controlled by the flbB operon gene products and are not required for the expression of other Fla operons; (class 3) the flbA, flaG, flaD, flaN, flaB, and flaA operons, which are under flbB operon control and are required for the expression of other fla operons; (class4) the flaZ operon, which is controlled by the gene products of the group 1 and 3 operons and is required for hag transcription; (class 5) the mocha and flaS operons, which are controlled by the gene products of the group 1 and 3 operons; and (class 6) the hag operon. These results are discussed with respect to the possible assembly sequence of the fla gene products. PMID:7037746

  5. TbFlabarin, a flagellar protein of Trypanosoma brucei, highlights differences between Leishmania and Trypanosoma flagellar-targeting signals.

    PubMed

    Tetaud, Emmanuel; Lefebvre, Michèle; M'Bang-Benet, Diane-Ethna; Crobu, Lucien; Blancard, Corinne; Sterkers, Yvon; Pages, Michel; Bastien, Patrick; Merlin, Gilles

    2016-07-01

    TbFlabarin is the Trypanosoma brucei orthologue of the Leishmania flagellar protein LdFlabarin but its sequence is 33% shorter than LdFlabarin, as it lacks a C-terminal domain that is indispensable for LdFlabarin to localize to the Leishmania flagellum. TbFlabarin is mainly expressed in the procyclic forms of the parasite and localized to the flagellum, but only when two palmitoylable cysteines at positions 3 and 4 are present. TbFlabarin is more strongly attached to the membrane fraction than its Leishmania counterpart, as it resists complete solubilization with as much as 0.5% NP-40. Expression ablation by RNA interference did not change parasite growth in culture, its morphology or apparent motility. Heterologous expression showed that neither TbFlabarin in L. amazonensis nor LdFlabarin in T. brucei localized to the flagellum, revealing non-cross-reacting targeting signals between the two species. PMID:27060615

  6. Real-Time Imaging of Fluorescent Flagellar Filaments of Rhizobium lupini H13-3: Flagellar Rotation and pH-Induced Polymorphic Transitions

    PubMed Central

    Scharf, Birgit

    2002-01-01

    The soil bacterium Rhizobium lupini H13-3 has complex right-handed flagellar filaments with unusual ridged, grooved surfaces. Clockwise (CW) rotation propels the cells forward, and course changes (tumbling) result from changes in filament speed instead of the more common change in direction of rotation. In view of these novelties, fluorescence labeling was used to analyze the behavior of single flagellar filaments during swimming and tumbling, leading to a model for directional changes in R. lupini. Also, flagellar filaments were investigated for helical conformational changes, which have not been previously shown for complex filaments. During full-speed CW rotation, the flagellar filaments form a propulsive bundle that pushes the cell on a straight path. Tumbling is caused by asynchronous deceleration and stops of individual filaments, resulting in dissociation of the propulsive bundle. R. lupini tumbles were not accompanied by helical conformational changes as are tumbles in other organisms including enteric bacteria. However, when pH was experimentally changed, four different polymorphic forms were observed. At a physiological pH of 7, normal flagellar helices were characterized by a pitch angle of 30°, a pitch of 1.36 μm, and a helical diameter of 0.50 μm. As pH increased from 9 to 11, the helices transformed from normal to semicoiled to straight. As pH decreased from 5 to 3, the helices transformed from normal to curly to straight. Transient conformational changes were also noted at high viscosity, suggesting that the R. lupini flagellar filament may adapt to high loads in viscous environments (soil) by assuming hydrodynamically favorable conformations. PMID:12374832

  7. Intact capture of hypervelocity particles

    NASA Technical Reports Server (NTRS)

    Tsou, P.; Brownlee, D. E.; Albee, A. L.

    1986-01-01

    Knowledge of the phase, structure, and crystallography of cosmic particles, as well as their elemental and isotopic compositions, would be very valuable information toward understanding the nature of our solar system. This information can be obtained from the intact capture of large mineral grains of cosmic particles from hypervelocity impacts. Hypervelocity experiments of intact capture in underdense media have indicated realistic potential in this endeaver. The recovery of the thermal blankets and louvers from the Solar Max spacecraft have independently verified this potential in the unintended capture of cosmic materials from hypervelocity impacts. Passive underdense media will permit relatively simple and inexpensive missions to capture cosmic particles intact, either by going to a planetary body or by waiting for the particles to come to the Shuttle or the Space Station. Experiments to explore the potential of using various underdense media for an intact comet sample capture up to 6.7 km/s were performed at NASA Ames Research Center Vertical Gun Range. Explorative hypervelocity experiments up to 7.9 km/s were also made at the Ernst Mach Institute. These experiments have proven that capturing intact particles at hypervelocity impacts is definitely possible. Further research is being conducted to achieve higher capture ratios at even higher hypervelocities for even smaller projectiles.

  8. Intact capture of hypervelocity particles

    NASA Astrophysics Data System (ADS)

    Tsou, P.; Brownlee, D. E.; Albee, A. L.

    Knowledge of the phase, structure, and crystallography of cosmic particles, as well as their elemental and isotopic compositions, would be very valuable information toward understanding the nature of our solar system. This information can be obtained from the intact capture of large mineral grains of cosmic particles from hypervelocity impacts. Hypervelocity experiments of intact capture in underdense media have indicated realistic potential in this endeaver. The recovery of the thermal blankets and louvers from the Solar Max spacecraft have independently verified this potential in the unintended capture of cosmic materials from hypervelocity impacts. Passive underdense media will permit relatively simple and inexpensive missions to capture cosmic particles intact, either by going to a planetary body or by waiting for the particles to come to the Shuttle or the Space Station. Experiments to explore the potential of using various underdense media for an intact comet sample capture up to 6.7 km/s were performed at NASA Ames Research Center Vertical Gun Range. Explorative hypervelocity experiments up to 7.9 km/s were also made at the Ernst Mach Institute. These experiments have proven that capturing intact particles at hypervelocity impacts is definitely possible. Further research is being conducted to achieve higher capture ratios at even higher hypervelocities for even smaller projectiles.

  9. Intact capture of cosmic dust

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1991-01-01

    The focus of this development effort is to capture dust particles at hypervelocities intact and unmelted in order to preserve volatile organics. At the same time, the capture process must minimize any organic elemental or compound contamination to prevent any compromise of exobiological analyses. Inorganic silicate aerogel has been developed as a successful capture medium to satisfy both requirements of intact capture and minimal organic contamination. Up to 6 km/s, silicate projectiles from a few microns up to 100 microns have been captured intact without any melting and with minimal loss of mass. Carbon in silicate aerogel can be reduced to less than 1 part in 1000 and hydrogen 3 parts in 1000 when baked in air. Under controlled inert gas environments, additional hydrocarbon reduction can be achieved.

  10. Vanadate-sensitized cleavage of dynein heavy chains by 365-nm irradiation of demembranated sperm flagella and its effect on the flagellar motility

    SciTech Connect

    Gibbons, B.H.; Gibbons, I.R.

    1987-06-15

    Irradiation of demembranated flagella of sea urchin sperm at 365 nm in the presence of 0.05-1 mM MgATP and 5-10 microM vanadate (Vi) cleaves the alpha and beta heavy chains of the outer arm dynein at the same site and at about the same rate as reported previously for the solubilized dynein. The decrease in intact alpha and beta heavy chain material is biphasic, with about 80% being lost with a half-time of 8-10 min, and the remainder more slowly. Five other axonemal polypeptides of Mr greater than 350,000 are lost similarly, concomitant with the appearance of at least 9 new peptides of Mr 150,000-250,000. The motility of irradiated sperm flagella upon subsequent dilution into reactivation medium containing 1 mM ATP and 2.5 mM catechol shows a progressive decrease in flagellar beat frequency for irradiation times that produce up to about 50% cleavage of the dynein heavy chains; more prolonged irradiation causes irreversible loss of motility. Competition between photocleaved and intact outer arm dynein for rebinding to dynein-depleted sperm flagella shows that cleavage has little effect upon the ability for rebinding, although the cleaved dynein partially inhibits subsequent motility. Substitution of MnATP for the MgATP in the irradiation medium prevents the loss of all of the axonemal polypeptides during irradiation for up to 60 min and also protects the potential for subsequent flagellar motility.

  11. Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions

    PubMed Central

    Juhas, Mario; Ajioka, James W

    2015-01-01

    The Gram-negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coli K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits. PMID:26074421

  12. Flagellar apparatus absolute orientations and the phylogeny of the green algae.

    PubMed

    O'Kelly, C J; Floyd, G L

    The absolute orientation of the flagellar apparatus in green algal motile cells is a feature of considerable value in studies of green algal systematics and phylogeny. The absolute orientation patterns found in those algae for which this feature is known or can be deduced are reviewed. Counterclockwise absolute orientation occurs in all classes except the Chlorophyceae and is considered primitive, while the clockwise absolute orientation present in most members of the Chlorophyceae is the result of progressive clockwise rotation of components during evolution. Extant intermediates documenting this rotation include Hafniomonas vegetative cells, which show counterclockwise absolute orientation, and Chaetopeltis quadriflagellate zoospores, in which the flagellar apparatus is strictly cruciate except for a slight clockwise offset of the microtubular rootlets. The V-shaped arrangement of the basal bodies in the flagellar apparatus, as well as the presence of proximal sheaths and of two layers of scales on the cell body, further identifies the Chaetopeltis zoospore as a primitive cell type within the Chlorophyceae . Trends towards the exsertion of basal bodies from a flagellar pit, either apically or laterally, the elimination of quadriflagellate cells, and, in the Chlorophyceae , an increasing amount of basal body offset, indicate advancement within the classes. Absolute orientation is conserved during flagellar apparatus replication and development. Events after flagellar apparatus division in the algae studied may be subdivided into component assembly, which is universal and preserves phylogenetically-useful features, and component reorientation, which occurs in relatively few green algae and adapts the flagellar apparatus to specialized functions. From these flagellar apparatus orientation studies, a major reevaluation of evolution within the Chlorophyceae is proposed, with weakly- thalloid algae possessing desmoschisis (e.g. Chaetopeltis ) considered primitive, and

  13. Giardia Flagellar Motility Is Not Directly Required to Maintain Attachment to Surfaces

    PubMed Central

    House, Susan A.; Richter, David J.; Pham, Jonathan K.; Dawson, Scott C.

    2011-01-01

    Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined “suction-based” mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing “hydrodynamic model” of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is

  14. Intact Procedural Motor Sequence Learning in Developmental Coordination Disorder

    ERIC Educational Resources Information Center

    Lejeune, Caroline; Catale, Corinne; Willems, Sylvie; Meulemans, Thierry

    2013-01-01

    The purpose of the present study was to explore the possibility of a procedural learning deficit among children with developmental coordination disorder (DCD). We tested 34 children aged 6-12 years with and without DCD using the serial reaction time task, in which the standard keyboard was replaced by a touch screen in order to minimize the impact…

  15. A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

    PubMed Central

    2015-01-01

    The parasite Trypanosoma brucei lives in the bloodstream of infected mammalian hosts, fully exposed to the adaptive immune system. It relies on a very high rate of endocytosis to clear bound antibodies from its cell surface. All endo- and exocytosis occurs at a single site on its plasma membrane, an intracellular invagination termed the flagellar pocket. Coiled around the neck of the flagellar pocket is a multiprotein complex containing the repeat motif protein T. brucei MORN1 (TbMORN1). In this study, the phenotypic effects of TbMORN1 depletion in the mammalian-infective form of T. brucei were analyzed. Depletion of TbMORN1 resulted in a rapid enlargement of the flagellar pocket. Dextran, a polysaccharide marker for fluid phase endocytosis, accumulated inside the enlarged flagellar pocket. Unexpectedly, however, the proteins concanavalin A and bovine serum albumin did not do so, and concanavalin A was instead found to concentrate outside it. This suggests that TbMORN1 may have a role in facilitating the entry of proteins into the flagellar pocket. PMID:26318396

  16. FlgM is secreted by the flagellar export apparatus in Bacillus subtilis.

    PubMed

    Calvo, Rebecca A; Kearns, Daniel B

    2015-01-01

    The bacterial flagellum is assembled from over 20 structural components, and flagellar gene regulation is morphogenetically coupled to the assembly state by control of the anti-sigma factor FlgM. In the Gram-negative bacterium Salmonella enterica, FlgM inhibits late-class flagellar gene expression until the hook-basal body structural intermediate is completed and FlgM is inhibited by secretion from the cytoplasm. Here we demonstrate that FlgM is also secreted in the Gram-positive bacterium Bacillus subtilis and is degraded extracellularly by the proteases Epr and WprA. We further demonstrate that, like in S. enterica, the structural genes required for the flagellar hook-basal body are required for robust activation of σ(D)-dependent gene expression and efficient secretion of FlgM. Finally, we determine that FlgM secretion is strongly enhanced by, but does not strictly require, hook-basal body completion and instead demands a minimal subset of flagellar proteins that includes the FliF/FliG basal body proteins, the flagellar type III export apparatus components FliO, FliP, FliQ, FliR, FlhA, and FlhB, and the substrate specificity switch regulator FliK. PMID:25313396

  17. High-speed holographic microscopy of malaria parasites reveals ambidextrous flagellar waveforms

    PubMed Central

    Wilson, Laurence G.; Carter, Lucy M.; Reece, Sarah E.

    2013-01-01

    Axonemes form the core of eukaryotic flagella and cilia, performing tasks ranging from transporting fluid in developing embryos to the propulsion of sperm. Despite their abundance across the eukaryotic domain, the mechanisms that regulate the beating action of axonemes remain unknown. The flagellar waveforms are 3D in general, but current understanding of how axoneme components interact stems from 2D data; comprehensive measurements of flagellar shape are beyond conventional microscopy. Moreover, current flagellar model systems (e.g., sea urchin, human sperm) contain accessory structures that impose mechanical constraints on movement, obscuring the “native” axoneme behavior. We address both problems by developing a high-speed holographic imaging scheme and applying it to the (male) microgametes of malaria (Plasmodium) parasites. These isolated flagella are a unique, mathematically tractable model system for the physics of microswimmers. We reveal the 3D flagellar waveforms of these microorganisms and map the differential shear between microtubules in their axonemes. Furthermore, we overturn claims that chirality in the structure of the axoneme governs the beat pattern [Hirokawa N, et al. (2009) Ann Rev Fluid Mech 41:53–72], because microgametes display a left- or right-handed character on alternate beats. This breaks the link between structural chirality in the axoneme and larger scale symmetry breaking (e.g., in developing embryos), leading us to conclude that accessory structures play a critical role in shaping the flagellar beat. PMID:24194551

  18. Flagellar pocket restructuring through the Leishmania life cycle involves a discrete flagellum attachment zone.

    PubMed

    Wheeler, Richard J; Sunter, Jack D; Gull, Keith

    2016-02-15

    Leishmania promastigote parasites have a flagellum, which protrudes from the flagellar pocket at the cell anterior, yet, surprisingly, have homologs of many flagellum attachment zone (FAZ) proteins--proteins used in the related Trypanosoma species to laterally attach the flagellum to the cell body from the flagellar pocket to the cell posterior. Here, we use seven Leishmania mexicana cell lines that expressed eYFP fusions of FAZ protein homologs to show that the Leishmania flagellar pocket includes a FAZ structure. Electron tomography revealed a precisely defined 3D organisation for both the flagellar pocket and FAZ, with striking similarities to those of Trypanosoma brucei. Expression of two T. brucei FAZ proteins in L. mexicana showed that T. brucei FAZ proteins can assemble into the Leishmania FAZ structure. Leishmania therefore have a previously unrecognised FAZ structure, which we show undergoes major structural reorganisation in the transition from the promastigote (sandfly vector) to amastigote (in mammalian macrophages). Morphogenesis of the Leishmania flagellar pocket, a structure important for pathogenicity, is therefore intimately associated with a FAZ; a finding with implications for understanding shape changes involving component modules during evolution. PMID:26746239

  19. Glucose induces delocalization of a flagellar biosynthesis protein from the flagellated pole.

    PubMed

    Park, Soyoung; Park, Young-Ha; Lee, Chang-Ro; Kim, Yeon-Ran; Seok, Yeong-Jae

    2016-09-01

    To survive in a continuously changing environment, bacteria sense concentration gradients of attractants or repellents, and purposefully migrate until a more favourable habitat is encountered. While glucose is known as the most effective attractant, the flagellar biosynthesis and hence chemotactic motility has been known to be repressed by glucose in some bacteria. To date, the only known regulatory mechanism of the repression of flagellar synthesis by glucose is via downregulation of the cAMP level, as shown in a few members of the family Enterobacteriaceae. Here we show that, in Vibrio vulnificus, the glucose-mediated inhibition of flagellar motility operates by a completely different mechanism. In the presence of glucose, EIIA(Glc) is dephosphorylated and inhibits the polar localization of FapA (flagellar assembly protein A) by sequestering it from the flagellated pole. A loss or delocalization of FapA results in a complete failure of the flagellar biosynthesis and motility. However, when glucose is depleted, EIIA(Glc) is phosphorylated and releases FapA such that free FapA can be localized back to the pole and trigger flagellation. Together, these data provide new insight into a bacterial strategy to reach and stay in the glucose-rich area. PMID:27218601

  20. Flagellar pocket restructuring through the Leishmania life cycle involves a discrete flagellum attachment zone

    PubMed Central

    Wheeler, Richard J.; Sunter, Jack D.; Gull, Keith

    2016-01-01

    ABSTRACT Leishmania promastigote parasites have a flagellum, which protrudes from the flagellar pocket at the cell anterior, yet, surprisingly, have homologs of many flagellum attachment zone (FAZ) proteins – proteins used in the related Trypanosoma species to laterally attach the flagellum to the cell body from the flagellar pocket to the cell posterior. Here, we use seven Leishmania mexicana cell lines that expressed eYFP fusions of FAZ protein homologs to show that the Leishmania flagellar pocket includes a FAZ structure. Electron tomography revealed a precisely defined 3D organisation for both the flagellar pocket and FAZ, with striking similarities to those of Trypanosoma brucei. Expression of two T. brucei FAZ proteins in L. mexicana showed that T. brucei FAZ proteins can assemble into the Leishmania FAZ structure. Leishmania therefore have a previously unrecognised FAZ structure, which we show undergoes major structural reorganisation in the transition from the promastigote (sandfly vector) to amastigote (in mammalian macrophages). Morphogenesis of the Leishmania flagellar pocket, a structure important for pathogenicity, is therefore intimately associated with a FAZ; a finding with implications for understanding shape changes involving component modules during evolution. PMID:26746239

  1. Entosiphon sulcatum (Euglenophyceae): flagellar roots of the basal body complex and reservoir region

    SciTech Connect

    Solomon, J.A.; Walne, P.L.; Kivic, P.A.

    1987-03-01

    The flagellar root system of Entosiphon sulcatum (Dujardin) Stein (Euglenophyceae) is described and compared with kinetoplastid and other euglenoid systems. An asymmetric pattern of three microtubular roots, one between the two flagellar basal bodies and one on either side (here called the intermediate, dorsal, and ventral roots), is consistent within the euglenoid flagellates studied thus far. The dorsal root is associated with the basal body of the anterior flagellum (F1) and lies on the left dorsal side of the basal body complex. Originating between the two flagellar basal bodies, and associated with the basal body of the trailing flagellum (F2), the intermediate root is morphologically distinguished by fibrils interconnecting the individual microtubules to one another and to the overlying reservoir membrane. The intermediate root is often borne on a ridge projecting into the reservoir. The ventral root originates near the F2 basal body and lies on the right ventral side of the cell. Fibrillar connections link the membrane of F2 with the reservoir membrane at the reservoir-canal transition level. A large cross-banded fiber joins the two flagellar basal bodies, and a series of smaller striated fibers links the anterior accessory and flagellar basal bodies. Large nonstriated fibers extend from the basal body complex posteriorly into the cytoplasm.

  2. Monitoring Intact Viruses Using Aptamers.

    PubMed

    Kumar, Penmetcha K R

    2016-01-01

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review. PMID:27527230

  3. FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies.

    PubMed Central

    Zhao, R; Amsler, C D; Matsumura, P; Khan, S

    1996-01-01

    Salmonella typhimurium FliG and FliM are two of three proteins known to be necessary for flagellar morphogenesis as well as energization and switching of flagellar rotation. We have determined FliG and FliM levels in cellular fractions and in extended flagellar basal bodies, using antibodies raised against the purified proteins. Both proteins were found predominantly in the detergent-solubilized particulate fraction containing flagellar structures. Basal flagellar fragments could be separated from partially constructed basal bodies by gel filtration chromatography. FliG and FliM were present in an approximately equimolar ration in all gel-filtered fractions. FliG and FliM copy numbers, estimated relative to that of the hook protein from the early fractions containing long, basal, flagellar fragments, were (means +/- standard errors) 41 +/- 10 and 37 +/- 13 per flagellum, respectively. Extended structures were present in the earliest identifiable basal bodies. Immunoelectron microscopy and immunoblot gel analysis suggested that the FliG and, to a less certain degree, the FliM contents of these structures were the same as those for the complete basal bodies. These facts are consistent with the postulate that FliG and FliM affect flagellar morphogenesis as part of the extended basal structure, formation of which is necessary for assembly of more-distal components of the flagellum. The determined stoichiometries will provide important constraints to modelling energization and switching of flagellar rotation. PMID:8550426

  4. Differentiation of Salmonella phase 1 flagellar antigen types by restriction of the amplified fliC gene.

    PubMed Central

    Kilger, G; Grimont, P A

    1993-01-01

    The large antigenic diversity (over 2,300 serotypes) expressed by Salmonella strains can probably be observed at the genetic level. The phase 1 flagellin gene fliC was amplified, and the amplified fragment was cleaved with a mixture of both endonucleases TaqI and ScaI. The restriction patterns observed allowed differentiation of flagellar types b, i, d, j, l,v, and z10. Flagellar group g (g,m, g,p, or g,m,s) could be differentiated from the other flagellar types. Flagellar types r and e,h could not be separated, although they could be distinguished from the other flagellar types studied. Practical applications of flagellar gene restriction are the distinction between serotype Gallinarum-Pullorum, which carries a cryptic gene for flagellar type g,m, and nonmotile Vi-negative variants of serotype Typhi, and the tentative assignation of nonmotile variants of Salmonella serotypes to a flagellar type. Images PMID:8388886

  5. Language and Williams syndrome: how intact is "intact"?

    PubMed

    Karmiloff-Smith, A; Grant, J; Berthoud, I; Davies, M; Howlin, P; Udwin, O

    1997-04-01

    It has been claimed that Williams syndrome (WS), a rare neurodevelopmental disorder, is characterized by serious cognitive deficits alongside intact language. The syndrome is often used as a prime example of the modularity of an innate faculty for morphosyntactic rules. We challenge this claim and hypothesize that morphosyntax, although surprisingly good given WS level of mental retardation, is by no means intact. We make an initial test of this hypothesis through an analysis of the receptive language of a group of English-speaking WS individuals on a standardized morphosyntactic test. We then present an experimental study of expressive language that examines grammatical gender assignment in French-speaking WS patients. Despite a Verbal Mental Age selected to be higher than the chronological age of the young control group, these people with WS continue even in adulthood to show clear-cut deficits in their production of an aspect of morphosyntax that normal children acquire effortlessly very early. The results of the 2 studies, one focusing on receptive language and the other on expressive language, challenge the notion that comprehension and use of morphosyntactic rules in WS individuals are intact. The Within-domain dissociations regarding the use of grammatical gender assignment across several sentence clements and their difficulties in understanding embedded sentences-two quintessentially linguistic skills-suggest that we must rethink the notion of spared, modular, language capacities in Williams syndrome. We conclude that WS language follows a different path to normal acquisition and may turn out to be more like second language learning. PMID:9180000

  6. Zipping and entanglement in flagellar bundle of E. coli: Role of motile cell body

    NASA Astrophysics Data System (ADS)

    Adhyapak, Tapan Chandra; Stark, Holger

    2015-11-01

    The course of a peritrichous bacterium, such as E. coli, crucially depends on the level of synchronization and self-organization of several rotating flagella. However, the rotation of each flagellum generates countermovements of the body which in turn affect the flagellar dynamics. Using a detailed numerical model of an E. coli, we demonstrate that flagellar entanglement, besides fluid flow relative to the moving body, dramatically changes the dynamics of flagella from that compared to anchored flagella. In particular, bundle formation occurs through a zipping motion in a remarkably rapid time, affected little by initial flagellar orientation. A simplified analytical model supports our observations. Finally, we illustrate how entanglement, hydrodynamic interactions, and body movement contribute to zipping and bundling.

  7. Rotational symmetry of the C ring and a mechanism for the flagellar rotary motor

    PubMed Central

    Thomas, Dennis R.; Morgan, David Gene; DeRosier, David J.

    1999-01-01

    FliG, FliM, and FliN, key proteins for torque generation, are located in two rings. The first protein is in the M ring and the last two are in the C ring. The rotational symmetries of the C and M rings have been determined to be about 34 (this paper) and 26 (previous work), respectively. The mechanism proposed here depends on the symmetry mismatch between the rings: the C ring extends 34 levers, of which 26 can bind to the 26 equivalent sites on the M ring. The remaining 8 levers bind to proton–pore complexes (studs) to form 8 torque generators. Movement results from the swapping of stud-bound levers with M ring-bound levers. The model predicts that both the M and C rings rotate in the same direction but at different speeds. PMID:10468575

  8. Isolation and characterization of flagellar filaments from Bacillus cereus ATCC 14579.

    PubMed

    Tagawa, Yuichi

    2014-12-01

    Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length. PMID:25227778

  9. Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment

    PubMed Central

    Wilson, Corinne S.; Chang, Alex J.; Greene, Rebecca; Machado, Sulynn; Parsons, Matthew W.; Takats, Taylor A.; Zambetti, Luke J.; Springer, Amy L.

    2015-01-01

    Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei. PMID:26555902

  10. Listeria monocytogenes DNA Glycosylase AdlP Affects Flagellar Motility, Biofilm Formation, Virulence, and Stress Responses

    PubMed Central

    Zhang, Ting; Bae, Dongryeoul

    2016-01-01

    ABSTRACT The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is structurally similar to the Bacillus cereus alkyl base DNA glycosylase (AlkD), was identified. This determinant was involved in the transcriptional repression of flagellar motility genes and was named adlP (encoding an AlkD-like protein [AdlP]). Deletion of adlP activated the expression of flagellar motility genes at 37°C and disrupted the temperature-dependent inhibition of L. monocytogenes motility. The adlP null strains demonstrated decreased survival in murine macrophage-like RAW264.7 cells and less virulence in mice. Furthermore, the deletion of adlP significantly decreased biofilm formation and impaired the survival of bacteria under several stress conditions, including the presence of a DNA alkylation compound (methyl methanesulfonate), an oxidative agent (H2O2), and aminoglycoside antibiotics. Our findings strongly suggest that adlP may encode a bifunctional protein that transcriptionally represses the expression of flagellar motility genes and influences stress responses through its DNA glycosylase activity. IMPORTANCE We discovered a novel protein that we named AlkD-like protein (AdlP). This protein affected flagellar motility, biofilm formation, and virulence. Our data suggest that AdlP may be a bifunctional protein that represses flagellar motility genes and influences stress responses through its DNA glycosylase activity. PMID:27316964

  11. The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export

    PubMed Central

    Minamino, Tohru; Morimoto, Yusuke V.; Hara, Noritaka; Aldridge, Phillip D.; Namba, Keiichi

    2016-01-01

    The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+–protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration. PMID:26943926

  12. Characterization of polymer release from the flagellar pocket of Leishmania mexicana promastigotes.

    PubMed

    Stierhof, Y D; Ilg, T; Russell, D G; Hohenberg, H; Overath, P

    1994-04-01

    Trypanosomatids contain a unique compartment, the flagellar pocket, formed by an invagination of the plasma membrane at the base of the flagellum, which is considered to be the sole cellular site for endocytosis and exocytosis of macromolecules. The culture supernatant of Leishmania mexicana promastigotes, the insect stage of this protozoan parasite, contains two types of polymers: a filamentous acid phosphatase (sAP) composed of a 100-kD phosphoglycoprotein with non-covalently associated proteo high molecular weight phosphoglycan (proteo-HMWPG) and fibrous material termed network consisting of complex phosphoglycans. Secretion of both polymers is investigated using mAbs and a combination of light and electron microscopic techniques. Long filaments of sAP are detectable in the lumen of the flagellar pocket. Both sAP filaments and network material emerge from the ostium of the flagellar pocket. While sAP filaments detach from the cells, the fibrous network frequently remains associated with the anterior end of the parasites and can be found in the center of cell aggregates. The related species L. major forms similar networks. Since polymeric structures cannot be detected in intracellular compartments, it is proposed that monomeric or, possibly, oligomeric subunits synthesized in the cells are secreted into the flagellar pocket. Polymer formation from subunits is suggested to occur in the lumen of the pocket before release into the culture medium or, naturally, into the gut of infected sandflies. PMID:8163549

  13. Modulation of toxin production by the flagellar regulon in Clostridium difficile.

    PubMed

    Aubry, Annie; Hussack, Greg; Chen, Wangxue; KuoLee, Rhonda; Twine, Susan M; Fulton, Kelly M; Foote, Simon; Carrillo, Catherine D; Tanha, Jamshid; Logan, Susan M

    2012-10-01

    We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile. PMID:22851750

  14. Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation.

    PubMed

    Ishijima, Sumio

    2016-01-01

    It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements. PMID:26863204

  15. Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation

    PubMed Central

    Ishijima, Sumio

    2016-01-01

    It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements. PMID:26863204

  16. A novel non-homologous recombination-mediated mechanism for Escherichia coli unilateral flagellar phase variation.

    PubMed

    Liu, Bin; Hu, Bo; Zhou, Zhemin; Guo, Dan; Guo, Xi; Ding, Peng; Feng, Lu; Wang, Lei

    2012-05-01

    Flagella contribute to the virulence of bacteria through chemotaxis, adhesion to and invasion of host surfaces. Flagellar phase variation is believed to facilitate bacterial evasion of the host immune response. In this study, the flnA gene that encodes Escherichia coli H17 flagellin was examined by whole genome sequencing and genetic deletion analysis. Unilateral flagellar phase variation has been reported in E. coli H3, H47 and H17 strains, although the mechanism for phase variation in the H17 strain has not been previously understood. Analysis of phase variants indicated that the flagellar phase variation in the H17 strain was caused by the deletion of an ∼35 kb DNA region containing the flnA gene from diverse excision sites. The presence of covalently closed extrachromosomal circular forms of this excised 35 kb region was confirmed by the two-step polymerase chain reaction. The deletion and complementation test revealed that the Int1157 integrase, a tyrosine recombinase, mediates the excision of this region. Unlike most tyrosine recombinases, Int1157 is suggested to recognize diverse sites and mediate recombination between non-homologous DNA sequences. This is the first report of non-homologous recombination mediating flagellar phase variation. PMID:22287625

  17. Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems.

    PubMed

    Quelas, J Ignacio; Althabegoiti, M Julia; Jimenez-Sanchez, Celia; Melgarejo, Augusto A; Marconi, Verónica I; Mongiardini, Elías J; Trejo, Sebastián A; Mengucci, Florencia; Ortega-Calvo, José-Julio; Lodeiro, Aníbal R

    2016-01-01

    Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species. PMID:27053439

  18. Identification of flagellar motility genes in Yersinia ruckeri by transposon mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible molecular mechanism for the emergence of non-mot...

  19. Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems

    PubMed Central

    Quelas, J. Ignacio; Althabegoiti, M. Julia; Jimenez-Sanchez, Celia; Melgarejo, Augusto A.; Marconi, Verónica I.; Mongiardini, Elías J.; Trejo, Sebastián A.; Mengucci, Florencia; Ortega-Calvo, José-Julio; Lodeiro, Aníbal R.

    2016-01-01

    Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species. PMID:27053439

  20. Xenocin Export by the Flagellar Type III Pathway in Xenorhabdus nematophila

    PubMed Central

    Singh, Preeti; Park, Dongjin; Forst, Steven

    2013-01-01

    The xenocin operon of Xenorhabdus nematophila consists of xciA and ximB genes encoding a 64-kDa xenocin and 42-kDa immunity protein to kill competing microbes in the insect larva. The catalytic domain of xenocin has RNase activity and is responsible for its cytotoxicity. Under SOS conditions, xenocin is produced with immunity protein as a complex. Here, we show that xenocin and immunity protein complex are exported through the flagellar type III system of X. nematophila. Secretion of xenocin complex was abolished in an flhA strain but not in an fliC strain. The xenocin operon is not linked to the flagellar operon transcriptionally. The immunity protein is produced alone from a second, constitutive promoter and is targeted to the periplasm in a flagellum-independent manner. For stable expression of xenocin, coexpression of immunity protein was necessary. To examine the role of immunity protein in xenocin export, an enzymatically inactive protein was produced by site-directed mutagenesis in the active site of the catalytic domain. Toxicity was abolished in D535A and H538A variants of xenocin, which were expressed alone without an immunity domain and secreted in the culture supernatant through flagellar export. Secretion of xenocin through the flagellar pathway has important implications in the evolutionary success of X. nematophila. PMID:23335409

  1. Listeria monocytogenes DNA glycosylase AdiP affects flagellar motility, biofilm formation, virulence, and stress responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The temperature-dependent alteration of flagellar motility gene expression is critical for the foodborne pathogen Listeria monocytogenes to respond to a changing environment. In this study, a genetic determinant, L. monocytogenes f2365_0220 (lmof2365_0220), encoding a putative protein that is struct...

  2. Intact capture of hypervelocity projectiles.

    PubMed

    Tsou, P

    1990-01-01

    The ability to capture projectiles intact at hypervelocities opens new applications in science and technology that would either not be possible or would be very costly by other means. This capability has been demonstrated in the laboratory for aluminum projectiles of 1.6 mm diameter, captured at 6 km/s, in one unmelted piece, and retaining up to 95% of the original mass. Furthermore, capture was accomplished passively using microcellular underdense polymer foam. Another advantage of capturing projectiles in an underdense medium is the ability of such a medium to preserve a record of the projectile's original velocity components of speed and direction. A survey of these experimental results is described in terms of a dozen parameters which characterize the amount of capture and the effect on the projectile due to different capture media. PMID:11538362

  3. Isolation and characterization of flagellar filament from zoospores of Dermatophilus congolensis.

    PubMed

    Hiraizumi, Mieko; Tagawa, Yuichi

    2014-09-17

    Highly motile zoospores from Dermatophilus congolensis bovine isolates from clinical dermatophilosis in Japan were obtained by culturing at 27°C in an ambient atmosphere on heart infusion agar supplemented with 5% defibrinated sheep blood for 72h or in heart infusion broth for 48h with gentle shaking. After vigorous mechanical agitation of the zoospore suspension, the flagellar filaments detached from motile zoospores and were isolated in the clear gelatinous part of the final pellet by differential centrifugation. Typical morphology of a flagellar filament, with a width of approximately 15nm, was observed in the isolated flagellar filament by electron microscopy. A single major protein (flagellin) band with an apparent molecular mass of 35kDa was detected in the flagellar filament of D. congolensis strain AM-1 and that of 33kDa was detected in strain IT-2 by SDS-PAGE. In immunoblot analysis of whole-cell proteins from seven isolates of D. congolensis, antiserum to strain AM-1 zoospores reacted with the 35-kDa antigen band of strain AM-1, but not with any antigen band of other strains in a similar molecular mass range. In contrast, antiserum to strain IT-2 zoospores reacted with antigen bands at 33kDa from six strains, except strain AM-1. Similar strain-specific reactions of these anti-zoospore sera with isolated flagellar filaments from strains AM-1 and IT-2 were confirmed by immunoblot, indicating the presence of antigenic variations of flagellins of D. congolensis zoospores. PMID:25132009

  4. Periplasmic Flagellar Export Apparatus Protein, FliH, Is Involved in Post-Transcriptional Regulation of FlaB, Motility and Virulence of the Relapsing Fever Spirochete Borrelia hermsii

    PubMed Central

    Guyard, Cyril; Raffel, Sandra J.; Schrumpf, Merry E.; Dahlstrom, Eric; Sturdevant, Daniel; Ricklefs, Stacy M.; Martens, Craig; Hayes, Stanley F.; Fischer, Elizabeth R.; Hansen, Bryan T.; Porcella, Stephen F.; Schwan, Tom G.

    2013-01-01

    Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their helical or flat-wave morphology and motility. While most other bacteria rely on a transcriptional cascade to regulate the expression of motility genes, spirochetes employ post-transcriptional mechanism(s) that are only partially known. In the present study, we characterize a spontaneous non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in periplasmic flagella. We used next generation DNA sequencing of the mutant’s genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcription analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major periplasmic flagellar filament core protein FlaB. Despite the lack of FlaB, the amount of FlaA produced by the fliH mutant was similar to the wild-type level. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the post-transcriptional level by influencing the synthesis of FlaB. PMID:24009690

  5. Variation in motor output and motor performance in a centrally generated motor pattern

    PubMed Central

    Norris, Brian J.; Doloc-Mihu, Anca; Calabrese, Ronald L.

    2014-01-01

    Central pattern generators (CPGs) produce motor patterns that ultimately drive motor outputs. We studied how functional motor performance is achieved, specifically, whether the variation seen in motor patterns is reflected in motor performance and whether fictive motor patterns differ from those in vivo. We used the leech heartbeat system in which a bilaterally symmetrical CPG coordinates segmental heart motor neurons and two segmented heart tubes into two mutually exclusive coordination modes: rear-to-front peristaltic on one side and nearly synchronous on the other, with regular side-to-side switches. We assessed individual variability of the motor pattern and the beat pattern in vivo. To quantify the beat pattern we imaged intact adults. To quantify the phase relations between motor neurons and heart constrictions we recorded extracellularly from two heart motor neurons and movement from the corresponding heart segments in minimally dissected leeches. Variation in the motor pattern was reflected in motor performance only in the peristaltic mode, where larger intersegmental phase differences in the motor neurons resulted in larger phase differences between heart constrictions. Fictive motor patterns differed from those in vivo only in the synchronous mode, where intersegmental phase differences in vivo had a larger front-to-rear bias and were more constrained. Additionally, load-influenced constriction timing might explain the amplification of the phase differences between heart segments in the peristaltic mode and the higher variability in motor output due to body shape assumed in this soft-bodied animal. The motor pattern determines the beat pattern, peristaltic or synchronous, but heart mechanics influence the phase relations achieved. PMID:24717348

  6. Retinal ganglion cell axons regenerate in the presence of intact sensory fibres.

    PubMed

    King, Carolyn; Bartlett, Carole; Sauvé, Yves; Lund, Ray; Dunlop, Sarah; Beazley, Lyn

    2006-02-01

    A novel allograft paradigm was used to test whether adult mammalian central axons regenerate within a peripheral nerve environment containing intact sensory axons. Retinal ganglion cell axon regeneration was compared following anastomosis of dorsal root ganglia grafts or conventional peripheral nerve grafts to the adult rat optic nerve. Dorsal root ganglia grafts comprised intact sensory and degenerate motor axons, whereas conventional grafts comprised both degenerating sensory and motor axons. Retinal ganglion cell axons were traced after 2 months. Dorsal root ganglia survived with their axons persisting throughout the graft. Comparable numbers of retinal ganglion cells regenerated axons into both dorsal root ganglia (1053+/-223) and conventional grafts (1323+/-881; P>0.05). The results indicate that an intact sensory environment supports central axon regeneration. PMID:16407770

  7. Mechanics of intact bone marrow.

    PubMed

    Jansen, Lauren E; Birch, Nathan P; Schiffman, Jessica D; Crosby, Alfred J; Peyton, Shelly R

    2015-10-01

    The current knowledge of bone marrow mechanics is limited to its viscous properties, neglecting the elastic contribution of the extracellular matrix. To get a more complete view of the mechanics of marrow, we characterized intact yellow porcine bone marrow using three different, but complementary techniques: rheology, indentation, and cavitation. Our analysis shows that bone marrow is elastic, and has a large amount of intra- and inter-sample heterogeneity, with an effective Young׳s modulus ranging from 0.25 to 24.7 kPa at physiological temperature. Each testing method was consistent across matched tissue samples, and each provided unique benefits depending on user needs. We recommend bulk rheology to capture the effects of temperature on tissue elasticity and moduli, indentation for quantifying local tissue heterogeneity, and cavitation rheology for mitigating destructive sample preparation. We anticipate the knowledge of bone marrow elastic properties for building in vitro models will elucidate mechanisms involved in disease progression and regenerative medicine. PMID:26189198

  8. Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori

    PubMed Central

    Tsang, Jennifer

    2015-01-01

    ABSTRACT Flagellar biogenesis in Helicobacter pylori is regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ80), RpoN (σ54), and FliA (σ28), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaB and flgE) and FliA-dependent (flaA) genes. The MS ring (encoded by fliF) is one of the earliest flagellar structures assembled. Deletion of fliF resulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease in flaA transcript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions of fliH and genes encoding C ring components (fliM and fliY) decreased transcript levels of flaB and flgE but had little or no effect on transcript levels of flaA. Transcript levels of flaB and flgE were elevated in mutants where genes encoding rod proteins (fliE and flgBC) were deleted, while transcript levels of flaA was reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure. IMPORTANCE The mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of the H. pylori flagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these

  9. RpoE may promote flagellar gene expression in Salmonella enterica serovar typhi under hyperosmotic stress.

    PubMed

    Du, Hong; Sheng, Xiumei; Zhang, Haifang; Zou, Xin; Ni, Bin; Xu, Shungao; Zhu, Xueming; Xu, Huaxi; Huang, Xinxiang

    2011-02-01

    Salmonella enterica serovar Typhi z66 positive strain contains a fljBA-like operon on a linear plasmid. The operon contains the gene fljB:z66 which encodes the z66 antigen. RpoE is a sigma factor σ(E) that initiates transcription of a series of genes in Escherichia and Salmonella under environmental stresses. To investigate whether the gene fljB:z66 is regulated by RpoE (σ(E)), a rpoE deletion mutant of S. enterica serovar Typhi (ΔrpoE) was prepared in this study. The defective motility of the ΔrpoE was confirmed firstly. Transcriptional expression of flagellar genes was screened using a genomic DNA microarray. Some class-2 and most class-3 flagellar genes were downregulated in the ΔrpoE after 30 min of hyperosmotic stress. The expression of fliA and fljB:z66, a class-2 flagellar gene and a class-3 flagellar gene, obviously decreased; however, expression of the class-1 flagellar genes flhDC did not change obviously in the ΔrpoE compared to the wild-type strain in the same conditions. Results of quantitative real-time PCR (qRT-PCR) showed that the expression levels of fliA and fljB:z66 in the ΔrpoE after 30 min of hyperosmotic stress decreased about five and eightfold, respectively, compared to the wild-type strain. Similar results were observed at 120 min of hyperosmotic stress. Western blotting and qRT-PCR analysis showed that expression of fliA and fljB:z66 was significantly increased after supplemental expression of rpoE with a recombinant plasmid pBADrpoE in the ΔrpoE strain. These results demonstrated that RpoE promoted the expression of class-3 flagellar genes and it might be performed by initiating the expression of fliA in S. enterica serovar Typhi under hyperosmotic stress. PMID:20717675

  10. Actin-interacting and flagellar proteins in Leishmania spp.: Bioinformatics predictions to functional assignments in phagosome formation

    PubMed Central

    2009-01-01

    Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented. PMID:21637533

  11. Actin-interacting and flagellar proteins in Leishmania spp.: Bioinformatics predictions to functional assignments in phagosome formation.

    PubMed

    Diniz, Michely C; Costa, Marcília P; Pacheco, Ana C L; Kamimura, Michel T; Silva, Samara C; Carneiro, Laura D G; Sousa, Ana P L; Soares, Carlos E A; Souza, Celeste S F; de Oliveira, Diana Magalhães

    2009-07-01

    Several motile processes are responsible for the movement of proteins into and within the flagellar membrane, but little is known about the process by which specific proteins (either actin-associated or not) are targeted to protozoan flagellar membranes. Actin is a major cytoskeleton protein, while polymerization and depolymerization of parasite actin and actin-interacting proteins (AIPs) during both processes of motility and host cell entry might be key events for successful infection. For a better understanding the eukaryotic flagellar dynamics, we have surveyed genomes, transcriptomes and proteomes of pathogenic Leishmania spp. to identify pertinent genes/proteins and to build in silico models to properly address their putative roles in trypanosomatid virulence. In a search for AIPs involved in flagellar activities, we applied computational biology and proteomic tools to infer from the biological meaning of coronins and Arp2/3, two important elements in phagosome formation after parasite phagocytosis by macrophages. Results presented here provide the first report of Leishmania coronin and Arp2/3 as flagellar proteins that also might be involved in phagosome formation through actin polymerization within the flagellar environment. This is an issue worthy of further in vitro examination that remains now as a direct, positive bioinformatics-derived inference to be presented. PMID:21637533

  12. Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amoebae into flagellates

    SciTech Connect

    Lee, J.H.; Walsh, C.J.

    1988-06-01

    The nuclear run-on technique was used to measure the rate of transcription of flagellar genes during the differentiation of Naegleria gruberi amebae into flagellates. Synthesis of mRNAs for the axonemal proteins ..cap alpha..- and BETA-tubulin and flagellar calmodulin, as well as a coordinately regulated poly(A)/sup +/ RNA that codes for an unidentified protein, showed transient increases averaging 22-fold. The rate of synthesis of two poly(A)/sup +/ RNAs common to ameobae and flagellates was low until the transcription of the flagellar genes began to decline, at which time synthesis of the RNAs found in ameobae increased 3- to 10-fold. The observed changes in the rate of transcription can account quantitatively for the 20-fold increase in flagellar mRNA concentration during the differentiation. The data for the flagellar calmodulin gene demonstrate transcriptional regulation for a nontubulin axonemal protein. The data also demonstrate at least two programs of transcriptional regulation during the differentiation and raise the intriguing possibility that some significant fraction of the nearly 200 different proteins of the flagellar axoneme is transcriptionally regulated during the 1 h it takes N. gruberi amebae to form visible flagella.

  13. Tubulin-dynein system in flagellar and ciliary movement

    PubMed Central

    MOHRI, Hideo; INABA, Kazuo; ISHIJIMA, Sumio; BABA, Shoji A.

    2012-01-01

    Eukaryotic flagella and cilia have attracted the attention of many researchers over the last century, since they are highly arranged organelles and show sophisticated bending movements. Two important cytoskeletal and motor proteins, tubulin and dynein, were first found and described in flagella and cilia. Half a century has passed since the discovery of these two proteins, and much information has been accumulated on their molecular structures and their roles in the mechanism of microtubule sliding, as well as on the architecture, the mechanism of bending movement and the regulation and signal transduction in flagella and cilia. Historical background and the recent advance in this field are described. PMID:23060230

  14. The phylogeny of swimming kinematics: The environment controls flagellar waveforms in sperm motility

    NASA Astrophysics Data System (ADS)

    Guasto, Jeffrey; Burton, Lisa; Zimmer, Richard; Hosoi, Anette; Stocker, Roman

    2013-11-01

    In recent years, phylogenetic and molecular analyses have dominated the study of ecology and evolution. However, physical interactions between organisms and their environment, a fundamental determinant of organism ecology and evolution, are mediated by organism form and function, highlighting the need to understand the mechanics of basic survival strategies, including locomotion. Focusing on spermatozoa, we combined high-speed video microscopy and singular value decomposition analysis to quantitatively compare the flagellar waveforms of eight species, ranging from marine invertebrates to humans. We found striking similarities in sperm swimming kinematics between genetically dissimilar organisms, which could not be uncovered by phylogenetic analysis. The emergence of dominant waveform patterns across species are suggestive of biological optimization for flagellar locomotion and point toward environmental cues as drivers of this convergence. These results reinforce the power of quantitative kinematic analysis to understand the physical drivers of evolution and as an approach to uncover new solutions for engineering applications, such as micro-robotics.

  15. Amphipathic helical ordering of the flagellar secretion signal of Salmonella flagellin.

    PubMed

    Tőke, Orsolya; Vonderviszt, Ferenc

    2016-08-01

    Export of external flagellar proteins requires a signal located within their N-terminal disordered part, however, these regions do not share any significant sequence similarity suggesting that the secondary/tertiary structure might be important for recognition by the export gate. NMR experiments were performed to reveal the conformational properties of the flagellin signal sequence in vitro. It assumed a largely disordered fluctuating structure in aqueous environment, but acquired a folded structure containing an amphipathic helical portion in 50% MeOH or upon addition of SDS micelles which are known to promote hydrophobic interactions. Our observations raise the possibility that the signal sequence may partially undergo amphipathic helical ordering upon interaction with the recognition unit of the flagellar export machinery in a similar way as revealed for protein import into intracellular eukaryotic organelles mediated by targeting signals of high diversity. PMID:27264954

  16. Flagellar filament bio-templated inorganic oxide materials - towards an efficient lithium battery anode

    NASA Astrophysics Data System (ADS)

    Beznosov, Sergei N.; Veluri, Pavan S.; Pyatibratov, Mikhail G.; Chatterjee, Abhijit; Macfarlane, Douglas R.; Fedorov, Oleg V.; Mitra, Sagar

    2015-01-01

    Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g-1 after 50 cycles and with high rate capability, delivering 770 mAh g-1 at 5 A g-1 (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future.

  17. Flagellar filament bio-templated inorganic oxide materials – towards an efficient lithium battery anode

    PubMed Central

    Beznosov, Sergei N.; Veluri, Pavan S.; Pyatibratov, Mikhail G.; Chatterjee, Abhijit; MacFarlane, Douglas R.; Fedorov, Oleg V.; Mitra, Sagar

    2015-01-01

    Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g−1 after 50 cycles and with high rate capability, delivering 770 mAh g−1 at 5 A g−1 (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future. PMID:25583370

  18. Flagellar filament bio-templated inorganic oxide materials - towards an efficient lithium battery anode.

    PubMed

    Beznosov, Sergei N; Veluri, Pavan S; Pyatibratov, Mikhail G; Chatterjee, Abhijit; MacFarlane, Douglas R; Fedorov, Oleg V; Mitra, Sagar

    2015-01-01

    Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g(-1) after 50 cycles and with high rate capability, delivering 770 mAh g(-1) at 5 A g(-1) (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future. PMID:25583370

  19. A chemotactic signaling surface on CheY defined by suppressors of flagellar switch mutations.

    PubMed Central

    Roman, S J; Meyers, M; Volz, K; Matsumura, P

    1992-01-01

    CheY is the response regulator protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The flagellar switch apparatus is a complex macromolecular structure composed of at least three gene products, FliG, FliM, and FliN. Genetic analysis of Escherichia coli has identified fliG and fliM as genes in which mutations occur that allele specifically suppress cheY mutations, indicating interactions among these gene products. We have generated a class of cheY mutations selected for dominant suppression of fliG mutations. Interestingly, these cheY mutations dominantly suppressed both fliG and fliM mutations; this is consistent with the idea that the CheY protein interacts with both switch gene products during signaling. Biochemical characterization of wild-type and suppressor CheY proteins did not reveal altered phosphorylation properties or evidence for phosphorylation-dependent CheY multimerization. These data indicate that suppressor CheY proteins are specifically altered in the ability to transduce chemotactic signals to the switch at some point subsequent to phosphorylation. Physical mapping of suppressor amino acid substitutions on the crystal structure of CheY revealed a high degree of spatial clustering, suggesting that this region of CheY is a signaling surface that transduces chemotactic signals to the switch. Images PMID:1400175

  20. Cross talk between type III secretion and flagellar assembly systems in Pseudomonas aeruginosa.

    PubMed

    Soscia, Chantal; Hachani, Abderrahman; Bernadac, Alain; Filloux, Alain; Bleves, Sophie

    2007-04-01

    Pseudomonas aeruginosa cytotoxicity is linked to a type III secretion system (T3SS) that delivers effectors into the host cell. We show here that a negative cross-control exists between T3SS and flagellar assembly. We observed that, in a strain lacking flagella, T3SS gene expression, effector secretion, and cytotoxicity were increased. Conversely, we revealed that flagellar-gene expression and motility were decreased in a strain overproducing ExsA, the T3SS master regulator. Interestingly, a nonmotile strain lacking the flagellar filament (DeltafliC) presented a hyperefficient T3SS and a nonmotile strain assembling flagella (DeltamotAB) did not. More intriguingly, a strain lacking motCD genes is a flagellated strain with a slight defect in swimming. However, in this strain, T3SS gene expression was up-regulated. These results suggest that flagellar assembly and/or mobility antagonizes the T3SS and that a negative cross talk exists between these two systems. An illustration of this is the visualization by electron microscopy of T3SS needles in a nonmotile P. aeruginosa strain, needles which otherwise are not detected. The molecular basis of the cross talk is complex and remains to be elucidated, but proteins like MotCD might have a crucial role in signaling between the two processes. In addition, we found that the GacA response regulator negatively affects the T3SS. In a gacA mutant, the T3SS effector ExoS is hypersecreted. Strikingly, GacA was previously reported as a positive regulator for motility. Globally, our data document the idea that some virulence factors are coordinately but inversely regulated, depending on the bacterial colonization phase and infection types. PMID:17307856

  1. A developmentally regulated Caulobacter flagellar promoter is activated by 3' enhancer and IHF binding elements.

    PubMed Central

    Gober, J W; Shapiro, L

    1992-01-01

    The transcription of a group of flagellar genes is temporally and spatially regulated during the Caulobacter crescentus cell cycle. These genes all share the same 5' cis-regulatory elements: a sigma 54 promoter, a binding site for integration host factor (IHF), and an enhancer sequence, known as the ftr element. We have partially purified the ftr-binding proteins, and we show that they require the same enhancer sequences for binding as are required for transcriptional activation. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. The IHF protein and the ftr-binding protein is primarily restricted to the predivisional cell, the cell type in which these promoters are transcribed. flaNQ promoter expression is localized to the swarmer pole of the predivisional cell, as are other flagellar promoters that possess these regulatory sequences 5' to the start site. The requirement for an IHF binding site and an ftr-enhancer element in spatially transcribed flagellar promoters indicates that a common mechanism may be responsible for both temporal and polar transcription. Images PMID:1392079

  2. Flagellar phenotypic plasticity in volvocalean algae correlates with Péclet number.

    PubMed

    Solari, Cristian A; Drescher, Knut; Ganguly, Sujoy; Kessler, John O; Michod, Richard E; Goldstein, Raymond E

    2011-10-01

    Flagella-generated fluid stirring has been suggested to enhance nutrient uptake for sufficiently large micro-organisms, and to have played a role in evolutionary transitions to multicellularity. A corollary to this predicted size-dependent benefit is a propensity for phenotypic plasticity in the flow-generating mechanism to appear in large species under nutrient deprivation. We examined four species of volvocalean algae whose radii and flow speeds differ greatly, with Péclet numbers (Pe) separated by several orders of magnitude. Populations of unicellular Chlamydomonas reinhardtii and one- to eight-celled Gonium pectorale (Pe ∼ 0.1-1) and multicellular Volvox carteri and Volvox barberi (Pe ∼ 100) were grown in diluted and undiluted media. For C. reinhardtii and G. pectorale, decreasing the nutrient concentration resulted in smaller cells, but had no effect on flagellar length and propulsion force. In contrast, these conditions induced Volvox colonies to grow larger and increase their flagellar length, separating the somatic cells further. Detailed studies on V. carteri found that the opposing effects of increasing beating force and flagellar spacing balance, so the fluid speed across the colony surface remains unchanged between nutrient conditions. These results lend further support to the hypothesized link between the Péclet number, nutrient uptake and the evolution of biological complexity in the Volvocales. PMID:21367778

  3. Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

    PubMed Central

    Stadler, Andreas M.; Unruh, Tobias; Namba, Keiichi; Samatey, Fadel; Zaccai, Giuseppe

    2013-01-01

    The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits. PMID:24209861

  4. Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

    PubMed Central

    Singer, Hanna M.; Erhardt, Marc; Steiner, Andrew M.; Zhang, Min-Min; Yoshikami, Doju; Bulaj, Grzegorz; Olivera, Baldomero M.; Hughes, Kelly T.

    2012-01-01

    ABSTRACT The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA. PMID:22647788

  5. Flagellar Biosynthesis Exerts Temporal Regulation of Secretion of Specific Campylobacter jejuni Colonization and Virulence Determinants

    PubMed Central

    Barrero-Tobon, Angelica M.; Hendrixson, David R.

    2014-01-01

    Summary The Campylobacter jejuni flagellum exports both proteins that form the flagellar organelle for swimming motility and colonization and virulence factors that promote commensal colonization of the avian intestinal tract or invasion of human intestinal cells, respectively. We explored how the C. jejuni flagellum is a versatile secretory organelle by examining molecular determinants that allow colonization and virulence factors to exploit the flagellum for their own secretion. Flagellar biogenesis was observed to exert temporal control of secretion of these proteins, indicating that a bolus of secretion of colonization and virulence factors occurs during hook biogenesis with filament polymerization itself reducing secretion of these factors. Furthermore, we found that intramolecular and intermolecular requirements for flagellar-dependent secretion of these proteins were most reminiscent to those for flagellin secretion. Importantly, we discovered that secretion of one colonization and virluence factor, CiaI, was not required for invasion of human colonic cells, which counters previous hypotheses for how this protein functions during invasion. Instead, secretion of CiaI was essential for C. jejuni to facilitate commensal colonization of the natural avian host. Our work provides insight into the versatility of the bacterial flagellum as a secretory machine that can export proteins promoting diverse biological processes. PMID:25041103

  6. Flagellar cells and ciliary cells in the renal tubule of elasmobranchs.

    PubMed

    Lacy, E R; Luciano, L; Reale, E

    1989-01-01

    Flagella or cilia are present on most epithelial cells in the renal tubule of elasmobranch fishes (little skate, spiny dogfish, smooth dogfish, Atlantic sharpnose, scalloped hammerhead, cow-nosed ray). Flagellar cells, those with numerous flagella ordered in one, two, or more rows on the luminal surface, are shown here for the first time in a vertebrate. The flagellar cells are intercalated among other epithelial cells, each bearing a single cilium, from Bowman's capsule to the third subdivision of the intermediate segment of the nephron. The flagella form undulated ribbons up to 55 microns long. In every ribbon the axis of the central pair of microtubules in the axoneme is oriented parallel to the long axis of the flagellar row. This suggests a beat perpendicular to these two axes. The arrangement of the flagella in ribbons most likely promotes movement of glomerular filtrate down the renal tubule. Cells bearing numerous cilia occur in the large collecting ducts of spiny dogfish but without apparent preferential orientation of the cilia. PMID:2575649

  7. Bimodal rheotactic behavior reflects flagellar beat asymmetry in human sperm cells

    PubMed Central

    Bukatin, Anton; Kukhtevich, Igor; Stoop, Norbert; Dunkel, Jörn; Kantsler, Vasily

    2015-01-01

    Rheotaxis, the directed response to fluid velocity gradients, has been shown to facilitate stable upstream swimming of mammalian sperm cells along solid surfaces, suggesting a robust physical mechanism for long-distance navigation during fertilization. However, the dynamics by which a human sperm orients itself relative to an ambient flow is poorly understood. Here, we combine microfluidic experiments with mathematical modeling and 3D flagellar beat reconstruction to quantify the response of individual sperm cells in time-varying flow fields. Single-cell tracking reveals two kinematically distinct swimming states that entail opposite turning behaviors under flow reversal. We constrain an effective 2D model for the turning dynamics through systematic large-scale parameter scans, and find good quantitative agreement with experiments at different shear rates and viscosities. Using a 3D reconstruction algorithm to identify the flagellar beat patterns causing left or right turning, we present comprehensive 3D data demonstrating the rolling dynamics of freely swimming sperm cells around their longitudinal axis. Contrary to current beliefs, this 3D analysis uncovers ambidextrous flagellar waveforms and shows that the cell’s turning direction is not defined by the rolling direction. Instead, the different rheotactic turning behaviors are linked to a broken mirror symmetry in the midpiece section, likely arising from a buckling instability. These results challenge current theoretical models of sperm locomotion. PMID:26655343

  8. Complex spatial organization and flagellin composition of flagellar propeller from marine magnetotactic ovoid strain MO-1.

    PubMed

    Zhang, Wei-Jia; Santini, Claire-Lise; Bernadac, Alain; Ruan, Juanfang; Zhang, Sheng-Da; Kato, Takayuki; Li, Ying; Namba, Keiichi; Wu, Long-Fei

    2012-03-01

    Marine magnetotactic ovoid bacterium MO-1 is capable of swimming along the geomagnetic field lines by means of its two sheathed flagellar bundles at a speed up to 300 μm/s. In this study, by using electron microscopy, we showed that, in each bundle, six individual flagella were organized in hexagon with a seventh in the middle. We identified 12 flagellin paralogs and 2 putative flagellins in the genome of MO-1. Among them, 13 were tandemly located on an ~ 17-kb segment while the 14th was on a separated locus. Using reverse transcription PCR and quantitative PCR, we found that all the 14 flagellin or putative flagellin genes were transcribed and that 2 of them were more abundantly expressed than others. A nLC (nanoliquid chromatography)-ESI (electrospray ionization)-MS/MS (mass spectrometry/mass spectrometry) mass spectrometry analysis identified all the 12 flagellin proteins in three glycosylated polypeptide bands resolved by one-dimensional denaturing polyacrylamide gel electrophoresis and 10 of them in 21 spots obtained by means of two-dimensional electrophoresis of flagellar extracts. Most spots contained more than one flagellin, and eight of the ten identified flagellins existed in multiple isoforms. Taken together, these results show unprecedented complexity in the spatial organization and flagellin composition of the flagellar propeller. Such architecture is observed only for ovoid-coccoid, bilophotrichously flagellated magnetotactic bacteria living in marine sediments, suggesting a species and environmental specificity. PMID:22245577

  9. Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly.

    PubMed

    Meshcheryakov, Vladimir A; Wolf, Matthias

    2016-06-01

    Archaeal flagella are unique structures that share functional similarity with bacterial flagella, but are structurally related to bacterial type IV pili. The flagellar accessory protein FlaH is one of the conserved components of the archaeal motility system. However, its function is not clearly understood. Here, we present the 2.2 Å resolution crystal structure of FlaH from the hyperthermophilic archaeon, Methanocaldococcus jannaschii. The protein has a characteristic RecA-like fold, which has been found previously both in archaea and bacteria. We show that FlaH binds to immobilized ATP-however, it lacks ATPase activity. Surface plasmon resonance analysis demonstrates that ATP affects the interaction between FlaH and the archaeal motor protein FlaI. In the presence of ATP, the FlaH-FlaI interaction becomes significantly weaker. A database search revealed similarity between FlaH and several DNA-binding proteins of the RecA superfamily. The closest structural homologs of FlaH are KaiC-like proteins, which are archaeal homologs of the circadian clock protein KaiC from cyanobacteria. We propose that one of the functions of FlaH may be the regulation of archaeal motor complex assembly. PMID:27060465

  10. The FLA3 KAP Subunit Is Required for Localization of Kinesin-2 to the Site of Flagellar Assembly and Processive Anterograde Intraflagellar TransportV⃞

    PubMed Central

    Mueller, Joshua; Perrone, Catherine A.; Bower, Raqual; Cole, Douglas G.; Porter, Mary E.

    2005-01-01

    Intraflagellar transport (IFT) is a bidirectional process required for assembly and maintenance of cilia and flagella. Kinesin-2 is the anterograde IFT motor, and Dhc1b/Dhc2 drives retrograde IFT. To understand how either motor interacts with the IFT particle or how their activities might be coordinated, we characterized a ts mutation in the Chlamydomonas gene encoding KAP, the nonmotor subunit of Kinesin-2. The fla3-1 mutation is an amino acid substitution in a conserved C-terminal domain. fla3-1 strains assemble flagella at 21°C, but cannot maintain them at 33°C. Although the Kinesin-2 complex is present at both 21 and 33°C, the fla3-1 Kinesin-2 complex is not efficiently targeted to or retained in the basal body region or flagella. Video-enhanced DIC microscopy of fla3-1 cells shows that the frequency of anterograde IFT particles is significantly reduced. Anterograde particles move at near wild-type velocities, but appear larger and pause more frequently in fla3-1. Transformation with an epitope-tagged KAP gene rescues all of the fla3-1 defects and results in preferential incorporation of tagged KAP complexes into flagella. KAP is therefore required for the localization of Kinesin-2 at the site of flagellar assembly and the efficient transport of anterograde IFT particles within flagella. PMID:15616187

  11. Conditional Granger Causality Analysis of Effective Connectivity during Motor Imagery and Motor Execution in Stroke Patients

    PubMed Central

    Wang, Li; Zhang, Jingna; Zhang, Ye; Yan, Rubing; Liu, Hongliang; Qiu, Mingguo

    2016-01-01

    Aims. Motor imagery has emerged as a promising technique for the improvement of motor function following stroke, but the mechanism of functional network reorganization in patients during this process remains unclear. The aim of this study is to evaluate the cortical motor network patterns of effective connectivity in stroke patients. Methods. Ten stroke patients with right hand hemiplegia and ten normal control subjects were recruited. We applied conditional Granger causality analysis (CGCA) to explore and compare the functional connectivity between motor execution and motor imagery. Results. Compared with the normal controls, the patient group showed lower effective connectivity to the primary motor cortex (M1), the premotor cortex (PMC), and the supplementary motor area (SMA) in the damaged hemisphere but stronger effective connectivity to the ipsilesional PMC and M1 in the intact hemisphere during motor execution. There were tighter connections in the cortical motor network in the patients than in the controls during motor imagery, and the patients showed more effective connectivity in the intact hemisphere. Conclusions. The increase in effective connectivity suggests that motor imagery enhances core corticocortical interactions, promotes internal interaction in damaged hemispheres in stroke patients, and may facilitate recovery of motor function. PMID:27200373

  12. Singly Flagellated Pseudomonas aeruginosa Chemotaxes Efficiently by Unbiased Motor Regulation

    PubMed Central

    Cai, Qiuxian; Li, Zhaojun; Ouyang, Qi

    2016-01-01

    ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen that has long been known to chemotax. More recently, it has been established that chemotaxis is an important factor in the ability of P. aeruginosa to make biofilms. Genes that allow P. aeruginosa to chemotax are homologous with genes in the paradigmatic model organism for chemotaxis, Escherichia coli. However, P. aeruginosa is singly flagellated and E. coli has multiple flagella. Therefore, the regulation of counterclockwise/clockwise flagellar motor bias that allows E. coli to efficiently chemotax by runs and tumbles would lead to inefficient chemotaxis by P. aeruginosa, as half of a randomly oriented population would respond to a chemoattractant gradient in the wrong sense. How P. aeruginosa regulates flagellar rotation to achieve chemotaxis is not known. Here, we analyze the swimming trajectories of single cells in microfluidic channels and the rotations of cells tethered by their flagella to the surface of a variable-environment flow cell. We show that P. aeruginosa chemotaxes by symmetrically increasing the durations of both counterclockwise and clockwise flagellar rotations when swimming up the chemoattractant gradient and symmetrically decreasing rotation durations when swimming down the chemoattractant gradient. Unlike the case for E. coli, the counterclockwise/clockwise bias stays constant for P. aeruginosa. We describe P. aeruginosa’s chemotaxis using an analytical model for symmetric motor regulation. We use this model to do simulations that show that, given P. aeruginosa’s physiological constraints on motility, its distinct, symmetric regulation of motor switching optimizes chemotaxis. PMID:27048795

  13. Evolved to fail: Bacteria induce flagellar buckling to reorient

    NASA Astrophysics Data System (ADS)

    Son, Kwangmin; Guasto, Jeffrey S.; Stocker, Roman

    2012-11-01

    Many marine bacteria swim with a single helical flagellum connected to a rotary motor via a 100 nm long universal joint called the ``hook.'' While these bacteria have seemingly just one degree of freedom, allowing them to swim only back and forth, they in fact exhibit large angular reorientations mediated by off-axis ``flicks'' of their flagellum. High-speed video microscopy revealed the mechanism underpinning this turning behavior: the buckling of the hook during the exceedingly brief (10 ms) forward run that follows a reversal. Direct measurements of the hook's mechanical properties corroborated this result, as the hook's structural stability is governed by the Sperm number, which compares the compressive load from propulsion to the elastic restoring force of the hook. Upon decreasing the Sperm number below a critical value by reducing the swimming speed, the frequency of flicks diminishes sharply, consistent with the criticality of buckling. This elegant, under-actuated turning mechanism appears widespread among marine bacteria and may provide a novel design concept in micro-robotics.

  14. Neural activation differences in amputees during imitation of intact versus amputee movements

    PubMed Central

    Cusack, William F.; Cope, Michael; Nathanson, Sheryl; Pirouz, Nikta; Kistenberg, Robert; Wheaton, Lewis A.

    2012-01-01

    The mirror neuron system (MNS) has been attributed with increased activation in motor-related cortical areas upon viewing of another's actions. Recent work suggests that limb movements that are similar and dissimilar in appearance to that of the viewer equivalently activate the MNS. It is unclear if this result can be observed in the action encoding areas in amputees who use prosthetic devices. Intact subjects and upper extremity amputee prosthesis users were recruited to view video demonstrations of tools being used by an intact actor and a prosthetic device user. All subjects pantomimed the movements seen in the video while recording electroencephalography (EEG). Intact subjects showed equivalent left parietofrontal activity during imitation planning after watching the intact or prosthetic arm. Likewise, when prosthesis users imitated prosthesis demonstrations, typical left parietofrontal activation was observed. When prosthesis users imitated intact actors, an additional pattern was revealed which showed greater activity in right parietal and occipital regions that are associated with the mentalizing system. This change may be required for prosthesis users to plan imitation movements in which the limb states between the observed and the observer do not match. The finding that prosthesis users imitating other prosthesis users showed typical left parietofrontal activation suggests that these subjects engage normal planning related activity when they are able to imitate a limb matching their own. This result has significant implications on rehabilitation, as standard therapy involves training with an intact occupational therapist, which could necessitate atypical planning mechanisms in amputees when learning to use their prosthesis. PMID:22754516

  15. Flagellar Cap Protein FliD Mediates Adherence of Atypical Enteropathogenic Escherichia coli to Enterocyte Microvilli.

    PubMed

    Sampaio, Suely C F; Luiz, Wilson B; Vieira, Mônica A M; Ferreira, Rita C C; Garcia, Bruna G; Sinigaglia-Coimbra, Rita; Sampaio, Jorge L M; Ferreira, Luís C S; Gomes, Tânia A T

    2016-04-01

    The expression of flagella correlates with different aspects of bacterial pathogenicity, ranging from adherence to host cells to activation of inflammatory responses by the innate immune system. In the present study, we investigated the role of flagella in the adherence of an atypical enteropathogenic Escherichia coli (aEPEC) strain (serotype O51:H40) to human enterocytes. Accordingly, isogenic mutants deficient in flagellin (FliC), the flagellar structural subunit; the flagellar cap protein (FliD); or the MotAB proteins, involved in the control of flagellar motion, were generated and tested for binding to differentiated Caco-2 cells. Binding of the aEPEC strain to enterocytes was significantly impaired in strains with the fliCa nd fliD genes deleted, both of which could not form flagella on the bacterial surface. A nonmotile but flagellated MotAB mutant also showed impaired adhesion to Caco-2 cells. In accordance with these observations, adhesion of a EPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of a EPEC bacteria with specific anti-FliD serum impaired binding to Caco-2 cells. Finally, incubation of Caco-2 cells with purified FliD, followed by immunolabeling, showed that the protein was specifically bound to the microvillus tips of differentiated Caco-2 cells. The a EPEC FliD or anti-FliD serum also reduced the adherence of prototype typical enteropathogenic, enterohemorrhagic, and enterotoxigenic E. coli strains to Caco-2 cells. In conclusion, our findings further strengthened the role of flagella in the adherence of a EPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process. PMID:26831466

  16. Two Distinct Ca2+ Signaling Pathways Modulate Sperm Flagellar Beating Patterns in Mice1

    PubMed Central

    Chang, Haixin; Suarez, Susan S.

    2011-01-01

    Hyperactivation, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization. It is characterized by asymmetrical flagellar beating and an increase of cytoplasmic Ca2+. We observed that some mouse sperm swimming in the oviduct produce high-amplitude pro-hook bends (bends in the direction of the hook on the head), whereas other sperm produce high-amplitude anti-hook bends. Switching direction of the major bends could serve to redirect sperm toward oocytes. We hypothesized that different Ca2+ signaling pathways produce high-amplitude pro-hook and anti-hook bends. In vitro, sperm that hyperactivated during capacitation (because of activation of CATSPER plasma membrane Ca2+ channels) developed high-amplitude pro-hook bends. The CATSPER activators procaine and 4-aminopyridine (4-AP) also induced high-amplitude pro-hook bends. Thimerosal, which triggers a Ca2+ release from internal stores, induced high-amplitude anti-hook bends. Activation of CATSPER channels is facilitated by a pH rise, so both Ca2+ and pH responses to treatments with 4-AP and thimerosal were monitored. Thimerosal triggered a Ca2+ increase that initiated at the base of the flagellum, whereas 4-AP initiated a rise in the proximal principal piece. Only 4-AP triggered a flagellar pH rise. Proteins were extracted from sperm for examination of phosphorylation patterns induced by Ca2+ signaling. Procaine and 4-AP induced phosphorylation of proteins on threonine and serine, whereas thimerosal primarily induced dephosphorylation of proteins. Tyrosine phosphorylation was unaffected. We concluded that hyperactivation, which is associated with capacitation, can be modulated by release of Ca2+ from intracellular stores to reverse the direction of the dominant flagellar bend and, thus, redirect sperm. PMID:21389347

  17. Adenine Nucleotide Metabolism and a Role for AMP in Modulating Flagellar Waveforms in Mouse Sperm1

    PubMed Central

    Vadnais, Melissa L.; Cao, Wenlei; Aghajanian, Haig K.; Haig-Ladewig, Lisa; Lin, Angel M.; Al-Alao, Osama; Gerton, George L.

    2014-01-01

    ABSTRACT While most ATP, the main energy source driving sperm motility, is derived from glycolysis and oxidative phosphorylation, the metabolic demands of the cell require the efficient use of power stored in high-energy phosphate bonds. In times of high energy consumption, adenylate kinase (AK) scavenges one ATP molecule by transphosphorylation of two molecules of ADP, simultaneously yielding one molecule of AMP as a by-product. Either ATP or ADP supported motility of detergent-modeled cauda epididymal mouse sperm, indicating that flagellar AKs are functional. However, the ensuing flagellar waveforms fueled by ATP or ADP were qualitatively different. Motility driven by ATP was rapid but restricted to the distal region of the sperm tail, whereas ADP produced slower and more fluid waves that propagated down the full flagellum. Characterization of wave patterns by tracing and superimposing the images of the flagella, quantifying the differences using digital image analysis, and computer-assisted sperm analysis revealed differences in the amplitude, periodicity, and propagation of the waves between detergent-modeled sperm treated with either ATP or ADP. Surprisingly, addition of AMP to the incubation medium containing ATP recapitulated the pattern of sperm motility seen with ADP alone. In addition to AK1 and AK2, which we previously demonstrated are present in outer dense fibers and mitochondrial sheath of the mouse sperm tail, we show that another AK, AK8, is present in a third flagellar compartment, the axoneme. These results extend the known regulators of sperm motility to include AMP, which may be operating through an AMP-activated protein kinase. PMID:24740601

  18. Analysis of an engineered Salmonella flagellar fusion protein, FliR-FlhB.

    PubMed

    Van Arnam, John S; McMurry, Jonathan L; Kihara, May; Macnab, Robert M

    2004-04-01

    Salmonella FliR and FlhB are membrane proteins necessary for flagellar export. In Clostridium a fliR-flhB fusion gene exists. We constructed a similar Salmonella fusion gene which is able to complement fliR, flhB, and fliR flhB null strains. Western blotting revealed that the FliR-FlhB fusion protein retains the FlhB protein's cleavage properties. We conclude that the FliR and FlhB proteins are physically associated in the wild-type Salmonella basal body, probably in a 1:1 ratio. PMID:15060055

  19. Basic timing abilities stay intact in patients with musician's dystonia.

    PubMed

    van der Steen, M C; van Vugt, Floris T; Keller, Peter E; Altenmüller, Eckart

    2014-01-01

    Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15) and a matched control group (N = 15) on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo). In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument. PMID:24667273

  20. Basic Timing Abilities Stay Intact in Patients with Musician's Dystonia

    PubMed Central

    van der Steen, M. C.; van Vugt, Floris T.; Keller, Peter E.; Altenmüller, Eckart

    2014-01-01

    Task-specific focal dystonia is a movement disorder that is characterized by the loss of voluntary motor control in extensively trained movements. Musician's dystonia is a type of task-specific dystonia that is elicited in professional musicians during instrumental playing. The disorder has been associated with deficits in timing. In order to test the hypothesis that basic timing abilities are affected by musician's dystonia, we investigated a group of patients (N = 15) and a matched control group (N = 15) on a battery of sensory and sensorimotor synchronization tasks. Results did not show any deficits in auditory-motor processing for patients relative to controls. Both groups benefited from a pacing sequence that adapted to their timing (in a sensorimotor synchronization task at a stable tempo). In a purely perceptual task, both groups were able to detect a misaligned metronome when it was late rather than early relative to a musical beat. Overall, the results suggest that basic timing abilities stay intact in patients with musician's dystonia. This supports the idea that musician's dystonia is a highly task-specific movement disorder in which patients are mostly impaired in tasks closely related to the demands of actually playing their instrument. PMID:24667273

  1. Chlamydomonas IFT70/CrDYF-1 is a core component of IFT particle complex B and is required for flagellar assembly.

    PubMed

    Fan, Zhen-Chuan; Behal, Robert H; Geimer, Stefan; Wang, Zhaohui; Williamson, Shana M; Zhang, Haili; Cole, Douglas G; Qin, Hongmin

    2010-08-01

    DYF-1 is a highly conserved protein essential for ciliogenesis in several model organisms. In Caenorhabditis elegans, DYF-1 serves as an essential activator for an anterograde motor OSM-3 of intraflagellar transport (IFT), the ciliogenesis-required motility that mediates the transport of flagellar precursors and removal of turnover products. In zebrafish and Tetrahymena DYF-1 influences the cilia tubulin posttranslational modification and may have more ubiquitous function in ciliogenesis than OSM-3. Here we address how DYF-1 biochemically interacts with the IFT machinery by using the model organism Chlamydomonas reinhardtii, in which the anterograde IFT does not depend on OSM-3. Our results show that this protein is a stoichiometric component of the IFT particle complex B and interacts directly with complex B subunit IFT46. In concurrence with the established IFT protein nomenclature, DYF-1 is also named IFT70 after the apparent size of the protein. IFT70/CrDYF-1 is essential for the function of IFT in building the flagellum because the flagella of IFT70/CrDYF-1-depleted cells were greatly shortened. Together, these results demonstrate that IFT70/CrDYF-1 is a canonical subunit of IFT particle complex B and strongly support the hypothesis that the IFT machinery has species- and tissue-specific variations with functional ramifications. PMID:20534810

  2. Metachronal waves in the flagellar beating of Volvox and their hydrodynamic origin.

    PubMed

    Brumley, Douglas R; Polin, Marco; Pedley, Timothy J; Goldstein, Raymond E

    2015-07-01

    Groups of eukaryotic cilia and flagella are capable of coordinating their beating over large scales, routinely exhibiting collective dynamics in the form of metachronal waves. The origin of this behavior--possibly influenced by both mechanical interactions and direct biological regulation--is poorly understood, in large part due to a lack of quantitative experimental studies. Here we characterize in detail flagellar coordination on the surface of the multicellular alga Volvox carteri, an emerging model organism for flagellar dynamics. Our studies reveal for the first time that the average metachronal coordination observed is punctuated by periodic phase defects during which synchrony is partial and limited to specific groups of cells. A minimal model of hydrodynamically coupled oscillators can reproduce semi-quantitatively the characteristics of the average metachronal dynamics, and the emergence of defects. We systematically study the model's behaviour by assessing the effect of changing intrinsic rotor characteristics, including oscillator stiffness and the nature of their internal driving force, as well as their geometric properties and spatial arrangement. Our results suggest that metachronal coordination follows from deformations in the oscillators' limit cycles induced by hydrodynamic stresses, and that defects result from sufficiently steep local biases in the oscillators' intrinsic frequencies. Additionally, we find that random variations in the intrinsic rotor frequencies increase the robustness of the average properties of the emergent metachronal waves. PMID:26040592

  3. Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

    PubMed

    Juhas, Mario; Evans, Lewis D B; Frost, Joe; Davenport, Peter W; Yarkoni, Orr; Fraser, Gillian M; Ajioka, James W

    2014-01-01

    E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site. PMID:25350000

  4. Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits

    PubMed Central

    Juhas, Mario; Evans, Lewis D. B.; Frost, Joe; Davenport, Peter W.; Yarkoni, Orr; Fraser, Gillian M.; Ajioka, James W.

    2014-01-01

    E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site. PMID:25350000

  5. Metachronal waves in the flagellar beating of Volvox and their hydrodynamic origin

    PubMed Central

    Brumley, Douglas R.; Polin, Marco; Pedley, Timothy J.; Goldstein, Raymond E.

    2015-01-01

    Groups of eukaryotic cilia and flagella are capable of coordinating their beating over large scales, routinely exhibiting collective dynamics in the form of metachronal waves. The origin of this behaviour—possibly influenced by both mechanical interactions and direct biological regulation—is poorly understood, in large part due to a lack of quantitative experimental studies. Here we characterize in detail flagellar coordination on the surface of the multicellular alga Volvox carteri, an emerging model organism for flagellar dynamics. Our studies reveal for the first time that the average metachronal coordination observed is punctuated by periodic phase defects during which synchrony is partial and limited to specific groups of cells. A minimal model of hydrodynamically coupled oscillators can reproduce semi-quantitatively the characteristics of the average metachronal dynamics, and the emergence of defects. We systematically study the model's behaviour by assessing the effect of changing intrinsic rotor characteristics, including oscillator stiffness and the nature of their internal driving force, as well as their geometric properties and spatial arrangement. Our results suggest that metachronal coordination follows from deformations in the oscillators' limit cycles induced by hydrodynamic stresses, and that defects result from sufficiently steep local biases in the oscillators' intrinsic frequencies. Additionally, we find that random variations in the intrinsic rotor frequencies increase the robustness of the average properties of the emergent metachronal waves. PMID:26040592

  6. Differentiation of the major flagellar antigens of Pseudomonas aeruginosa by the slide coagglutination technique.

    PubMed

    Ansorg, R A; Knoche, M E; Spies, A F; Kraus, C J

    1984-07-01

    Antisera against the two major flagellar antigens of Pseudomonas aeruginosa were obtained by immunization of rabbits with isolated flagella and absorption of contaminating antisomatic antibodies. In the conventional slide agglutination test, the pure H antisera did not agglutinate the flagellated cells of the homologous strains. The addition of protein A-bearing staphylococci to H antiserum and homologous flagellated cells, the so-called slide coagglutination, results in a rapid development of flaky clumps. H coagglutination tests of reference strains, which formerly have been H typed by long-term tube agglutination and by the indirect fluorescent-antibody technique, yielded exactly the same subdivision of the strains in H type a and H type b as the more laborious and time-consuming methods. O grouping and H typing of 181 isolates from clinical specimens revealed a free combination of the somatic and flagellar antigens. 25 OH serovars were found. The simple and rapid coagglutination technique can promote the serovar determination of P. aeruginosa, particularly for the purpose of hospital infection control. PMID:6430957

  7. DksA and ppGpp Directly Regulate Transcription of the Escherichia coli Flagellar Cascade

    PubMed Central

    Lemke, Justin J.; Durfee, Tim; Gourse, Richard L.

    2009-01-01

    The components of the Escherichia coli flagella apparatus are synthesized in a three-level transcriptional cascade activated by the master regulator FlhDC. The cascade coordinates the synthesis rates of a large number of gene products with each other and with nutritional conditions. Recent genome-wide studies have reported that flagellar transcription is altered in cells lacking the transcription regulators DksA or ppGpp, but some or all reported effects could be indirect, and some are contradictory. We report here that the activities of promoters at all three levels of the cascade are much higher in strains lacking dksA, resulting in overproduction of flagellin and hyperflagellated cells. In vitro, DksA/ppGpp inhibits the flhDC promoter and the σ70-dependent fliA promoter transcribing the gene for σ28. However, DksA and ppGpp do not affect the σ28-dependent fliA promoter or the σ28-dependent fliC promoter in vitro, suggesting that the dramatic effects on expression of those genes in vivo are mediated indirectly through direct effects of DksA/ppGpp on FlhDC and σ28 expression. We conclude that DksA/ppGpp inhibits expression of the flagellar cascade during stationary phase and following starvation, thereby coordinating flagella and ribosome assembly and preventing expenditure of scarce energy resources on synthesis of two of the cell’s largest macromolecular complexes. PMID:19889089

  8. The Trypanosome Flagellar Pocket Collar and Its Ring Forming Protein—TbBILBO1

    PubMed Central

    Perdomo, Doranda; Bonhivers, Mélanie; Robinson, Derrick R.

    2016-01-01

    Sub-species of Trypanosoma brucei are the causal agents of human African sleeping sickness and Nagana in domesticated livestock. These pathogens have developed an organelle-like compartment called the flagellar pocket (FP). The FP carries out endo- and exocytosis and is the only structure this parasite has evolved to do so. The FP is essential for parasite viability, making it an interesting structure to evaluate as a drug target, especially since it has an indispensible cytoskeleton component called the flagellar pocket collar (FPC). The FPC is located at the neck of the FP where the flagellum exits the cell. The FPC has a complex architecture and division cycle, but little is known concerning its organization. Recent work has focused on understanding how the FP and the FPC are formed and as a result of these studies an important calcium-binding, polymer-forming protein named TbBILBO1 was identified. Cellular biology analysis of TbBILBO1 has demonstrated its uniqueness as a FPC component and until recently, it was unknown what structural role it played in forming the FPC. This review summarizes the recent data on the polymer forming properties of TbBILBO1 and how these are correlated to the FP cytoskeleton. PMID:26950156

  9. The Trypanosome Flagellar Pocket Collar and Its Ring Forming Protein-TbBILBO1.

    PubMed

    Perdomo, Doranda; Bonhivers, Mélanie; Robinson, Derrick R

    2016-01-01

    Sub-species of Trypanosoma brucei are the causal agents of human African sleeping sickness and Nagana in domesticated livestock. These pathogens have developed an organelle-like compartment called the flagellar pocket (FP). The FP carries out endo- and exocytosis and is the only structure this parasite has evolved to do so. The FP is essential for parasite viability, making it an interesting structure to evaluate as a drug target, especially since it has an indispensible cytoskeleton component called the flagellar pocket collar (FPC). The FPC is located at the neck of the FP where the flagellum exits the cell. The FPC has a complex architecture and division cycle, but little is known concerning its organization. Recent work has focused on understanding how the FP and the FPC are formed and as a result of these studies an important calcium-binding, polymer-forming protein named TbBILBO1 was identified. Cellular biology analysis of TbBILBO1 has demonstrated its uniqueness as a FPC component and until recently, it was unknown what structural role it played in forming the FPC. This review summarizes the recent data on the polymer forming properties of TbBILBO1 and how these are correlated to the FP cytoskeleton. PMID:26950156

  10. A “Mechanistic” Explanation of the Multiple Helical Forms Adopted by Bacterial Flagellar Filaments

    PubMed Central

    Calladine, C.R.; Luisi, B.F.; Pratap, J.V.

    2013-01-01

    The corkscrew-like flagellar filaments emerging from the surface of bacteria such as Salmonella typhimurium propel the cells toward nutrient and away from repellents. This kind of motility depends upon the ability of the flagellar filaments to adopt a range of distinct helical forms. A filament is typically constructed from ~ 30,000 identical flagellin molecules, which self-assemble into a tubular structure containing 11 near-longitudinal protofilaments. A “mechanical” model, in which the flagellin building block has the capacity to switch between two principal interfacial states, predicts that the filament can assemble into a “canonical” family of 12 distinct helical forms, each having unique curvature and twist: these include two “extreme” straight forms having left- and right-handed twists, respectively, and 10 intermediate helical forms. Measured shapes of the filaments correspond well with predictions of the model. This report is concerned with two unanswered questions. First, what properties of the flagellin determine which of the 12 discrete forms is preferred? Second, how does the interfacial “switch” work, at a molecular level? Our proposed solution of these problems is based mainly on a detailed examination of differences between the available electron cryo-microscopy structures of the straight L and R filaments, respectively. PMID:23274110