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1.
Terasawa S Fukuoka H Inoue Y Sagawa T Takahashi H Ishijima A 《Biophysical journal》2011,(9):2193-2200
An Escherichia coli cell transduces extracellular stimuli sensed by chemoreceptors to the state of an intracellular signal molecule, which regulates the switching of the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) and from CW back to CCW. Here, we performed high-speed imaging of flagellar motor rotation and show that the switching of two different motors on a cell is controlled coordinatedly by an intracellular signal protein, phosphorylated CheY (CheY-P). The switching is highly coordinated with a subsecond delay between motors in clear correlation with the distance of each motor from the chemoreceptor patch localized at a cell pole, which would be explained by the diffusive motion of CheY-P molecules in the cell. The coordinated switching becomes disordered by the expression of a constitutively active CheY mutant that mimics the CW-rotation stimulating function. The coordinated switching requires CheZ, which is the phosphatase for CheY-P. Our results suggest that a transient increase and decrease in the concentration of CheY-P caused by a spontaneous burst of its production by the chemoreceptor patch followed by its dephosphorylation by CheZ, which is probably a wavelike propagation in a subsecond timescale, triggers and regulates the coordinated switching of flagellar motors. 相似文献
2.
Isolation of the polar and lateral flagellum-defective mutants in Vibrio alginolyticus and identification of their flagellar driving energy sources. 总被引:7,自引:4,他引:3
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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. 相似文献
3.
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. 相似文献
4.
Effect of viscosity on swimming by the lateral and polar flagella of Vibrio alginolyticus. 总被引:3,自引:2,他引:1
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T Atsumi Y Maekawa T Yamada I Kawagishi Y Imae M Homma 《Journal of bacteriology》1996,178(16):5024-5026
By using mutants of Vibrio alginolyticus with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+), we examined the relationship between swimming speed and the viscosity of the medium for each flagellar system. Pof+ Laf- cells could not swim in the high-viscosity environment (ca. 200 cP) in which Pof- Laf+ cells swam at 20 microns/s. The Pof- Laf+ cells swam at about 20 microns/s at normal viscosity (1 cP) without the viscous agent, and the speed increased to 40 microns/s at about 5 cP and then decreased gradually as the viscosity was increased further. These results show the functional difference between polar and lateral flagella in viscous environments. 相似文献
5.
Identification of a bacterial sensing protein and effects of its elevated expression. 总被引:19,自引:13,他引:6
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The Escherichia coli flaA gene product (also called cheC) plays a crucial role in switching flagellar rotational direction during chemotactic responses. Wild-type and mutant alleles have been cloned onto plasmid vectors, and the gene product has been identified as a 37,000-dalton protein. The flaA product appeared as a soluble protein in the cytoplasm when overproduced in minicells and maxicells. The protein could not be detected in flagellar basal structures purified from a wild-type strain. To assess the effects of altered flaA expression, the gene was fused to a synthetic tac promoter that could be regulated by the addition of an inducer. Overproduction resulted in strong counterclockwise flagellar rotational bias and partial paralysis of flagellar motors. These results suggest that the flaA protein provides the interface between the flagellar machinery and the chemotaxis signaling system in a motor structure external to the basal body. 相似文献
6.
Phosphorylation-independent bacterial chemoresponses correlate with changes in the cytoplasmic level of fumarate. 总被引:4,自引:0,他引:4
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Bacterial chemotaxis is based on modulation of the probability to switch the direction of flagellar rotation. Responses to many stimuli are transduced by a two-component system via reversible phosphorylation of CheY, a small cytoplasmic protein that directly interacts with the switch complex at the flagellar motor. We found that the chemorepellents indole and benzoate induce motor switching in Escherichia coli cells with a disabled phosphorylation cascade. This phosphorylation-independent chemoresponse is explained by reversible inhibition of fumarase by indole or benzoate which leads to an increased level of cellular fumarate, a compound involved in motor switching for bacteria and archaea. Genetic deletion of fumarase increased the intracellular concentration of fumarate and enhanced the switching frequency of the flagellar motors irrespective of the presence or absence of the phosphorylation cascade. These correlations provide evidence for fumarate-dependent metabolic signal transduction in bacterial chemosensing. 相似文献
7.
Only one of the five CheY homologs in Vibrio cholerae directly switches flagellar rotation
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Hyakutake A Homma M Austin MJ Boin MA Häse CC Kawagishi I 《Journal of bacteriology》2005,187(24):8403-8410
Vibrio cholerae has three sets of chemotaxis (Che) proteins, including three histidine kinases (CheA) and four response regulators (CheY) that are encoded by three che gene clusters. We deleted the cheY genes individually or in combination and found that only the cheY3 deletion impaired chemotaxis, reinforcing the previous conclusion that che cluster II is involved in chemotaxis. However, this does not exclude the involvement of the other clusters in chemotaxis. In other bacteria, phospho-CheY binds directly to the flagellar motor to modulate its rotation, and CheY overexpression, even without CheA, causes extremely biased swimming behavior. We reasoned that a V. cholerae CheY homolog, if it directly controls flagellar rotation, should also induce extreme swimming behavior when overproduced. This was the case for CheY3 (che cluster II). However, no other CheY homolog, including the putative CheY (CheY0) protein encoded outside the che clusters, affected swimming, demonstrating that these CheY homologs cannot act directly on the flagellar motor. CheY4 very slightly enhanced the spreading of an Escherichia coli cheZ mutant in semisolid agar, raising the possibility that it can affect chemotaxis by removing a phosphoryl group from CheY3. We also found that V. cholerae CheY3 and E. coli CheY are only partially exchangeable. Mutagenic analyses suggested that this may come from coevolution of the interacting pair of proteins, CheY and the motor protein FliM. Taken together, it is likely that the principal roles of che clusters I and III as well as cheY0 are to control functions other than chemotaxis. 相似文献
8.
Martin Schuster Walid N. Abouhamad Ruth E. Silversmith & Robert B. Bourret 《Molecular microbiology》1998,27(5):1065-1075
CheY, a response regulator protein in bacterial chemotaxis, mediates swimming behaviour through interaction with the flagellar switch protein, FliM. In its active, phosphorylated state, CheY binds to the motor switch complex and induces a change from counterclockwise (CCW) to clockwise (CW) flagellar rotation. The conformation of a conserved aromatic residue, tyrosine 106, has been proposed to play an important role in this signalling process. Here, we show that an isoleucine to valine substitution in CheY at position 95 — in close proximity to residue 106 — results in an extremely CW, hyperactive phenotype that is dependent on phosphorylation. Further biochemical characterization of this mutant protein revealed phosphorylation and dephosphorylation rates that were indistinguishable from those of wild-type CheY. CheY95IV, however, exhibited an increased binding affinity to FliM. Taken together, these results show for the first time a correlation between enhanced switch binding and constitutive signalling in bacterial chemotaxis. Considering present structural information, we also propose possible models for the role of residue 95 in the mechanism of CheY signal transduction. 相似文献
9.
Intracellular Na+ kinetically interferes with the rotation of the Na(+)-driven flagellar motors of Vibrio alginolyticus 总被引:3,自引:0,他引:3
S Yoshida S Sugiyama Y Hojo H Tokuda Y Imae 《The Journal of biological chemistry》1990,265(33):20346-20350
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. 相似文献
10.
Using self-trapped Escherichia coli bacteria that have intact flagellar bundles on glass surfaces, we study statistical fluctuations of cell-body rotation in a steady (unstimulated) state. These fluctuations underline direction randomization of bacterial swimming trajectories and plays a fundamental role in bacterial chemotaxis. A parallel study is also conducted using a classical rotation assay in which cell-body rotation is driven by a single flagellar motor. These investigations allow us to draw the important conclusion that during periods of counterclockwise motor rotation, which contributes to a run, all flagellar motors are strongly correlated, but during the clockwise period, which contributes to a tumble, individual motors are uncorrelated in long times. Our observation is consistent with the physical picture that formation and maintenance of a coherent flagellar bundle is provided by a single dominant flagellum in the bundle. 相似文献
11.
Putative channel components for the fast-rotating sodium-driven flagellar motor of a marine bacterium. 总被引:14,自引:7,他引:7
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Y Asai S Kojima H Kato N Nishioka I Kawagishi M Homma 《Journal of bacteriology》1997,179(16):5104-5110
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. 相似文献
12.
Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. Bacteria move using rotating helical flagellar filaments. The flagellar motor is located at the base of the filament and is buried in the cytoplasmic membrane. Flagellar motors are classified into two types according to the coupling ion: namely the H(+)-driven motor and the Na(+)-driven motor. Analysis of the flagellar motor at the molecular level is far more advanced in the H(+)-driven motor than in the Na(+)-driven motor. Recently, the genes of the Na(+)-driven motor have been cloned from a marine bacterium of Vibrio sp. and some of the motor proteins have been purified and characterized. In this review, we summarize recent studies of the Na(+)-driven flagellar motor. 相似文献
13.
Bacterial flagellar motors use specific ion gradients to drive their rotation. It has been suggested that the electrostatic interactions between charged residues of the stator and rotor proteins are important for rotation in Escherichia coli. Mutational studies have indicated that the Na(+)-driven motor of Vibrio alginolyticus may incorporate interactions similar to those of the E. coli motor, but the other electrostatic interactions between the rotor and stator proteins may occur in the Na(+)-driven motor. Thus, we investigated the C-terminal charged residues of the stator protein, PomA, in the Na(+)-driven motor. Three of eight charge-reversing mutations, PomA(K203E), PomA(R215E), and PomA(D220K), did not confer motility either with the motor of V. alginolyticus or with the Na(+)-driven chimeric motor of E. coli. Overproduction of the R215E and D220K mutant proteins but not overproduction of the K203E mutant protein impaired the motility of wild-type V. alginolyticus. The R207E mutant conferred motility with the motor of V. alginolyticus but not with the chimeric motor of E. coli. The motility with the E211K and R232E mutants was similar to that with wild-type PomA in V. alginolyticus but was greatly reduced in E. coli. Suppressor analysis suggested that R215 may participate in PomA-PomA interactions or PomA intramolecular interactions to form the stator complex. 相似文献
14.
Genetic evidence for a switching and energy-transducing complex in the flagellar motor of Salmonella typhimurium. 总被引:54,自引:36,他引:18
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S Yamaguchi S Aizawa M Kihara M Isomura C J Jones R M Macnab 《Journal of bacteriology》1986,168(3):1172-1179
The flaAII.2, flaQ, and flaN genes of Salmonella typhimurium are important for assembly, rotation, and counterclockwise-clockwise switching of the flagellar motor. Paralyzed and nonchemotactic mutants were subjected to selection pressure for partial acquisition of motility and chemotaxis, and the suppressor mutations of the resulting pseudorevertants were mapped and isolated. Many of the intergenic suppressor mutations were in one of the other two genes. Others were in genes for cytoplasmic components of the chemotaxis system, notably cheY and cheZ; one of the mutations was found in the cheA gene and one in a motility gene, motB. Suppression among the three fla genes was allele specific, and many of the pseudorevertants were either cold sensitive or heat sensitive. We conclude that the FlaAII.2, FlaQ, and FlaN proteins form a complex which determines the rotational sense, either counterclockwise or clockwise, of the motor and also participates in the conversion of proton energy into mechanical work of rotation. This switch complex is probably mounted to the base of the flagellar basal body and, via binding of the CheY and CheZ proteins, receives sensory information and uses it to control flagellar operation. 相似文献
15.
Control of bacterial chemotaxis 总被引:8,自引:3,他引:5
Michael Eisenbach 《Molecular microbiology》1996,20(5):903-910
Bacterial chemotaxis, which has been extensively studied for three decades, is the most prominent model system for signal transduction in bacteria. Chemotaxis is achieved by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein, CheY. This protein is activated by a stimulus-dependent phosphorylation mediated by an autophosphorylatable kinase (CheA) whose activity is controlled by chemoreceptors. Upon phosphorylation, CheY dissociates from its kinase, binds to the switch at the base of the flagellar motor, and changes the motor rotation from the default direction (counter-clockwise) to clockwise. Phosphorylation may also be involved in terminating the response. Phosphorylated CheY binds to the phosphatase CheZ and modulates its oligomeric state and thereby its dephosphorylating activity. Thus CheY phosphorylation appears to be involved in controlling both the excitation and adaptation mechanisms of bacterial chemotaxis. Additional control sites might be involved in bacterial chemotaxis, e.g. lateral control at the receptor level, control at the motor level, or control by metabolites that link central metabolism with chemotaxis. 相似文献
16.
Collin M. Dyer 《Journal of molecular biology》2009,388(1):71-17176
The high-resolution structures of nearly all the proteins that comprise the bacterial flagellar motor switch complex have been solved; yet a clear picture of the switching mechanism has not emerged. Here, we used NMR to characterize the interaction modes and solution properties of a number of these proteins, including several soluble fragments of the flagellar motor proteins FliM and FliG, and the response-regulator CheY. We find that activated CheY, the switch signal, binds to a previously unidentified region of FliM, adjacent to the FliM-FliM interface. We also find that activated CheY and FliG bind with mutual exclusivity to this site on FliM, because their respective binding surfaces partially overlap. These data support a model of CheY-driven motor switching wherein the binding of activated CheY to FliM displaces the carboxy-terminal domain of FliG (FliGC) from FliM, modulating the FliGC-MotA interaction, and causing the motor to switch rotational sense as required for chemotaxis. 相似文献
17.
Regulation of Switching Frequency and Bias of the Bacterial Flagellar Motor by CheY and Fumarate 总被引:5,自引:1,他引:4
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Marco Montrone Michael Eisenbach Dieter Oesterhelt Wolfgang Marwan 《Journal of bacteriology》1998,180(13):3375-3380
The effect of CheY and fumarate on switching frequency and rotational bias of the bacterial flagellar motor was analyzed by computer-aided tracking of tethered Escherichia coli. Plots of cells overexpressing CheY in a gutted background showed a bell-shaped correlation curve of switching frequency and bias centering at about 50% clockwise rotation. Gutted cells (i.e., with cheA to cheZ deleted) with a low CheY level but a high cytoplasmic fumarate concentration displayed the same correlation of switching frequency and bias as cells overexpressing CheY at the wild-type fumarate level. Hence, a high fumarate level can phenotypically mimic CheY overexpression by simultaneously changing the switching frequency and the bias. A linear correlation of cytoplasmic fumarate concentration and clockwise rotation bias was found and predicts exclusively counterclockwise rotation without switching when fumarate is absent. This suggests that (i) fumarate is essential for clockwise rotation in vivo and (ii) any metabolically induced fluctuation of its cytoplasmic concentration will result in a transient change in bias and switching probability. A high fumarate level resulted in a dose-response curve linking bias and cytoplasmic CheY concentration that was offset but with a slope similar to that for a low fumarate level. It is concluded that fumarate and CheY act additively presumably at different reaction steps in the conformational transition of the switch complex from counterclockwise to clockwise motor rotation. 相似文献
18.
Many kinds of peritrichous bacteria that repeat runs and tumbles by using multiple flagella exhibit chemotaxis by sensing a difference in the concentration of the attractant or repellent between two adjacent time points. If a cell senses that the concentration of an attractant has increased, their flagellar motors decrease the switching frequency from counterclockwise to clockwise direction of rotation, which causes a longer run in swimming up the concentration gradient than swimming down. We investigated the turn angle in tumbles of peritrichous bacteria swimming across the concentration gradient of a chemoattractant because the change in the switching frequency in the rotational direction may affect the way tumbles. We tracked several hundreds of runs and tumbles of single cells of Salmonella enterica serovar Typhimurium in the concentration gradient of L-serine and found that the turn angle depends on the concentration gradient that the cell senses just before the tumble. The turn angle is biased toward a smaller value when the cells swim up the concentration gradient, whereas the distribution of the angle is almost uniform (random direction) when the cells swim down the gradient. The effect of the observed bias in the turn angle on the degree of chemotaxis was investigated by random walk simulation. In the concentration field where attractants diffuse concentrically from the point source, we found that this angular distribution clearly affects the reduction of the mean-square displacement of the cell that has started at the attractant source, that is, the bias in the turn angle distribution contributes to chemotaxis in peritrichous bacteria. 相似文献
19.
Strains missing several genes required for chemotaxis toward amino acids, peptides, and certain sugars were tethered and their rotational behavior was analyzed. Null strains (called gutted) were deleted for genes that code for the transducers Tsr, Tar, Tap, and Trg and for the cytoplasmic proteins CheA, CheW, CheR, CheB, CheY, and CheZ. Motor switch components were wild type, flaAII(cheC), or flaBII(cheV). Gutted cells with wild-type motors spun exclusively counterclockwise, while those with mutant motors changed their directions of rotation. CheY reduced the bias (the fraction of time that cells spun counterclockwise) in either case. CheZ offset the effect of CheY to an extent that varied with switch allele but did not change the bias when tested alone. Transducers also increased the bias in the presence of CheY but not when tested alone. However, cells containing transducers and CheY failed to respond to attractants or repellents normally detected in the periplasm. This sensitivity was restored by addition of CheA and CheW. Thus, CheY both enhances clockwise rotation and couples the transducers to the flagella. CheZ acts, at the level of the motor, as a CheY antagonist. CheA or CheW or both are required to complete the signal pathway. A model is presented that explains these results and is consistent with other data found in the literature. 相似文献
20.
The chemotaxis system, but not chemotaxis, is essential for swarming motility in Salmonella enterica serovar Typhimurium. Mutants in the chemotaxis pathway exhibit fewer and shorter flagella, downregulate class 3 or 'late' motility genes, and appear to be less hydrated when propagated on a surface. We show here that the output of the chemotaxis system, CheY approximately P, modulates motor bias during swarming as it does during chemotaxis, but for a distinctly different end. A constitutively active form of CheY was found to promote swarming in the absence of several upstream chemotaxis components. Two point mutations that suppressed the swarming defect of a cheY null mutation mapped to FliM, a protein in the motor switch complex with which CheY approximately P interacts. A common property of these suppressors was their increased frequency of motor reversal. These and other data suggest that the ability to switch motor direction is important for promoting optimal surface wetness. If the surface is sufficiently wet, exclusively clockwise or counterclockwise directions of motor rotation will support swarming, suggesting also that the bacteria can move on a surface with flagellar bundles of either handedness. 相似文献