Direct imaging of intracellular signaling components that regulate bacterial chemotaxis.

RESEARCH ARTICLE MICROBIOLOGY

Direct Imaging of Intracellular Signaling Components That Regulate Bacterial Chemotaxis Hajime Fukuoka,1,2 Takashi Sagawa,2 Yuichi Inoue,1,2 Hiroto Takahashi,1 Akihiko Ishijima1,2* The bacterial chemotaxis system regulates the rotational direction of flagellar motors through an intracellular signaling molecule, the phosphorylated form of CheY (CheY-P). The binding of CheY-P to a motor is believed to switch the motor’s rotational direction from counterclockwise to clockwise. We demonstrated that the rotational switch of a motor was directly regulated by the binding and dissociation of CheY-P by simultaneously visualizing CheY tagged with green fluorescent protein and the rotational switching of a motor in live cells. The binding of 13 ± 7 CheY-P molecules was sufficient to induce clockwise rotation, and CheY-P molecules bound to and dissociated from a motor within ~100 ms during switching. Thus, we have directly measured the regulation of the output from a signal transduction pathway by intracellular signaling proteins.

The chemotaxis signaling system of Escherichia coli, a well-studied signal transduction system, modulates the function of the cell’s flagellar motors. E. coli cells migrate toward favorable environments in response to extracellular signals by controlling the rotational direction of flagellar motors (1). Chemoreceptors modulate the autophosphorylation activity of a histidine protein kinase, CheA, and the phosphoryl group on CheA is rapidly transferred to a response regulator, CheY (2, 3). CheY-P functions as an activated intracellular signaling molecule; its binding to FliM subunits in the motor is believed to induce a switch in rotation from a counterclockwise (CCW) to a clockwise (CW) direction (4, 5). The CheY-P concentration is thought to fluctuate around its average in the absence of stimuli and that the flagellar motor stochastically switches rotational directions, so that the CheY-P concentration determines only the probability of rotational switching (6–9). The CW bias (the fraction of time spent rotating in CW direction) shows a cooperative response to the concentration of the response regulator CheY-P, contributing to gain of bacterial chemotaxis (8, 9). However, the relationships among the rotational direction of a motor, the number of CheY-P molecules bound to a single functioning motor, and the CheY-P concentration in cytoplasm are not clear. It also remains elusive the cause of strong cooperativity between CW bias and the CheY-P concentration, and whether differences in binding affinity of CheY-P molecules for CCW- and CW-state motors affect this cooperativity. We have proposed that the steady-state concentration of CheY-P undergoes transient increases and decreases, and that these dynamic changes function as an intracellular signal to regulate the rotational switching of the motors (10). Here, we sought to test this proposed mechanism in vivo by directly determining whether the binding and dissociation of CheY-P regulates the rotational direction of a functioning motor. By direct imaging, we demonstrated that the binding and dissociation of CheY-P molecules is essential to induce CW and CCW rotation, respectively. We also quantitatively estimated some parameters to understand the mechanisms in rotational switching, such as the number of CheY-P molecules bound to a motor to induce CW rotation, the time span of the binding and dissociation of CheY-P molecules during rotational switching, and the difference of binding affinity for CheY-P between CCW- and CW-state motors. 1 Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai 980-8577, Japan. 2Graduate School of Life Sciences, Tohoku University, Aobak-ku, Sendai 980-8577, Japan. *Corresponding author. E-mail: [email protected]

RESULTS

Intracellular concentration of CheY-GFP in E. coli cells To observe the binding of CheY proteins to a functioning motor, we produced enhanced green fluorescent protein (EGFP)–labeled CheY (CheYGFP) in a cheY gene–deleted strain (table S1). We measured the intracellular concentration of CheY-GFP in this strain by quantitative Western blotting analysis (fig. S1) (Materials and Methods). By comparing the intensity of the CheY-GFP and purified enhanced GFP (EGFP) bands, we estimated that the applied cell suspension for immunoblotting contained 0.07 mg of CheY-GFP. The cell suspension that was immunoblotted contained 1.5 × 108 cells, and the average cellular volume was 0.87 ± 0.36 mm3 (mean ± SD, n = 60 cells). Therefore, the concentration of CheY-GFP in a single cell was 13 ± 3 mM (mean ± SD derived from three independent experiments). The concentration of CheY-GFP in a single E. coli cell was comparable to that of CheY produced in a wild-type cell (11).

Simultaneous observation of the binding of CheY-GFP to a functioning motor and switching of the motor’s rotational direction To simultaneously observe CheY-GFP localization and the rotational switching of the flagellar motor, we recorded fluorescence images of CheY-GFP visualized by total internal reflection fluorescence (TIRF) microscopy and bright-field images of tethered rotating cells with electron-multiplying charge-coupled device (EMCCD) and high-speed CCD cameras, respectively (Fig. 1A). The fluorescent spot derived from CheY-GFP was detected at the rotational center of the tethered cell during CW rotation but not during CCW rotation (Fig. 1B and movie S1). The fluorescence intensity changed depending on the rotational direction of the motor; the intensity immediately increased with CCW-to-CW switching and was sustained during CW rotation (Fig. 1C). In contrast, the intensity immediately decreased with CW-to-CCW switching. Cross-correlation analysis between the change in fluorescence intensity and the rotational direction of the motor showed significant correlation in all cells examined (Fig. 1D). Thus, the increase and decrease in fluorescence intensity represent the binding and dissociation of CheY-GFP molecules to and from a motor, respectively. No peak shift was detected in the correlation profile, indicating that the rotational switching of the motor occurred within our sampling time (20 ms), after the binding or dissociation of CheY-GFP molecules. The intensity of excitation light under the TIRF microscope decays exponentially, depending on the distance from a glass surface. Therefore, it

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Vol 7 Issue 319 ra32

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INTRODUCTION

RESEARCH ARTICLE A

Halogen lamp

Tethered cell

Long-pass filter Cold filter Flagellum

E. coli

Dichroic mirror

0 ms

FliM

Laser

EM-CCD camera Emission filter High-speed camera

20 ms

40 ms

60 ms

80 ms

100 ms

120 ms

140 ms

160 ms

20

20,000

10

0

0

Number of CheY molecules

Fluorescence intensity (A.U.)

40,000

20 Rotational velocity (Hz)

220 ms

–2

2

CW

C

10

CCW

0

D 0.4 0.2 0 –0.2 –0.4 –0.6

–10 –20

200 ms


CCW

180 ms

Correlation

B

Evanescent field

Laser ( = 488 nm)

Dichroic mirror

Long-pass filter

Flagellar motor CheY-GFP

CW 0

1

2

3

4

5 Time (s)

6

7

8

–0.8

9

–4

0 τ (s)

4

Fig. 1. Simultaneous visualization of CheY-GFP and flagellar motor rotation. (A) Schematic diagram of the microscope and method used for simultaneously viewing CheY-GFP localization and flagellar motor rotation. (B) Sequential fluorescence (top) and bright-field (bottom) images of a tethered cell expressing CheY-GFP. Arrowhead, CheY-GFP localized at a motor. Bar, 1 mm. (C) Fluorescence intensity at the rotational

center (top) and rotational velocity (bottom) of a tethered cell. Plus and minus values in rotational velocity indicate CCW and CW rotation, respectively. A.U., arbitrary units. (D) Cross-correlation profiles between fluorescence intensity and rotational direction. Gray and red lines indicate an individual correlation profile for each of eight cells and the average trace, respectively.

was possible that the change in fluorescence intensity in a cell producing CheY-GFP was due to a change in distance between the glass surface and the cell body owing to the rotational direction of the tethered cell. To exclude this possibility, we investigated the fluorescence intensity during CCWto-CW and CW-to-CCW switching using a motor composed of FliM-GFP (12). The intensity of the motor composed of FliM-GFP decayed exponentially, and the fluorescence intensity did not depend on the rotational direction (fig. S2). Therefore, the change in fluorescence intensity that was observed in tethered cells producing CheY-GFP was not an artifact, but a result of the binding and dissociation of CheY-GFP.

Binding of phosphorylated CheY-GFP to a motor in the presence of chemotactic signals To confirm whether the localization of CheY-GFP represented the binding of activated CheY (CheY-P) to a flagellar motor, we investigated the localization of CheY-GFP at a functioning motor in the presence of attractant or repellent. In the presence of the attractant aMeAsp, the activity of the chemoreceptor-kinase clusters is inhibited, and CheY-P is dephosphorylated by the phosphatase CheZ (13), resulting in a reduced intracellular concentration of CheY-P. In the presence of the repellent glycerol, the chemoreceptorkinase clusters are activated, leading to the phosphorylation of CheY and


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RESEARCH ARTICLE an increased intracellular concentration of CheY-P. We used the CW-locked mutant motor (14) in this experiment to remove the effect of the different motor structures (CCW and CW states) and to assess the binding of CheY-P molecules to a flagellar motor. The CW-locked motor lacks three amino acid residues (Pro169-Ala171) in its FliG subunits and rotates only in the CW direction, independently of CheY-P binding. In tethered cells producing CW-locked mutant motors, CheY-GFP localized to the rotational center of the cell in motility medium (Fig. 2A, top, and movie S2). When motility medium containing a-methyl aspartate (aMeAsp) was applied by perfusion, CheY-GFP dispersed and did not localize to the motor (Fig. 2A, middle, and movie S3). In contrast, when the solution was A CW-locked motor

Buffer

CW

Estimation of the number of GFP-fusion proteins in a functioning flagellar motor

+Aspartate (attractant)

+Glycerol (repellent)

0 ms

40 ms

B WT motor

80 ms

120 ms

160 ms

Buffer

CW

+Aspartate (attractant)

CCW

+Glycerol (repellent)

CW 0 ms

40 ms

80 ms

120 ms

To estimate the number of GFP-fusion proteins in a flagellar motor, we used the fluorescently labeled flagellar motor containing FliM-GFP, which is one of the rotor components of the flagellar motor. We used the same TIRF microscopy method used to observe CheY-GFP localization to observe the subcellular localization of FliM-GFP (Fig. 3A). The time course of fluorescence intensity derived from the photobleaching of FliM-GFP molecules was fitted by a single exponential function (Eq. 2) (Fig. 3B). The initial fluorescence intensity (I0, Fig. 3B) as estimated from the curve was 64,500 ± 5900 (table S2), which corresponds to the total intensity from FliM-GFP molecules in a single flagellar motor. The number of FliM subunits in a single flagellar motor has been estimated by cryo-electron microscopy images to be 32 to 36 (15–17), and a similar number has been estimated from fluorescence microscopic images (18, 19). The number of FliM subunits also changes because of the adaptation of the motor to extracellular signals (20). The number of CheY-GFP molecules bound to a motor was estimated by Eq. 3, which considers the distribution in the number of FliM subunits. The fluorescence intensities from CheY-GFP during CW and CCW rotation were 24,900 ± 12,300 and 4600 ± 8200, respectively. Therefore, on average, 13 ± 7 and 2 ± 4 CheY-GFP molecules bind to a flagellar motor during CW and CCW rotation, respectively (Fig. 3C) (table S2). Therefore, CheY-P molecules are not required to bind to all the FliM subunits to induce a CW directional change, because each motor contains ~34 FliM subunits (17–19).

Estimation of the time spans for increasing and decreasing the number of bound CheY-P molecules during switching 160 ms

Fig. 2. Binding of CheY-GFP to a flagellar motor in the presence of chemotactic signals. (A and B) Subcellular localization of CheY-GFP to CW-locked mutant (A) and wild-type (B) motors in the motility buffer in the absence of chemotactic signal (upper) and in the presence of 100 mM a-methyl aspartate as an attractant (middle) or 16% glycerol as a repellent (bottom). Arrowhead, CheY-GFP localized to a motor. Each series of images is representative of three cells (CW-locked mutant) or four cells (wild type).

To further investigate the process by which CheY-P binds to and dissociates from a flagellar motor during rotational switching, we examined the time course of the binding and dissociation phases. The binding phase lasted ~120 ms (Fig. 4, left, blue area), in agreement with the time span of the increase of CheY-P concentration by CheA-mediated phosphorylation after the addition of repellent (3). The dissociation phase lasted ~70 ms (Fig. 4, right, blue area), which was much shorter than the time span of the decrease of CheY-P concentration by the dephosphorylation of CheY-P by CheZ (~1 s) after the addition of attractant (3). These results suggest that the dissociation of CheY-P during CW-to-CCW switching may accelerate as the concentration of CheY-P decreases.


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replaced with motility medium containing glycerol, the localization of CheY-GFP to the motor was recovered (Fig. 2A, bottom, and movie S4). Similar results were observed in a cell producing wild-type motors. In cells producing wild-type motors, a cell rotating in the CW direction was chosen for observation. Initially, the cell rotated in the CW direction and CheY-GFP localized to the rotational center (Fig. 2B, top, and movie S5). When motility medium containing aMeAsp was applied by perfusion, the rotational direction switched to the CCW direction and the localization of CheY-GFP at the motor was dispersed (Fig. 2B, middle, and movie S6). When the solution was changed to motility medium containing glycerol, the rotational direction switched back to the CW direction and the localization of CheYGFP to the motor was recovered (Fig. 2B, bottom, and movie S7). These results indicate that the localization of CheY-GFP directly represents the binding of phosphorylated CheY to a flagellar motor and that dephosphorylated CheY does not bind to a CW-state motor. Thus, CheY binding to a functioning motor is not promoted by the CW state of the motor but by the phosphorylation of CheY by receptor-kinase clusters activated in response to chemotactic signals.

RESEARCH ARTICLE Number of CheY molecules

FliM-GFP (34 molecules in a motor)

A

Evanescent field

Laser B

10 5 0 –0.4 –0.2

60,000 40,000

I0

20,000

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0

10 Time (s)

15

600

Counts

15

80,000

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C

20

20

0.4 –0.4

–0.2

0 0.2 Time (s)

0.4

Fig. 4. Estimation of the time spans for increasing and decreasing the number of bound CheY-P molecules during rotational switching. Curves representing the CheY-GFP binding phase during CCW-to-CW switching (left, blue hatched area) and the CheY-GFP dissociation phase during CW-toCCW switching (right, blue hatched area) over time. Blue lines are fitted curves based on Eq. 5. Curves of the CCW-to-CW switching and CW-toCCW switching are derived from 59 and 55 switches from seven cells, respectively.

CCW CW

The binding affinity of CCW- and CW-state motors for CheY-P

400

200

0 –10

0 0.2 Time (s)

0

10 20 30 Number of CheY molecules

40

Fig. 3. Estimation of the number of CheY-GFP molecules bound to a functioning flagellar motor. (A) Conceptual illustration of the experimental method. Tethered cells containing flagellar motors composed of FliM-GFP were observed under TIRF microscopy using the method in Fig. 1A. (B) Typical photobleaching profile of a motor composed of FliM-GFP, representative of 17 cells. Blue and red lines indicate the fluorescence intensity of the FliM-GFPlabeled motor and the fitted curve of the photobleaching profile using a single exponential function, respectively. The value of the fitted curve at time 0 (I0) corresponds to the intensity from all FliM-GFP molecules incorporated into flagellar motors. (C) Histograms representing the number of bound CheYGFP molecules during CCW (blue) and CW (red) rotation, respectively.

We also simulated the dynamic change in CheY-P concentration due to the activation and inactivation of the activity of CheA in the receptorkinase cluster (Fig. 5A). The simulation showed that the CheY-P concentration immediately increased and saturated within ~300 ms, which was consistent with the time span of the binding phase of CheY-P during CCW-to-CW switching (Fig. 5B). In contrast, the decrease in CheY-P concentration was estimated to take a few seconds after stopping the activity of CheA. The rapid subsecond decrease in CheY-P concentration was reproduced only when an unusually high number of CheZ molecules (32,000 molecules) was assumed (Fig. 5C). These results suggest that the ~70-ms time span of the CheY-P dissociation phase during CW-to-CCW switching could not be explained only by the decrease in CheY-P concentration by the activity of CheZ.

A previous theoretical model assumed that the CW-rotating motor has a higher affinity for CheY-P than does the CCW-rotating one (21). We thus hypothesized that CCW and CW motors have different binding affinities for CheY-P, a difference that plays a role in signal transduction. To investigate this possibility, we measured the binding of a constitutively active mutant of CheY (caCheY-GFP) to wild-type or CW-locked mutant motors (14). caCheY has a double–amino acid substitution (D13K/Y106W) that mimics the CW rotation–stimulating function of CheY-P in a phosphorylationindependent manner (8). Thus, this mutant enables investigation of the binding affinity of the motor to CheY molecules without the CheAmediated phosphorylation of CheY. To compare the binding affinity of the two motors, we confirmed the same concentration of caCheY-GFP between two strains producing wild-type and CW-locked mutant motors (Fig. 6A). When caCheY-GFP expression was low, caCheY-GFP did not localize to wild-type motor [Fig. 6, B (top) and C (top left), and movie S8] and the motor rotated in the CCW direction (Fig. 6C, bottom left). However, caCheY-GFP at the same concentration was localized to the CW-locked motor [Fig. 6, B (bottom) and C (top right), and movie S9] and rotated only in the CW direction (Fig. 6C, bottom right). The CW-locked motor bound 6 ± 5 caCheY molecules, whereas the CCW state of the wild-type motor bound 1 ± 4 molecules (Fig. 6D). The dissociation constant of the CCW motor for CheY-P was estimated to be 4.7-fold greater than that for the CW motor. Thus, the CW motor has a higher affinity than the CCW motor for CheY-P. DISCUSSION

Traditional signal transduction research has focused on clarifying the cascade in signaling pathways and the activity and subcellular localization of proteins in the pathway; however, the mechanism of how each signaling molecule dynamically behaves and is propagated to the targeted machinery remains elusive. We demonstrated that the binding and dissociation of CheY-P molecules directly induces the CW and CCW rotation of a functioning


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100,000

25

RESEARCH ARTICLE A

A

Receptor-kinase cluster (CheA, CheZ) 20 nm 0.2 µm

B

WT motor (CCW-state) + caCheY-GFP

caCheY-GFP GFP

kD

CCW

CWWT locked

50

CW-locked motor + caCheY-GFP

37

Motor 0.8 µm

25

CW

0 ms

C

0

5 0 –5 –10

0

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1

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2 2.5 Time (s)

3

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4 D

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1

1.5

2 2.5 Time (s)

3

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4

Fig. 5. Simulation of the dynamic change in CheY-P concentration. (A) Scheme depicting the method used to estimate the CheY-P concentration around a flagellar motor. The motor was positioned 1 mm from the left side of the rectangle. The number of CheY-P molecules within a 0.2-mm-long and 0.8-mm-wide area surrounding a motor (red dotted area) was counted, and the number of molecules was converted to the concentration. (B) The CheY-P concentration when assumed values of 2500 CheA and 3200 CheZ molecules were used in the calculation. One of the typical traces from five independent calculations is shown. (C) The CheY-P concentration when assumed values of 2500 CheA and 32,000 CheZ molecules were used in the calculation. One of the typical traces from five independent calculations is shown. Under both conditions, the CheY molecules were phosphorylated from 0 to 2 s; in contrast, CheY-P was dephosphorylated constantly from 0 to 4 s.

flagellar motor, respectively, indicating that we have directly observed the functional output of signaling by CheY-P in a living E. coli cell. Also, we have previously shown that two flagellar motors on the same cell coordi-

6 4 Time (s)

8

10 0

2

4 6 Time (s)

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10

WT motor CW-locked motor

1000

0

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Counts

14 12 10 8 6 4 2 0

20,000

10 Rotational velocity (Hz)

14 12 10 8 6 4 2 0

CW-locked motor

–10

0 10 20 Number of CheY molecules

30

Fig. 6. Ability of CCW- and CW-rotating motors to bind caCheY. (A) Immunoblotting of caCheY-GFP in cells producing wild-type (WT) and CW-locked motors using an anti-GFP antibody. Representative of three Western blots. (B) Fluorescence images of caCheY-GFP localization in a cell producing WT (top) and CW-locked mutant (bottom) motors. Arrowhead, caCheY-GFP localized to a motor. Bar, 1 mm. (C) Time course of the fluorescence intensity at the rotational center (top row) and the rotational velocity (bottom row) of tethered cells. Results of cells producing WT (left column) and CW-locked mutant (right column) motors are shown. (D) Histograms representing the number of caCheY-GFP molecules bound to WT (blue, n = 20 cells) and CW-locked mutant (red, n = 22 cells) motors, respectively.

nately switch their rotational direction (10). Our studies indicate that CheY-P molecules bind to two flagellar motors with similar timing. To produce such a situation, the CheY-P concentration should dynamically change with the rotational switching under steady-state conditions in an E. coli cell (Fig. 7). The findings in our present study fill a blank in our recently proposed model, in which the rotational switching of flagellar motors is


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C

CheY-P concentration (µM)

B

CheY-P concentration (µM)

2 µm

WT motor (CCW-state)

40,000


1 µm

20 ms 40 ms 60 ms 80 ms 100 ms

RESEARCH ARTICLE A

CheA Receptor

P

P

CheY Motor 1

Motor 2

P

CheY-P

CheW CheZ CheAshort

Activity of CheA

B

CheA ON

CheA OFF

Diffusive propagation of CheY-P concentration CheA ON

CheA OFF

Time

Number of bound CheY-P molecules

Time

Rotational direction

Time

C

CCW

Time CW

CW-state motor CheY-P

CCW-state motor CheY-P

FliMCW

FliMCCW

Fig. 7. Model for intracellular signaling. (A) Schematic of the bacterial chemotaxis signaling process. (B) Schematic diagrams illustrating the signaling process. Receptor-kinase clusters spontaneously and cooperatively activate and inactivate CheA (row 1). The CheY-P concentration increases and decreases, depending on the activities of receptor-kinase clusters and CheZ (row 2). CheY-P binds to and dissociates from a motor with increasing and decreasing CheY-P concentrations (row 3). Binding and dissociation of CheY-P induces CW and CCW rotation, respectively (row 4). (C) Regulation of CheY-P binding by the CW- and CCW-rotating states. CCW- and CW-rotational motors have relatively lower (gray broken arrows) and higher (red arrows) binding affinities for CheY-P, respectively. Therefore, CCWrotating motors maintain their CCW structure by reducing the probability of CheY-P binding, and CW-rotating motors maintain their CW structure by increasing the probability of CheY-P binding.


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CheY-P concentration

Motor 1 Motor 2

regulated by dynamic increases and decreases in the intracellular concentration of CheY-P (Fig. 7, A and B) (10). On the basis of direct measurements in a single E. coli cell, we propose the following mechanism for the intracellular signaling process initiated by the activation of receptor-kinase clusters and resulting in the functional output of a signal transduction pathway. Under steady-state conditions, the intracellular CheY-P concentration dynamically increases and decreases by the spontaneous and cooperative activation of receptor-kinase clusters (Fig. 7B, row 1), and the change in CheY-P concentration is propagated by diffusion through the cytoplasm (Fig. 7B, row 2). As the CheY-P concentration increases, CheY-P molecules directly bind to the motors to induce CCW-to-CW switching (Fig. 7B, rows 3 and 4). In contrast, with decreasing CheY-P concentration, CheY-P molecules dissociate from the motor to induce CW-to-CCW switching. Therefore, the activation and inactivation of the receptor-kinase cluster are directly reflected in each rotational switch of the flagellar motors under steady-state conditions in E. coli. A previously published paper has proposed that the switching coordination is produced by extracellular components, such as hydrodynamic interaction between flagella (22). However, we found that two motors on different cells did not show switching coordination even if the distance between two motors was close enough (10). Switching coordination is also absent in cells expressing the caCheY mutant (10, 23), suggesting that the switching coordination is generated by changes in the CheY-P concentration produced from chemoreceptor clusters rather than a hydrodynamic interaction between flagella. Moreover, a hydrodynamic interaction cannot explain the switching delay between two motors, which correlates with the distance of each motor from the chemoreceptor cluster (10). Therefore, we argue that switching coordination is produced by the signaling molecule CheY-P in E. coli cells. We measured the number of CheY-P molecules bound to CW-rotating motor plateaus at about 13 molecules, instead of saturating the motor with 34 molecules that is the number of FliM subunits. This is probably explained by a conventional kinetic scheme using the secondary rate constant of CheY-P binding (5 × 106 M−1 s−1) and the dissociation rate of CheY-P from FliM subunits in the motor (20 s−1), as previously reported (3, 24). Even if all of CheY molecules in the E. coli cell (13 mM) were fully phosphorylated, the number of CheY-P molecules bound to FliM did not saturate with 34 molecules of FliM. This estimation indicates that the binding and dissociation of CheY-P molecules would be repeated in several tens of milliseconds at the motor during CW rotation. Some theoretical models have also proposed that the conformational change of FliM subunits by binding of CheY-P in a flagellar motor spreads to the other subunits without CheY-P binding during the process of the rotational switching (25, 26), suggesting that CheY-P molecules are not necessary to bind to all FliM subunits to induce CW rotation. This is consistent with our experimental estimation that the binding of 13 CheY-P molecules induces CW rotation. Therefore, the cooperative conformational change of FliM subunits derived from binding of CheY-P might occur in a functioning motor. By directly observing functioning motors, we showed that the dissociation of CheY-P during CW-to-CCW switching took ~70 ms. In contrast, the decrease of CheY-P concentration by CheZ has been reported to take a few seconds, as assessed by ensemble FRET analysis between chemotactic proteins in live cell populations (3, 27). We also simulated the change in CheY-P concentration by assuming equilibrium equations of chemotactic reactions and the diffusion of chemotactic proteins, but the time span for the decrease of CheY-P concentration in several tens of milliseconds was not reproduced (Fig. 5). Moreover, the dissociation phase of CheY-P has been estimated to be a few seconds by previously described computational models (24). Therefore, we propose that a cooperative mechanism accelerates

RESEARCH ARTICLE perspective on the dynamic behaviors of more complex signal transduction networks.

MATERIALS AND METHODS

E. coli strains, plasmids, and growth conditions

The E. coli strains and plasmids are listed in table S1. All E. coli strains were derived from E. coli K12 strain RP437, which is considered to be wild type for chemotaxis (31). The gene deletion procedures were carried out with the l red recombinase and tetracycline sensitivity selection method (32, 33). The strain EFS037 was constructed from EFS031 (10) by deletion of the cheY gene, and strain EFS039 was constructed from EFS037 by deletion of the fliG gene. L broth (1% bactotryptone, 0.5% yeast extract, 0.5% NaCl) was used for culture growth, transformations, and plasmid isolation. Tryptone broth (TB) (1% bactotryptone, 0.5% NaCl) was used to cultivate cells for observations of motor rotation and localization of GFP-labeled proteins. Ampicillin, chloramphenicol, kanamycin, and tetracycline were added to final concentrations of 50, 25, 50, and 15 µg/ml, respectively. To express CheY-GFP from pHF6602, we fused the egfp gene to the 3′ end of cheY gene, and the cheY-egfp fusion gene was placed under the control of an isopropyl-b-D-thiogalactopyranoside (IPTG)–inducible promoter in pMMB206. To observe the localization of CheY-GFP, we cultured EFS037 cells harboring pHF6602 in TB containing 5 mM IPTG and chloramphenicol (25 mg/ml) at 30°C for 5.25 hours. To observe the localization of FliMGFP, we cultured EFS012 cells harboring pFS6003m in TB containing 0.002% arabinose and ampicillin (50 mg/ml) at 30°C for 5.25 hours. To examine the localization of caCheY-GFP [CheY(D13K/Y106W)] (8) in a cell producing wild-type or CW-locked mutant motors (14), we cultured EFS039 cells harboring pHF6603 and pYF3 or EFS039 cells harboring pHF6603 and pYF3DPAA in TB containing 5 mM IPTG, 0.002% arabinose, chloramphenicol (25 mg/ml), and ampicillin (50 mg/ml) at 30°C for 5.25 hours. To observe control cells producing EGFP, we cultured EFS031 cells harboring pBAD24-EGFP and pTH2300 in TB containing 0.002% arabinose, 30 mM IPTG, ampicillin (50 mg/ml), and chloramphenicol (25 mg/ml) at 30°C for 5 hours.

Microscope system for the simultaneous observation of fluorescence and bright-field images of cells expressing GFP-fusion proteins To measure the localization of GFP-fusion proteins and the rotational switching of flagellar motors simultaneously, we constructed a microscope system to observe the fluorescence and bright-field images simultaneously (Fig. 1A). For the excitation of GFP-fusion proteins, a blue laser beam (sapphire 488-20-SV, Coherent) was inserted into the microscope (IX71, Olympus) from the right-side port. The blue laser beam was reflected by a primary dichroic mirror (FF495-Di02, Semrock) and focused on the back focal plane of the objective lens (APON 60XOTIRF, numerical aperture 1.49, Olympus). To use objective-type TIRF microscopy (34), we adjusted the incident angle of the laser beam to the objective lens. The fluorescence from the GFP-fusion protein was passed through the primary dichroic mirror and was reflected by a secondary dichroic mirror (FF593-Di03, Semrock). The fluorescence was then passed through an emission filter (FF01-520/35, Semrock) and was focused on an EMCCD camera (DU860DCS0-BV, Andor Technology). The fluorescence images were recorded at 50 frames/s. Long-pass (R60, Hoya) and cold (SC751, Asahi Spectra) filters were set in front of the halogen lamp to observe the bright-field image using red light and to remove heat generated by the lamp. The brightfield image of the cell was passed through the primary and secondary


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the dissociation of CheY-P during CW-to-CCW switching. (We could not determine whether binding cooperativity occurs, because the time spans for the increase in CheY-P concentration and the binding phase of CheY-P were comparable.) A previous study has demonstrated strong cooperativity between the CheY-P concentration and the CW bias (the fraction of time spent rotating in CW direction) by comparing the CW bias among the E. coli cells that have different CheY-P concentrations (8, 9). This relationship suggests the possibility that the motor itself confers strong cooperativity on the CheY-P concentration. On the other hand, our present study suggested that there is cooperativity in the binding and dissociation of CheY-P molecules to and from a motor. Therefore, we think that the binding and/or dissociation cooperativity of CheY-P molecules is one of the components to generate the strong cooperativity between the CheY-P concentration and the motor bias. However, it would be difficult to explain the high Hill coefficient only by the different affinity between CCW- and CW-state motors to CheY-P molecules. Therefore, we should consider some mechanisms that are theoretically proposed, including the Monod-Wyman-Changeux (MWC) and spreading models (21, 25, 26). The different binding affinities of the CCW and CW motors suggest that, during signal transduction, the motor itself can regulate the binding and dissociation of CheY-P molecules. The CW-rotating motor would maintain its CW structure by increasing the probability of CheY-P binding owing to its higher affinity for CheY-P (Fig. 7C, left). In contrast, the CCW-rotating motor would maintain its CCW structure by reducing the probability of CheY-P binding owing to its lower affinity for CheY-P (Fig. 7C, right). By this property, the rotational direction of a motor acquires a robustness against the local fluctuations in CheY-P concentration, so that the motors more effectively respond to the change in the CheY-P concentration produced by the receptor-kinase cluster. Direct imaging of CheY-GFP in a living E. coli cell showed that the binding and dissociation of CheY-P molecules is essential to induce CW and CCW rotation, indicating that CheY-P molecules bind to and dissociate from multiple motors with similar timing to generate switching coordination among motors. Therefore, we proposed a dynamic change in CheY-P concentration by the spontaneous and cooperative activation and/or inactivation of a receptor cluster that is composed of more than 10,000 protein molecules. This phenomenon in a steady state of E. coli cells (in which chemotaxis is “idling”) might be useful for a rapid response to sudden changes in environmental conditions. To verify the spontaneous and cooperative activation of the receptor-kinase cluster, further investigations would be required, such as the detection of fluorescence resonance energy transfer (FRET) between proteins composing the receptor-kinase cluster. By the single-cell measurement of signal transduction through the chemotaxis signaling system, we can have a more detailed discussion about the behavior and the dynamics of protein molecules at the moment of cellular response, such as the dissociation (and/or binding) cooperativity of CheY-P during rotational switching (this study), the dependence of the binding affinity for CheY-P on the rotational direction (this study), the remodeling of the number of motor components through adaptation of the motor (20), and the turnover of motor components (18). The cooperative manner, the diffusive motion, and the fluctuation of protein molecules in the signal transduction pathway might play a key role in signal transduction; however, these properties are difficult to detect by bulk measurements such as biochemical analyses. The basic mechanisms of signal transduction networks through phosphotransfer are shared not only by bacterial chemotactic systems but also by many organisms, including eukaryotes (28–30). Our findings provide insight into the mechanism of a signal transduction system in prokaryotic cells, as well as what we believe to be a new

RESEARCH ARTICLE dichroic mirrors and was focused on a high-speed CCD camera (ICLB0610M-KC, Imperx). The long-pass filter (BLP01-488R-25, Semrock) was set in front of the high-speed CCD camera to block the excitation laser light leaking through the dichroic mirrors. The bright-field images of tethered cells were recorded at 200 frames/s.

Simultaneous observation of GFP-fusion proteins and the rotation of tethered cells

Estimation of the intracellular concentration of CheY-GFP in a cell for microscopic observation

E. coli JM109 cells harboring pTrcHisB-EGFP, which encodes His6-tagged EGFP, were grown in LB supplemented with ampicillin (50 mg/ml) at 37°C overnight. The overnight culture was diluted 1:100 in 40 ml of LB supplemented with ampicillin (50 mg/ml) and 0.1 mM IPTG and incubated at 37°C for 10 hours. The cells were harvested by centrifugation and then suspended in suspension buffer [50 mM tris-HCl (pH 8.0), 300 mM NaCl]. The suspended cells were centrifuged, and the pellets were stored at −30°C overnight. Frozen cells were thawed and suspended in suspension buffer. Lysozyme solution was added at 0.5 mg/ml, and the cell suspension was incubated on ice for 1 hour. Deoxyribonuclease I solution was added at 5 mg/ml, and the cell suspension was subjected to three freeze-thaw cycles. The cell suspension was ultracentrifuged (150,000g for 0.5 hour at 4°C), and the supernatant was recovered. Ni-NTA agarose solution (100 ml) (Life Technologies) was added to the supernatant, and the solution was gently mixed for 2 hours at 4°C. The Ni-NTA agarose with bound His-tagged EGFP was washed with wash buffer [50 mM tris-HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole] three times, and then bound His-tagged EGFP was eluted with elution buffer [50 mM tris-HCl (pH 8.0), 300 mM NaCl, 200 mM imidazole]. To remove imidazole, the purified GFP solution was diluted with suspension buffer and condensed by ultrafiltration (Vivaspin20 10 kD, Sartorius) three times. The concentration of purified GFP was estimated to be 1.2 mg/ml with the MicroBCA protein Assay Kit (Thermo Scientific). The intracellular concentration of CheY-GFP in EFS037 cells harboring pHF6602 was measured by quantitative Western blotting analysis. EFS037 cells harboring pHF6602 were grown in TB containing 5 mM IPTG and chloramphenicol (25 mg/ml) at 30°C for 5.25 hours. The cells were harvested by centrifugation and then condensed by resuspending in 10 mM sodium phosphate buffer (pH 7.0) so that the optical density at 660 nm was 20. Cell suspensions were diluted 1:1 in 5× SDS loading buffer containing b-mercaptoethanol and were boiled at 100°C for 5 min. Whole-cell lysates and purified GFP were separated by SDS-PAGE, and the proteins were detected by immunoblotting with an anti-GFP antibody (Clontech). Bands were detected with an alkaline phosphatase (AP)–conjugated anti-

Estimation of fluorescence intensity from the GFP-fusion proteins on a flagellar motor under TIRF microscopy We applied two regions of interest (ROIs) surrounding the fluorescent spot on the tethered cell (fig. S3A); the small ROI (ROI 1) was 7 × 7 pixels (560 × 560 nm) and the large one (ROI 2) was 9 × 9 pixels (720 × 720 nm). The fluorescence intensity was estimated with the following equation: Fmotor ¼ F1 − C

F2 − F1 P1 P2 − P1

ð1Þ

where F1 and F2 are the total fluorescence intensities of ROI 1 and ROI 2, and P1 and P2 are the total number of pixels in ROI 1 and ROI 2, respectively. C is a correction factor for the estimation of fluorescence intensity using the tethered cell method and TIRF microscopy. In the analytical method shown in Eq. 1, it is important to estimate the background intensity of the cell producing cytoplasmic GFP-fusion proteins because the intensity of excitation light under TIRF microscopy exponentially decays from the glass surface and the shape of the E. coli cell is like a rod. We assumed a rod-shape structure and that fluorescence molecules were distributed uniformly, and calculated the intensity of the rod rotating under TIRF microscopy (fig. S3B). When the rod was rotated like a tethered cell, the rotational center had a Gaussian-like profile of fluorescence intensity (fig. S3C). This profile affects the estimation of the background intensity of cells expressing GFP-fusion proteins. The effect of excitation light under TIRF microscopy on the E. coli cell was experimentally investigated by using a GFP-producing strain. Two ROIs were applied at the rotational center of the tethered cell producing EGFP (fig. S3D); the sizes of ROI 1 and ROI 2 were the same as in the analysis for the fluorescent spot of GFP-fusion proteins. The calculation of F2 − F1 P2 − P1 P1 in Eq. 1 corresponds to the apparent background intensity (ABg) of the cell. If the background intensity is uniformly distributed throughout the cell, F1 and ABg should be the same. However, F1 was about 1.3 times higher than the experimentally estimated ABg (fig. S3E, row 4), which was consistent with theoretical estimation (fig. S3C), most likely because of the rod shape of the E. coli cell and its effect on the fluorescence profile. Therefore, we applied ~1.3 as correction factor (C) in the estimation of fluorescence intensity (Eq. 1).

Estimation of the number of GFP-fusion proteins incorporated into a functioning flagellar motor To investigate the relationship between the fluorescence intensity and the number of GFP-fusion proteins incorporated into a flagellar motor, we measured the time course of fluorescence intensity derived from the FliMGFP molecules using Eq. 1, and the photobleaching profile of the FliMGFP-labeled motor was fitted by a single exponential function (Eq. 2). FFliMGFP ¼ I0 e−lt

ð2Þ

where FFliM-GFP is the fluorescence intensity of a motor labeled by FliM-GFP, t is the laser exposure time, l is a rate constant, and I0 is the initial intensity of the motor when t = 0.


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E. coli cells harboring plasmids encoding GFP-fusion proteins were grown in TB supplemented with the appropriate antibiotics and inducers at 30°C for 5.25 hours. To shorten flagellar filaments, cells in l ml of culture were passed through a narrow polystyrene tube connected between two syringes more than 100 times. Cells were harvested by centrifugation and suspended in 1 ml of motility medium, called 10NaMB [10 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA-2K (pH 7.0), 10 mM NaCl, and 75 mM KCl]. Cells were harvested by centrifugation and resuspended in 350 ml of 10NaMB. The cell suspension was loaded into the space between the glass slide and coverslip with spacers and incubated for 10 min. The space between the coverslips was gently perfused with additional 10NaMB to remove the remaining unattached cells. To simultaneously visualize the localization of GFP-fusion proteins and the rotational switching of a flagellar motor, we recorded fluorescence and bright-field images of a tethered cell at 50 frames/s by an EMCCD camera and at 200 frames/s by a highspeed CCD camera, respectively.

mouse immunoglobulin G antibody and the AP conjugate substrate kit (Bio-Rad). To estimate the total amount of CheY-GFP in a cell, the immunoblot image was captured, and the total intensity within each discrete band was estimated using ImageJ (http://rsb.info.nih.gov/ij/). By comparing the intensity of the CheY-GFP and purified EGFP bands, the amount of CheY-GFP in the cell suspension applied for immunoblotting was estimated. The intracellular concentration of CheY-GFP was estimated from the average cellular volume and the number of cells in the cell suspension applied for immunoblotting.

RESEARCH ARTICLE The number of CheY-GFP molecules bound to a motor was estimated by Eq. 3. NcheYGFP ¼ ð34 þ − 2Þ

FcheYGFP I0

ð3Þ

where NCheY-GFP is the number of CheY-GFP bound to a flagellar motor, FCheY-GFP is the fluorescence intensity of a motor labeled by CheY-GFP, and I0 is the initial intensity of the motor labeled by FliM-GFP when t = 0. We applied 34 ± 2 as the number of FliM molecules in a single flagellar motor because the number of FliM subunits has been proposed to distribute around 34 (15–17, 20).

Correlation analysis The correlation analysis was performed by applying Eq. 4 (10) to the time traces of the rotational direction of the motor and to the fluorescence intensity at the motor.

NCheYGFP ¼

t m ð1 − e−jtj=t Þ þ b jtj

ð5Þ

where NCheY-GFP is the number of CheY-GFP molecules bound to a flagellar motor, t is time (t ≠ 0), t is a time constant, m is the magnification ratio, and b is the base for fitting. Here, we defined “t × 2” as the time span for increasing and decreasing the number of bound CheY-P molecules during CCW-to-CW and CW-to-CCW switching, respectively.

ð4Þ

Estimation of the dynamic change in CheY-P concentration

where Z is the function used for the correlation analysis, t is time, t is a time difference, and N is the total number of sampling points. x(t) and y(t) are the time traces of the fluorescence intensity at the motor and of the rotational direction of the motor, respectively. Before performing the correlation analysis, the time trace of the rotational velocity was filtered by the Chug-Kennedy filtering algorithm (35), using an analytical window of 5 data points and a weight of 10.

To investigate whether the change in CheY concentration explains the subsecond time scale of the CheY-P binding and dissociation phases (Fig. 4), we simulated the change in CheY-P concentration considering the chemotactic reactions and the diffusion of chemotactic proteins. To calculate the change of CheY-P concentration around a flagellar motor, we performed a particle-based Monte Carlo simulation to estimate the diffusion of CheY and CheY-P molecules in the cytoplasm. Ten thousand CheY particles were placed in a 2-mm-long and 0.8-mm-wide rectangle (Fig. 5A), which corresponds to 13 mM CheY. These particles can freely and two-dimensionally diffuse in the rectangle with a diffusion coefficient of 11.7 mm2/s, as estimated in our previous report (10). The phosphorylation of CheY and dephosphorylation of CheY-P occur by the following reaction scheme:

t¼1

t¼1

Estimation of the number of CheY-P molecules bound to a flagellar motor during CW rotation by a conventional kinetic scheme We estimated the number of CheY-P molecules bound to a flagellar motor during CW rotation using a conventional kinetic scheme and parameters reported in previous studies (3, 24). Therefore, we hypothesized that the binding and dissociation reactions occur independently among 34 binding sites by the following reaction scheme:

M

p⋅ ½YP → ← q

M⋅ YP

where M is a FliM subunit, [YP] is the CheY-P concentration, M·YP is a FliM subunit that binds CheY-P, p is the secondary rate constant of CheY-P binding (5 × 106 M−1 s−1), and q is the dissociation rate of CheY-P from the motor (20 s−1), as previously reported (3, 24).

Estimation of the time spans for increasing and decreasing the number of bound CheY-P molecules during switching To estimate the time for the number of CheY-P molecules bound to a motor to increase or decrease, we extracted individually the changes in fluorescence intensity during CCW-to-CW and CW-to-CCW switching (fig. S4, A and B). The extracted fluorescence profile was applied to cross-correlation analysis with the reference trace by using Eq. 4. The reference trace was composed of the same data points of the extracted fluorescence profile and constructed by −1 and +1 (fig. S4C). For the CCW-to-CW switching, −1 was changed to +1 at the half of the data points of the reference trace. On

Y þ A2 P YP þ Z2

k1 YP þ A2 → k2 → Y þ Z2

where Y is a CheY, YP is a CheY-P, A2 is a CheA dimer, A2P is an autophosphorylated CheA, and Z2 is a CheZ dimer. k1 and k2 are the rate constants of CheY phosphorylation (1.0 × 108 M−1 s−1) and CheY-P dephosphorylation (1.6 × 106 M−1 s−1), respectively, as previously reported (24). Values of 2500 CheA molecules and 3200 CheZ molecules were assumed and used in this calculation to represent physiological conditions (11). All the CheA and CheZ molecules were located at the left side of this rectangle as reported previously (1, 13) (within 20 nm from the edge), and the CheYand CheY-P molecules were phosphorylated and dephosphorylated in this area. CheY molecules were phosphorylated from 0 to 2 s, and CheY-P was dephosphorylated constantly from 0 to 4 s. The motor was positioned 1 mm from the left side of the rectangle. The number of CheY-P molecules within a 0.2-mm-long and 0.8-mm-wide area surrounding a motor was counted, and the number of molecules was converted to the concentration (Fig. 5, B and C).

Estimation of the ratio of the dissociation constants of the CW- and CCW-state motors for CheY-P To discuss the ratio of the dissociation constant of the CW- and CCWstate motors for CheY-P, we assumed the following reaction schemes: → MCCW ⋅ YP MCCW þ YPCCW ← → MCW ⋅ YP MCW þ YPCW ←


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N ‐‐ ∑ ½xðtÞ⋅ yðt þ tÞ − xðtÞ⋅ yðtÞ ZðtÞ = t¼1 sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi N N ‐ ‐ 1 ∑ ½xðtÞ − xðtÞ2 ⋅ N1 ∑ ½yðtÞ − yðtÞ2 N 1 N

the other hand, for the CW-to-CCW switching, +1 was changed to −1 at the half of the data points of the reference trace. The calculated correlation profile was fitted with a Gaussian function (fig. S4D). Dt value was estimated from the time in the peak of fitted curve, and time 0 for the extracted profile of intensity was estimated from Dt value (fig. S4E). The same procedure was performed to all extracted fluorescence profiles. These adjusted profiles were averaged, and the intensity values were converted to “the number of CheY-P molecules bound to a flagellar motor” (Fig. 4). The averaged profile of fluorescence intensity during switching (times of binding and dissociation phases) (Fig. 4) was fitted by Eq. 5.

RESEARCH ARTICLE where MCCW is a FliM subunit in a CCW-state motor, YP is CheY-P, and MCCW·YP is a FliM subunit in a CCW-state motor that binds CheY-P. MCW is a FliM subunit in a CW-state motor, and MCW·YP is a FliM subunit in a CW-state motor that binds CheY-P. The ratio of the dissociation constants of the CW- and CCW-state motors for CheY-P (RKd) is expressed as follows: ½MCW ½YPCW

RKd ¼

KdCW ½MCW ⋅YP ¼ KdCCW ½MCCW ½YPCCW ½MCCW ⋅YP

Because the number of FliM subunits in a motor is sufficiently small compared to that of CheY-P molecules in a cell under both conditions of the motor, RKd is rewritten as follows: RKd ¼

½MCW ½MCCW ⋅YP ½MCCW ½MCW ⋅YP

½MCCW þ ½MCCW ⋅YP ¼ ½M0 ½MCW þ ½MCW ⋅YP ¼ ½M0 The number of bound caCheY-GFP molecules in the CCW- and CWstate motors was estimated to be 1.4 and 5.7, respectively. Therefore, the ratio of CCW- and CW-state motors that bind CheY-P is expressed as follows: RBoundCheY ¼

½MCW ⋅YP 5:7 ¼ ½MCCW ⋅YP 1:4

Alternatively, the ratio of FliM subunits that bind CheY-P in a motor (RCCW or RCW) is expressed as follows: ½MCCW ⋅YP 1:4 ¼ ½M0 34 ½MCW ⋅YP 5:7 ¼ ¼ ½M0 34

RCCW ¼ RCW

where 34 indicates the number of FliM subunits in a motor. Therefore, the ratio of the dissociation constants of the CW- and CCW-state motors for CheY-P (RKd) is rewritten as follows: ½MCW ½MCCW ⋅YP ½MCCW ½MCW ⋅YP ½M0 − ½MCW ⋅YP ½MCCW ⋅YP ¼ ½M0 − ½MCCW ⋅YP ½MCW ⋅YP ½MCW ⋅YP 1− ½MCCW ⋅YP ½M0 ¼ ½MCCW ⋅YP ½MCW ⋅YP 1− ½M0 1 − RCW 1 ¼ 1 − RCCW RBoundCheY

RKd ¼

Therefore, RKd ¼

KdCW ¼ 0:21 KdCCW

SUPPLEMENTARY MATERIALS www.sciencesignaling.org/cgi/content/full/7/319/ra32/DC1 Fig. S1. Immunoblotting of CheY-GFP in whole-cell extracts with an anti-GFP antibody. Fig. S2. Fluorescence intensity and rotational direction of a motor composed of FliM-GFP.

REFERENCES AND NOTES 1. G. H. Wadhams, J. P. Armitage, Making sense of it all: Bacterial chemotaxis. Nat. Rev. Mol. Cell Biol. 5, 1024–1037 (2004). 2. R. C. Stewart, Kinetic characterization of phosphotransfer between CheA and CheY in the bacterial chemotaxis signal transduction pathway. Biochemistry 36, 2030–2040 (1997). 3. V. Sourjik, H. C. Berg, Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. U.S.A. 99, 12669–12674 (2002). 4. A. Bren, M. Eisenbach, The N terminus of the flagellar switch protein, FliM, is the binding domain for the chemotactic response regulator, CheY. J. Mol. Biol. 278, 507–514 (1998). 5. M. Welch, K. Oosawa, S. Aizawa, M. Eisenbach, Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc. Natl. Acad. Sci. U.S.A. 90, 8787–8791 (1993). 6. A. Ishihara, J. E. Segall, S. M. Block, H. C. Berg, Coordination of flagella on filamentous cells of Escherichia coli. J. Bacteriol. 155, 228–237 (1983). 7. R. M. Macnab, D. P. Han, Asynchronous switching of flagellar motors on a single bacterial cell. Cell 32, 109–117 (1983). 8. B. E. Scharf, K. A. Fahrner, L. Turner, H. C. Berg, Control of direction of flagellar rotation in bacterial chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 95, 201–206 (1998). 9. P. Cluzel, M. Surette, S. Leibler, An ultrasensitive bacterial motor revealed by monitoring signaling proteins in single cells. Science 287, 1652–1655 (2000). 10. S. Terasawa, H. Fukuoka, Y. Inoue, T. Sagawa, H. Takahashi, A. Ishijima, Coordinated reversal of flagellar motors on a single Escherichia coli cell. Biophys. J. 100, 2193–2200 (2011). 11. M. Li, G. L. Hazelbauer, Cellular stoichiometry of the components of the chemotaxis signaling complex. J. Bacteriol. 186, 3687–3694 (2004). 12. H. Fukuoka, Y. Inoue, S. Terasawa, H. Takahashi, A. Ishijima, Exchange of rotor components in functioning bacterial flagellar motor. Biochem. Biophys. Res. Commun. 394, 130–135 (2010). 13. B. J. Cantwell, R. R. Draheim, R. B. Weart, C. Nguyen, R. C. Stewart, M. D. Manson, CheZ phosphatase localizes to chemoreceptor patches via CheA-short. J. Bacteriol. 185, 2354–2361 (2003). 14. F. Togashi, S. Yamaguchi, M. Kihara, S. I. Aizawa, R. M. Macnab, An extreme clockwise switch bias mutation in fliG of Salmonella typhimurium and its suppression by slow-motile mutations in motA and motB. J. Bacteriol. 179, 2994–3003 (1997). 15. H. Suzuki, K. Yonekura, K. Namba, Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis. J. Mol. Biol. 337, 105–113 (2004). 16. D. R. Thomas, D. G. Morgan, D. J. DeRosier, Rotational symmetry of the C ring and a mechanism for the flagellar rotary motor. Proc. Natl. Acad. Sci. U.S.A. 96, 10134–10139 (1999). 17. D. R. Thomas, N. R. Francis, C. Xu, D. J. DeRosier, The three-dimensional structure of the flagellar rotor from a clockwise-locked mutant of Salmonella enterica serovar Typhimurium. J. Bacteriol. 188, 7039–7048 (2006). 18. N. J. Delalez, G. H. Wadhams, G. Rosser, Q. Xue, M. T. Brown, I. M. Dobbie, R. M. Berry, M. C. Leake, J. P. Armitage, Signal-dependent turnover of the bacterial flagellar switch protein FliM. Proc. Natl. Acad. Sci. U.S.A. 107, 11347–11351 (2010).


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Here, we assumed that the total concentration of FliM subunits in a motor [M0] is constant regardless of the state of motor.

Fig. S3. Estimation of fluorescence intensity at the rotational center of a tethered cell. Fig. S4. Extraction of the fluorescence intensity profile during switching events and time adjustment of the extracted profile. Table S1. Bacterial strains and plasmids. Table S2. Number of CheY-GFP molecules bound to flagellar motor. Movie S1. Bright-field and fluorescence imaging of a tethered cell producing CheY-GFP. Movie S2. Fluorescence imaging of a tethered cell expressing CheY-GFP and producing CW-locked mutant motor. Movie S3. Fluorescence imaging of a tethered cell expressing CheY-GFP, producing CW-locked mutant motor, and treated with attractant. Movie S4. Fluorescence imaging of a tethered cell expressing CheY-GFP, producing CW-locked mutant motor, and treated with repellent. Movie S5. Fluorescence imaging of a tethered cell expressing CheY-GFP and producing wild-type motor. Movie S6. Fluorescence imaging of a tethered cell expressing CheY-GFP, producing wildtype motor, and treated with attractant. Movie S7. Fluorescence imaging of a tethered cell expressing CheY-GFP, producing wildtype mutant motor, and treated with repellent. Movie S8. Fluorescence imaging of a tethered cell expressing caCheY-GFP and producing wild-type motor. Movie S9. Fluorescence imaging of a tethered cell expressing caCheY-GFP and producing CW-locked mutant motor. References (36, 37)

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Direct Imaging of Intracellular Signaling Components That Regulate Bacterial Chemotaxis Hajime Fukuoka, Takashi Sagawa, Yuichi Inoue, Hiroto Takahashi and Akihiko Ishijima (April 1, 2014) Science Signaling 7 (319), ra32. [doi: 10.1126/scisignal.2004963]

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"Supplementary Materials" http://stke.sciencemag.org/content/suppl/2014/03/28/7.319.ra32.DC1.html The editors suggest related resources on Science's sites: http://stke.sciencemag.org/content/sigtrans/3/128/ra50.full.html http://www.sciencemag.org/content/sci/344/6181/236.3.full.html This article cites 37 articles, 19 of which you can access for free at: http://stke.sciencemag.org/content/7/319/ra32#BIBL Look up definitions for abbreviations and terms found in this article: http://stke.sciencemag.org/cgi/glossarylookup Obtain information about reproducing this article: http://www.sciencemag.org/about/permissions.dtl

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Chemosensory signaling systems that control bacterial survival.

Bacterial chemotaxis and the molecular logic of intracellular signal transduction networks.

Chamber for bacterial chemotaxis experiments.

Bacterial chemotaxis without gradient-sensing.

Macroscopic equations for bacterial chemotaxis: integration of detailed biochemistry of cell signaling.

CheW complex enhances autophosphorylation and affinity for CheY.

Evidence that the methylesterase of bacterial chemotaxis may be a serine hydrolase.

Increasing and decreasing the ultrastability of bacterial chemotaxis core signaling complexes by modifying protein-protein contacts.

Limits of feedback control in bacterial chemotaxis.

Signaling Pathways Controlling Microglia Chemotaxis.

Statistical measures of bacterial motility and chemotaxis.

Exponential signaling gain at the receptor level enhances signal-to-noise ratio in bacterial chemotaxis.

Deciphering the components that coordinately regulate virulence factors of the soft rot pathogen Dickeya dadantii.

Components of bacterial polysaccharides.

Stochastic gene expression: bacterial elites in chemotaxis.

Universal response-adaptation relation in bacterial chemotaxis.

Change in membrane potential during bacterial chemotaxis.

Bacterial chemotaxis in a fixed attractant gradient.

Relevance of intracellular polarity to accuracy of eukaryotic chemotaxis.

Polymorphonulcear leukocyte chemotaxis toward oxidized lipid components of cell membranes.

Suppression of leukocyte chemotaxis by human IgA myeloma components.

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells.

Direct measurement of intracellular pressure.

Intracellular trafficking of bacterial toxins.