Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor.

Phosphorylation of the chemotaxis protein CheY by its kinase CheA appears to play a central role in the process of signal transduction in bacterial chemotaxis. It is presumed that the role is activation of CheY which results in clockwise (CW) flagellar rotation. The aim of this study was to determine whether this activity of CheY indeed depends on the protein being phosphorylated. Since the phosphorylation of CheY can be detected only in vitro, we studied the ability of CheY to cause CW rotation in an in vitro system, consisting of cytoplasm-free envelopes of Salmonella typhimurium or Escherichia coli having functional flagella. Envelopes containing just buffer rotated only counterclockwise. Inclusion of CheY caused 14% of the rotating envelopes to go CW. This fraction of CW-rotating envelopes was not altered when the phosphate potential in the envelopes was lowered by inclusion of ADP together with CheY in them, indicating that CheY has a certain degree of activity even without being phosphorylated. Attempts to increase the activity of CheY in the envelopes by phosphorylation were not successful. However, when CheY was inserted into partially-lysed cells (semienvelopes) under phosphorylating conditions, the number of CW-rotating cells increased 3-fold. This corresponds to more than a 100-fold increase in the activity of a single CheY molecule upon phosphorylation. It is concluded that nonphosphorylated CheY can interact with the flagellar switch and cause CW rotation, but that this activity is increased by at least 2 orders of magnitude by phosphorylation. This increase in activity requires additional cytoplasmic constituents, the identity of which is not yet known.     

Structural diversity of bacterial flagellar motors

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.     

Temperature dependence of switching of the bacterial flagellar motor by the protein CheY(13DK106YW).

The behavior of the bacterium Escherichia coli is controlled by switching of the flagellar rotary motor between the two rotational states, clockwise (CW) and counterclockwise (CCW). The molecular mechanism for switching remains unknown, but binding of the response regulator CheY-P to the motor component FliM enhances CW rotation. This effect is mimicked by the unphosphorylated double mutant CheY13DK106YW (CheY**). To learn more about switching, we measured the fraction of time that a motor spends in the CW state (the CW bias) at different concentrations of CheY** and at different temperatures. From the CW bias, we computed the standard free energy change of switching. In the absence of CheY, this free energy change is a linear function of temperature (. Biophys. J. 71:2227-2233). In the presence of CheY**, it is nonlinear. However, the data can be fit by models in which binding of each molecule of CheY** shifts the difference in free energy between CW and CCW states by a fixed amount. The shift increases linearly from approximately 0.3kT per molecule at 5 degrees C to approximately 0.9kT at 25 degrees C, where k is Boltzmann's constant and T is 289 Kelvin (= 16 degrees C). The entropy and enthalpy contributions to this shift are about -0. 031kT/ degrees C and 0.10kT, respectively.     

Mechanical limits of bacterial flagellar motors probed by electrorotation.

We used the technique of electrorotation to apply steadily increasing external torque to tethered cells of the bacterium Escherichia coli while continuously recording the speed of cell rotation. We found that the bacterial flagellar motor generates constant torque when rotating forward at low speeds and constant but considerably higher torque when rotating backward. At intermediate torques, the motor stalls. The torque-speed relationship is the same in both directional modes of switching motors. Motors forced backward usually break, either suddenly and irreversibly or progressively. Motors broken progressively rotate predominantly at integral multiples of a unitary speed during the course of both breaking and subsequent recovery, as expected if progressive breaking affects individual torque-generating units. Torque is reduced by the same factor at all speeds in partially broken motors, implying that the torque-speed relationship is a property of the individual torque-generating units.     

Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch.

CheY, a key protein in the mechanism of bacterial chemotaxis, is known to interact with the flagellar switch and thereby cause clockwise rotation. This activity of CheY was significantly increased by producing acetyladenylate (AcAMP) within cytoplasm-free bacterial envelopes containing purified CheY. This was achieved by including in the envelopes the enzyme acetyl-CoA synthetase (ACS) and ATP, and adding acetate externally. The fraction of clockwise-rotating envelopes, tethered to glass by their flagella, increased from 14% to 58% by the presence of AcAMP (or its derivative). In parallel experiments carried out with [14C]acetate under similar conditions, CheY became acetylated: [1-14C]acetate was as effective as [2-14C]acetate in labeling CheY, and ACS-dependent labeling of CheY by [alpha-32P]ATP was not detected. The switch proteins, FliG, FliM, and FliN, isolated to purity, were not acetylated. The acetylation was specific for CheY and dependent on its native conformation. The acetylated form the CheY was estimated to be more active than its nonacetylated form by 4-5 orders of magnitude. Acetylated CheY was stable in the presence of the strong nucleophiles hydroxylamine or ethanolamine, indicative of N-acetylation. There was a correlation between the activity of CheY in vivo and its ability to be acetylated in vitro. Thus, proteins with a single substitution at their active site, CheY57DE and CheY109KR, are not active in vivo and accordingly were not acetylated in vitro; in contrast, the protein CheY13DK is active in vivo and was normally acetylated in vitro. The possibility that CheY acetylation plays a role in bacterial chemotaxis is discussed.     

Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis

Signal transduction in bacterial chemotaxis involves transfer of a phosphoryl group between the cytoplasmic proteins CheA and CheY. In addition to the established metal ion requirement for autophosphorylation of CheA, divalent magnesium ions are necessary for the transfer of phosphate from CheA to CheY. The work described here demonstrates via fluorescence studies that CheY contains a magnesium ion binding site. This site is a strong candidate for the metal ion site required to facilitate phosphotransfer from phospho-CheA to CheY. The diminished magnesium ion interaction with CheY mutant D13N and the lack of metal ion binding to D57N along with significant reduction in phosphotransfer to these two mutants are in direct contrast to the behavior of wild-type CheY. This supports the hypothesis that the acidic pocket formed by Asp13 and Asp57 is essential to metal binding and phosphotransfer activity. Metal ion is also required for the dephosphorylation reaction, raising the possibility that the phosphotransfer and hydrolysis reactions occur by a common metal-phosphoprotein transition-state intermediate. The highly conserved nature of the proposed metal ion binding site and site of phosphorylation within the large family of phosphorylated regulatory proteins that are homologous to CheY supports the hypothesis that all these proteins function by a similar catalytic mechanism.     

Binding of the chemotaxis response regulator CheY to the isolated,intact switch complex of the bacterial flagellar motor: lack of cooperativity

In bacteria, the chemotactic signal is greatly amplified between the chemotaxis receptors and the flagellar motor. In Escherichia coli, part of this amplification occurs at the flagellar switch. However, it is not known whether the amplification results from cooperativity of CheY binding to the switch or from a post-binding step. To address this question, we purified the intact switch complex (constituting the switch proteins FliG, FliM, and FliN and the scaffolding protein FliF) in quantities sufficient for biochemical work and used it to investigate whether the binding of CheY to the switch complex is cooperative. As a negative control, we used complexes of switchless basal bodies, formed from the proteins FliF and FliG and similarly isolated. Using double-labeling centrifugation assays for binding, we found that CheY binds to the isolated, intact switch complex in a phosphorylation-dependent manner. We observed no significant phosphorylation-dependent binding to the negative control of the switchless basal body. The dissociation constant for the binding between the switch complex and phosphorylated CheY (CheY approximately P) was 4.0 +/- 1.1 microm, well in line with the published range of CheY approximately P concentrations to which the flagellar motor is responsive. Furthermore, the binding was not cooperative (Hill coefficient approximately 1). This lack of CheY approximately P-switch complex binding cooperativity, taken together with earlier in vivo studies suggesting that the dependence of the rotational state of the motor on the fraction of occupied sites at the switch is sigmoidal and very steep (Bren, A., and Eisenbach, M. (2001) J. Mol. Biol. 312, 699-709), indicates that the chemotactic signal is amplified within the switch, subsequent to the CheY approximately P binding.     

Lighting up the cell surface with evanescent wave microscopy.

Evanescent wave microscopy, also termed total internal reflection fluorescence microscopy (TIR-FM), has shed new light on important cellular processes taking place near the plasma membrane. For example, this technique can enable the direct observation of membrane fusion of synaptic vesicles and the movement of single molecules during signal transduction. There has been a recent surge in the popularity of this technique with the advent of green-fluorescent protein (GFP) as a fluorescent marker and new technical developments. These technical developments and some of the latest applications of TIR-FM are the subject of this review.     

Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy

Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spatial and temporal resolution. Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules. In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light. We have used a prism type TIR setup with a penetration depth of approximately 50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein. Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature. TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion. Combining TIR and wide-field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips.     

The bidirectional polar and unidirectional lateral flagellar motors of Vibrio alginolyticus are controlled by a single CheY species

The bacterial flagellar motor is an elaborate molecular machine that converts ion-motive force into mechanical force (rotation). One of its remarkable features is its swift switching of the rotational direction or speed upon binding of the response regulator phospho-CheY, which causes the changes in swimming that achieve chemotaxis. Vibrio alginolyticus has dual flagellar systems: the Na(+)-driven polar flagellum (Pof) and the H(+)-driven lateral flagella (Laf), which are used for swimming in liquid and swarming over surfaces respectively. Here we show that both swimming and surface-swarming of V. alginolyticus involve chemotaxis and are regulated by a single CheY species. Some of the substitutions of CheY residues conserved in various bacteria have different effects on the Pof and Laf motors, implying that CheY interacts with the two motors differently. Furthermore, analyses of tethered cells revealed that their switching modes are different: the Laf motor rotates exclusively counterclockwise and is slowed down by CheY, whereas the Pof motor turns both counterclockwise and clockwise, and CheY controls its rotational direction.     

Acetylation of the response regulator, CheY, is involved in bacterial chemotaxis

It is well established that the response regulator of the chemotaxis system of Escherichia coli, CheY, can undergo acetylation at lysine residues 92 and 109 via a reaction mediated by acetyl-CoA synthetase (Acs). The outcome is activation of CheY, which results in increased clockwise rotation. Nevertheless, it has not been known whether CheY acetylation is involved in chemotaxis. To address this question, we examined the chemotactic behaviour of two mutants, one lacking the acetylating enzyme Acs, and the other having an arginine-for-lysine substitution at residue 92 of CheY - one of the acetylation sites. The Deltaacs mutant exhibited much reduced sensitivity to chemotactic stimuli (both attractants and repellents) in tethering assays and greatly reduced responses in ring-forming, plug and capillary assays. Likewise, the cheY(92KR) mutant had reduced sensitivity to repellents in tethering assays and a reduced response in capillary assays. However, its response to the addition or removal of attractants was normal. These observations suggest that Acs-mediated acetylation of CheY is involved in chemotaxis and that the acetylation site Lys-92 is only involved in the response to repellents. The observation that, in the cheY(92KR) mutant, the addition of a repellent was not chemotactically equivalent to the removal of an attractant also suggests that there are different signalling pathways for attractants and repellents in E. coli.     

Asynchronous switching of flagellar motors on a single bacterial cell

Salmonella possesses several flagella, each capable of counterclockwise and clockwise rotation. Counterclockwise rotation produces swimming, clockwise rotation produces tumbling. Switching between senses occurs stochastically. The rotational sense of individual flagella on a single cell could be monitored under special conditions (partially de-energized cells of cheC and cheZ mutants). Switching was totally asynchronous, indicating that the stochastic process operates at the level of the individual organelle. Coordinated rotation in the flagellar bundle during swimming may therefore derive simply from a high counterclockwise probability enhanced by mechanical interactions, and not from a synchronizing switch mechanism. Different flagella on a given cell had different switching probabilities, on a time scale (greater than 2 min) spanning many switching events. This heterogeneity may reflect permanent structural differences, or slow fluctuations in some regulatory process.     

Functions of the flagellar modes of rotation in bacterial motility and chemotaxis

Bacteria swim by rotating their flagella, the rotation being due to a motor located at the base of each flagellum. In this paper the correlation between motor function and mode of swimming is reviewed, with special emphasis on recent data that indicate that the motor is a three-state device. Novel findings with regard to the motor function and bioenergetics are surveyed, and mechanisms are proposed to account for these findings.     

Multiple forms of "kiss-and-run" exocytosis revealed by evanescent wave microscopy

Exocytotic release of neuropeptides and hormones is generally believed to involve the complete merger of the secretory vesicle with the plasma membrane. However, recent data have suggested that "kiss-and-run" mechanisms may also play a role. Here, we have examined the dynamics of exocytosis in pancreatic MIN6 beta cells by imaging lumen- (neuropeptide Y/pH-insensitive yellow fluorescent protein; NPY.Venus) or vesicle membrane-targeted fluorescent probes (synaptobrevin-2/enhanced green fluorescent protein; synapto.pHluorin, or phosphatase on the granule of insulinoma-enhanced green fluorescent protein, phogrin.EGFP) by evanescent wave microscopy. Unexpectedly, NPY.Venus release events occurred much less frequently (13%-40% maximal rate) than those of synapto.pHluorin, even though the latter molecule, but not phogrin.EGFP, usually diffused away from the site of fusion. Thus, the majority of exocytosis occurs in these cells by kiss-and-run events that involve either the release of small molecules only, small molecules and selected membrane proteins, or all soluble cargoes ("pure," "mixed," and "full" kiss-and-run, respectively). Changes in the activity of synaptotagmin IV, achieved here by overexpression of the wild-type protein, may allow different stimuli to alter the ratio of these events, and thus the release of selected vesicle cargoes.     

Effect of intracellular pH on rotational speed of bacterial flagellar motors

Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of Escherichia coli and Salmonella at an external pH of 5.5. In this study, we examined in more detail the effect of weak acids on motility at various external pH values. A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of E. coli cells in the absence of 34 mM potassium acetate. In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate. The total proton motive force of E. coli cells was not changed greatly by the presence of acetate. We measured the rotational rate of tethered E. coli cells as a function of external pH. Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely. When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H+) concentration does not irreversibly abolish flagellar motor function. Both the swimming speeds and rotation rates of tethered cells of Salmonella also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate. We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors.     

Structures of bacterial flagellar motors from two FliF-FliG gene fusion mutants

Flagella purified from Salmonella enterica serovar Typhimurium contain FliG, FliM, and FliN, cytoplasmic proteins that are important in torque generation and switching, and FliF, a transmembrane structural protein. The motor portion of the flagellum (the basal body complex) has a cytoplasmic C ring and a transmembrane M ring. Incubation of purified basal bodies at pH 4.5 removed FliM and FliN but not FliG or FliF. These basal bodies lacked C rings but had intact M rings, suggesting that FliM and FliN are part of the C ring but not a detectable part of the M ring. Incubation of basal bodies at pH 2.5 removed FliG, FliM, and FliN but not FliF. These basal bodies lacked the C ring, and the cytoplasmic face of the M ring was altered, suggesting that FliG makes up at least part of the cytoplasmic face of the M ring. Further insights into FliG were obtained from cells expressing a fusion protein of FliF and FliG. Flagella from these mutants still rotated but cells were not chemotactic. One mutant is a full-length fusion of FliF and FliG; the second mutant has a deletion lacking the last 56 residues of FliF and the first 94 residues of FliG. In the former, C rings appeared complete, but a portion of the M ring was shifted to higher radius. The C-ring-M-ring interaction appeared to be altered. In basal bodies with the fusion-deletion protein, the C ring was smaller in diameter, and one of its domains occupied space vacated by missing portions of FliF and FliG.     

Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY

The protein (Escherichia coli CheY) that controls the direction of flagellar rotation during bacterial chemotaxis has been shown to be phosphorylated on the aspartate 57 residue. The residue phosphorylated is present within a conserved sequence in every member of a family of bacterial regulatory proteins. The phosphorylation is transient, with a much shorter half-life than that expected of a simple acyl phosphate intermediate, indicating that the sequence and conformation of the protein is designed to achieve a rapid hydrolysis. The CheY-phosphate linkage can be reductively cleaved by sodium borohydride. High-performance tandem mass-spectrometric analysis of proteolytic peptides derived from [3H]borohydride-reduced phosphorylated CheY protein was used to identify the position of phosphorylation. Mutants with altered aspartate 57 exhibited no chemotaxis. When aspartate 13, another conserved residue, was changed, greatly reduced chemotaxis was observed, suggesting an important role for aspartate 13. The rate-determining step of chemotactic signaling is governed by the kinetics of formation and hydrolysis of the CheY protein phosphoaspartate bond. The CheY protein apparently functions as a protein phosphatase that possesses a transient covalent intermediate. Transient phosphorylation of an aspartate residue is an effective mechanism for producing a biochemical signal with a short concentration-independent half-life. The duration of the signal can be controlled by small structural elements within the phosphorylated protein.     

CobB regulates Escherichia coli chemotaxis by deacetylating the response regulator CheY

The silent information regulator (Sir2) family proteins are NAD⁺‐dependent deacetylases. Although a few substrates have been identified, functions of the bacteria Sir2‐like protein (CobB) still remain unclear. Here the role of CobB on Escherichia coli chemotaxis was investigated. We used Western blotting and mass spectrometry to show that the response regulator CheY is a substrate of CobB. Surface plasmon resonance (SPR) indicated that acetylation affects the interaction between CheY and the flagellar switch protein FliM. The presence of intact flagella in knockout strains ΔcobB, Δacs, Δ(cobB) Δ(acs), Δ(cheA) Δ(cheZ), Δ(cheA) Δ(cheZ) Δ(cobB) and Δ(cheA) Δ(cheZ) Δ(acs) was confirmed by electron microscopy. Genetic analysis of these knockout strains showed that: (i) the ΔcobB mutant exhibited reduced responses to chemotactic stimuli in chemotactic assays, whereas the Δacs mutant was indistinguishable from the parental strain, (ii) CheY from the ΔcobB mutant showed a higher level of acetylation, indicating that CobB can mediate the deacetylation of CheY in vivo, and (iii) deletion of cobB reversed the phenotype of Δ(cheA) Δ(cheZ). Our findings suggest that CobB regulates E. coli chemotaxis by deacetylating CheY. Thus a new function of bacterial cobB was identified and also new insights of regulation of bacterial chemotaxis were provided.     

Different roles of CheY1 and CheY2 in the chemotaxis of Rhizobium meliloti

Cells of Rhizobium meliloti swim by the unidirectional, clockwise rotation of their right-handed helical flagella and respond to tactic stimuli by modulating the flagellar rotary speed. We have shown that wild-type cells respond to the addition of proline, a strong chemoattractant, by a sustained increase in free-swimming speed (chemokinesis). We have examined the role of two response regulators, CheY1 and CheY2, and of CheA autokinase in the chemotaxis and chemokinesis of R. meliloti by comparing wild-type and mutant strains that carry deletions in the corresponding genes. Swarm tests, capillary assays, and computerized motion analysis revealed that (i) CheY2 alone mediates 60 to 70% of wild-type taxis, whereas CheY1 alone mediates no taxis, but is needed for the full tactic response; (ii) CheY2 is the main response regulator directing chemokinesis and smooth swimming in response to attractant, whereas CheY1 contributes little to chemokinesis, but interferes with smooth swimming; (iii) in a CheY2-overproducing strain, flagellar rotary speed increases upon addition and decreases upon removal of attractant; (iv) both CheY2 and CheY1 require phosphorylation by CheA for activity. We conclude that addition of attractant causes inhibition of CheA kinase and removal causes activation, and that consequent production of CheY1-P and CheY2-P acts to slow the flagellar motor. The action of the chief regulator, CheY2-P, on flagellar rotation is modulated by CheY1, probably by competition for phosphate from CheA.