
==== Front
Sci Adv
Sci Adv
sciadv
advances
Science Advances
2375-2548
American Association for the Advancement of Science

adp5636
10.1126/sciadv.adp5636
Research Article
Biomedicine and Life Sciences
SciAdv r-articles
Microbiology
CheB localizes to polar receptor arrays during repellent adaptation
CheB localization depends on receptor’s activity
https://orcid.org/0000-0002-9060-4347
Fukuoka Hajime Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing - original draft Writing - review & editing *
Nishitani Keisuke Investigation Validation
Deguchi Taiga Investigation Validation
Oshima Taketo Investigation Validation
Uchida Yumiko Investigation Writing - review & editing
https://orcid.org/0000-0002-8654-9322
Hamamoto Tatsuki Investigation †
Che Yong-Suk Writing - review & editing
https://orcid.org/0000-0002-8919-9268
Ishijima Akihiko Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing - review & editing
Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
* Corresponding author. Email: fukuoka.hajime.fbs@osaka-u.ac.jp
† Present address: Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna-son, Kunigami-gun, Okinawa 904-0495, Japan.

20 9 2024
20 9 2024
10 38 eadp563601 4 2024
14 8 2024
Copyright © 2024 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).
2024
The Authors
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Adaptation of the response to stimuli is a fundamental process for all organisms. Here, we show that the adaptation enzyme CheB methylesterase of Escherichia coli assembles to the ON state receptor array after exposure to the repellent l-isoleucine and dissociates from the array after adaptation is complete. The duration of increased CheB localization and the time of highly clockwise-biased flagellar rotation were similar and depended on the strength of the stimulus. The increase in CheB at the receptor array and the decrease in cytoplasmic CheB were both ~100 molecules, which represents 15 to 20% of the total cellular content of CheB. We confirmed that the main binding site for CheB in the ON state array is the P2 domain of phosphorylated CheA, with a second minor site being the carboxyl-terminal pentapeptide of the serine chemoreceptor. Thus, we have been able to quantify the regulation of the signal output of the receptor array by the intracellular dynamics of an adaptation enzyme.

Intracellular distribution of CheB and behavioral response of cell to repellent stimuli are quantified in a single E. coli cell.

http://dx.doi.org/10.13039/501100001691 Japan Society for the Promotion of Science JP19H05797 http://dx.doi.org/10.13039/501100001691 Japan Society for the Promotion of Science JP21K06097
==== Body
pmcINTRODUCTION

Escherichia coli cells swim by rotating left-handed helical flagellar filaments. Cells can navigate chemical gradients in a liquid medium with high precision, a behavior termed chemotaxis (1, 2). To monitor attractant or repellent chemicals in its environment, E. coli uses transmembrane chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). E. coli has four canonical MCPs for sensing attractant nutrients: Tsr (serine), Tar (aspartate and maltose), Tap (dipeptides), and Trg (ribose and galactose). These chemoreceptors have a common functional architecture and transmit sensory messages to the flagellar motors through a two-component phosphorelay (3). This phosphorelay directs cellular swimming by regulating the rotational direction of the flagellar motor; counterclockwise (CCW) rotation produces forward swimming; clockwise (CW) rotation triggers random reorientations in the swimming direction, called tumbles (4). In this study, we simultaneously measured the behavioral response to a repellent stimulus and the intracellular distribution of the adaptation enzyme CheB in single E. coli cells. We show that CheB is recruited to the ON state receptor array generated by a repellent stimulus and dissociates from the OFF state array once adaptation is complete. We quantified changes in the distribution of CheB and show that they correlate with the behavioral response.

MCPs assemble into large signaling arrays at the cell pole(s) through binding interactions with two cytoplasmic proteins (Fig. 1A): a histidine autokinase CheA and a scaffolding protein CheW that couples CheA activity to receptor control (3, 5–7). Receptor arrays modulate the autophosphorylation activity of CheA to control the flux of phosphoryl groups from CheA to the response regulator CheY. Phosphorylated CheY (CheY-P) works as the intracellular signaling molecule that binds to a flagellar motor to induce CW rotation (8–13). CheY-P molecules reach the flagellar motors through intracellular diffusion (14). CheZ, a CheY-P phosphatase, degrades the CW signal.

Fig. 1. An overview of the E. coli sensory adaptation system and the method used to follow the localization of CheB-GFP and the switching of flagellar motors.

(A) E. coli sensory adaptation system. The signaling activity of the polar chemoreceptor (MCP) array at the cell pole modulates the autophosphorylation of the His48 residue in the P1 domain of the CheA (A) dimer, which, in turn, donates its phosphoryl groups (P) to CheY (Y-P) and CheB (B-P). CheY-P interacts with the flagellar motors to promote CW rotation. CheB deamidates and demethylates adaptation-site residues in the receptor signaling domain; phosphorylation of CheB enhances these activities. CheB preferentially acts on receptors in the kinase-ON state to shift their output toward the OFF state. CheR (R) preferentially methylates available adaptation sites on receptors in the kinase-OFF state to shift their output toward the ON state. CheW (W) is a scaffolding protein that couples CheA activity to receptor control. ADP, adenosine 5′-diphosphate. (B) Schematic diagram of the microscope and method used for simultaneously viewing polar-localized CheB-GFP and flagellar motor rotation. Cells were stuck to a coverslip via sticky flagellar filaments, and polystyrene beads (ϕ = 1.0 μm) were allowed to attach to the stubs of freely rotating sticky flagellar filaments extending into the medium. The angular velocity of each motor could be determined from the movement of the attached bead. Obj, objective lens; LP, long-pass filter; CF, cold filter; DM, dichroic mirror; Em, emission filter.

Two chemotaxis proteins, CheR and CheB, mediate sensory adaptation in the E. coli chemotaxis system (Fig. 1A). CheR is a methyltransferase that converts specific glutamate (E) residues in MCPs to methylated glutamate (Em) residues (15). CheR preferentially acts on receptors in the kinase-OFF state and shifts their output to the kinase-ON state. CheB is a methylesterase that demethylates Em residues to E residues (15); its activity is enhanced upon phosphorylation to phosphorylated CheB (CheB-P) by CheA. CheB preferentially acts on receptors in the kinase-ON state and shifts their output to the OFF state. The methylation level of MCP is feedback-controlled in response to its activity. Therefore, the interplay of the activities of CheR and CheB comprises a feedback system of sensory adaptation that controls the signaling activity of the receptor array (16–18). The interplay of CheR and CheB action in the absence of external stimuli produces spontaneous changes in the output activity of the chemoreceptor array that generate fluctuations in the intracellular CheY-P concentration (19). These fluctuations produce the alternating periods of running (CCW flagellar rotation) and tumbling (CW flagellar rotation) that constitute the random walk of E. coli motility.

CheB and CheR are known to localize to the cell pole under steady-state conditions (20, 21). A C-terminal pentapeptide sequence (NWETF) present on Tsr and Tar is known to bind CheR and CheB. Receptor binding substantially enhances the ability of CheR and CheB to encounter and modify the E residues that are the targets for adaptation (22–25). The P2 domain of CheA is known to be a binding target for CheB and CheY (20, 26). The stable steady-state turnover of these two sensory adaptation enzymes has been reported previously (27).

Tsr recognizes some compounds, including l-leucine and l-isoleucine, as repellents (28). They may bind to the same site as the attractant serine (29) but with a different stereochemistry. The repellent response is important, but how chemotaxis proteins function during repellent responses is largely unknown. Here, we address this question by observing the localization of adaptation proteins fused with green fluorescent protein (GFP) and the pattern of flagellar rotation in single E. coli cells exposed to the repellent isoleucine.

RESULTS

CheB localizes to receptor arrays after a strong repellent stimulus

To measure the intracellular dynamics of the CheB demethylase and motor rotation simultaneously, we used a strain (EFS073) with chromosomal in-frame deletions of the genes encoding all four MCPs, aer (a redox sensor), and the adjacent cheR and cheB genes. CheB fused with GFP at the C terminus (CheB-GFP) and wild-type CheR were supplied from an expression plasmid, pFSRB-GFP, and wild-type Tsr was expressed from a plasmid, pPA114. The subcellular localization of CheB-GFP in glass surface–attached cells was monitored with an electron-multiplying charge-coupled device (EMCCD) camera, and rotation of the flagellar motor was monitored with a high-speed camera by following the rotation of a small bead (ϕ = 1 μm) attached to a sticky flagellar filament (Fig. 1B) (30, 31). We note that the CheB-GFP fusion protein remains fully functional. Cells producing CheB-GFP had the same ability to support chemotaxis in semisolid agar as cells producing wild-type CheB, and the cells showed coordinated directional switching between two flagellar motors on the same cell (fig. S1, A to D).

To investigate the intracellular dynamics of CheB after exposure to repellent, we used a hydrophobic amino acid, l-isoleucine (28). The sequential images of CheB localization before and after the addition of 25 mM isoleucine are shown in Fig. 2A. The polar localization of CheB-GFP increased transiently, and then CheB-GFP dispersed (Fig. 2A and movie S1). The time traces of the fluorescence intensities at a cell pole and in the cytoplasm of this cell are shown in Fig. 2B. The intensity of GFP fluorescence at the cell pole increased over approximately 15 s (20 to 35 s) after the addition of isoleucine and then decreased to its original level over the next approximately 10 s (35 to 45 s) (Fig. 2B, red trace). Although the increase in fluorescence intensity (FI) and the duration of its localization varied from cell to cell, the same trend was seen in all 20 cells that we observed (Fig. 2C). The changes in FI in the cytoplasm were inversely correlated with the FI at the cell poles; the fluorescence in the cytoplasm decreased immediately after the addition of isoleucine and then returned to its original level (Fig. 2B, blue trace).

Fig. 2. Simultaneous visualization of CheB-GFP and flagellar motor rotation after addition of 25 mM l-isoleucine.

(A) Sequential fluorescence images of an EFS073 cell harboring plasmids pPA114 [wild-type Tsr (WTTsr)] and pFSRB-GFP (CheR and CheB-GFP) before and after the addition of isoleucine. The arrowhead indicates increased CheB-GFP localization at a cell pole. Scale bar, 1 μm. The average images for every 10 s are shown. (B) Time traces of fluorescence intensities at the cell pole (red) and cytoplasm (blue) and the rotational velocity (green) of the cell shown in (A). The plus and the minus values in the rotational velocity represent CCW and CW rotation, respectively. The vertical dashed line marks the time of isoleucine addition. In this trial, the rotation of the motor stalled for a few seconds during the flow that introduced isoleucine. a.u., arbitrary units. (C) The gray lines represent the change in FI at the pole of individual 20 cells. Each trace was filtered by moving average, using an analytical window of 20 data points, which corresponds to 1 s; the red line shows the average of those traces. In these traces, the x-axis values were adjusted so that the start of the increase in FI was 0 s. All the time traces were adjusted so that the average intensity before addition of isoleucine (maximum of 20 s) was zero. (D) Sequential fluorescence images of an EFS073 cell carrying plasmids pUCI30a (TsrΔNWETF) and pFSRB-GFP before and after the addition of isoleucine. Scale bar, 1 μm. (E) Time traces of fluorescence intensities at the cell pole and in the cytoplasm and of the rotational velocity of the cell shown in (D). The rotation stalled briefly several times by the flow that introduced isoleucine. (F) The change in FI at the pole of 22 individual cells (gray) and the average of those traces (red).

The motor of this cell frequently switched its rotational direction before the addition of isoleucine (Fig. 2B, green trace). After the addition of isoleucine, the motor rotated exclusively CW for about 20 s in a repellent response. Then, the motor resumed switching and showed stable CCW rotation over time, indicating that the activity of the receptor array in producing CheY-P returned to prestimulus levels. This behavior of the motor and the changes in localization of CheB were in good agreement. These results indicate that the polar localization of CheB accurately reports the activity of the receptor array in producing CheY-P.

To confirm that the decrease in CheB-GFP at the cell poles to the original level is due to adaptation, we examined the response to l-isoleucine in cells producing a mutant Tsr lacking the C-terminal NWETF pentapeptide (TsrΔNWETF). The NWETF pentapeptide is known to be a site for binding CheB and is essential for the demethylation of the chemoreceptor that promotes adaptation (Fig. 1A) (22–25, 32, 33). TsrΔNWETF was expressed from a plasmid, pUCI30a, under the same expression condition as wild-type Tsr with CheB-GFP and CheR. The expression level that is comparable to wild-type Tsr has been observed (19). In cells producing TsrΔNWETF, after the addition of 25 mM isoleucine, the fluorescence at the cell pole increased (Fig. 2, D and E, red trace, and movie S2), but it did not subsequently decrease as it did in the presence of wild-type Tsr. The intensity of fluorescence in the cytoplasm decreased upon the addition of isoleucine and remained at a lower level for an extended period (Fig. 2E, blue trace). These cells responded to isoleucine with frequent switching to CW flagellar rotation that was sustained for a long time (Fig. 2E, green trace). This phenomenon was observed in all 22 cells that we examined (Fig. 2F). Thus, the receptor array containing TsrΔNWETF is activated by isoleucine stimulation but retains its high activity without adapting because the interaction of CheB with NWETF required for the demethylation of Tsr (32, 33) does not occur and the adaptation system does not function properly. Thus, CheB remains bound to the receptor array in its ON state. Together, these results show that CheB is recruited to the receptor array at the cell pole after repellent stimulation but disperses after adaptation dependent on the decreased activity of the receptor array. In the absence of normal adaptation, the receptor array remains active, and CheB remains localized at the cell pole.

We note that, in many cells containing wild-type Tsr, exclusively CCW rotation was observed after adaptation. This prolonged CCW rotation could be due to adaptation overshoot (34).

Dependence of the magnitude of the repellent response on isoleucine concentration

We measured the duration of polar localization of CheB and of high CW bias on the isoleucine concentration. The duration was defined as the time after the addition of isoleucine until CheB localization or CW bias fell to less than half of its maximum value (Fig. 3A, top right schematic). Both times increased with isoleucine concentration from 1 to 25 mM and saturated above 25 mM (Fig. 3A). A plot of the duration of CheB localization versus the duration of CW rotation was linear (Fig. 3B).

Fig. 3. Relationship between the strength of the repellent stimulus and the magnitude of the response.

(A) Duration of CheB-GFP localization (top) and CW rotation (bottom) of EFS073 cells harboring plasmids pPA114 (wild-type Tsr) and pFSRB-GFP (CheR and CheB-GFP) as a function of isoleucine concentration: 1 mM (pink; n = 8 cells), 5 mM (red; n = 13 cells), 15 mM (yellow ochre; n = 19 cells), 25 mM (green; n = 20 cells), 50 mM (light blue; n = 22 cells), and 100 mM (violet; n = 14 cells). Each data point represents a single cell. Blue diamonds show the average of duration in each isoleucine concentration. P > 0.05 [not significant (ns)], *P < 0.05, **P < 0.01, and ***P < 0.001 by Welch’s t test. (B) The relationship between the CW duration and the duration of localization. Colors are the same in (A). The black line shows the best-fit slope of the plot of the duration of polar localization against the duration of CW rotation. The slope of the approximate line and SE were 0.97 and 0.027. The Pearson’s correlation coefficient between these durations was 0.96.

The CW bias represents the intracellular concentration of CheY-P (35, 36). Thus, the linear relationship between the duration of polar localization of CheB and the duration of CW rotation suggests that the localization of CheB reflects the activity level of the polar receptor array. The change in FI at the cell pole (ΔFI) also increased with increasing isoleucine concentration up to 25 mM (fig. S2). Therefore, the time of polar localization of CheB-GFP, ΔFI at the cell pole, and the duration of the CW flagellar rotation all vary in a consistent dose-dependent manner.

At saturating concentrations of isoleucine, there was greater cell-to-cell variation in the durations of CheB localization and CW rotation (Fig. 3A and fig. S3). A negative correlation was observed between the duration of localization and the intensity of fluorescence for the entire cell (fig. S5). Thus, it is likely that cell-to-cell variation in the total amount of CheB present leads to cell-to-cell variation in the duration of CheB localization and the time required adapting to saturating concentrations of repellent.

Quantifying the number of CheB molecules at the cell pole and in the cytoplasm during a repellent response to isoleucine

To quantify the dynamic behavior of CheB, we estimated the number of polar-localized and cytoplasmic CheB-GFP molecules before and after the addition of 25 mM isoleucine (see Materials and Methods, Supplementary Methods, and figs. S10 and S11). There were 22 ± 4 (means ± SE) molecules of CheB-GFP localized at the cell pole before stimulation. This number increased by about 100 to 123 ± 14 molecules after stimulation (Fig. 4, left). In the cytoplasm, 375 ± 36 molecules of CheB-GFP were present before stimulation, and the number decreased by about 140 to 237 ± 29 molecules after stimulation (Fig. 4, right). These data demonstrate that CheB moves from the cytoplasm to the cell pole upon repellent stimulation. The total number of CheB-GFP molecules per cell was 398 ± 38 before the addition of isoleucine and 360 ± 35 after. Thus, almost equal numbers of CheB-GFP molecules showed different patterns of distribution within the cell before and after the addition of repellent. The number of CheB-GFP molecules per cell was similar to previously reported values for the amount of CheB present in wild-type cells (37). This correspondence in protein levels suggests that the behavior of CheB-GFP in our experiments should reflect what occurs in wild-type cells. We note that the culture condition, such as the concentration of inducer for gene expression, was determined by confirming chemotaxis similar to that of the wild-type cell (see Materials and Methods) (19, 38).

Fig. 4. Number of CheB-GFP molecules at cell pole and in the cytoplasm before and after the addition of 25 mM l-isoleucine.

Average numbers of CheB-GFP molecules at the cell pole (left) and in the cytoplasm (right) in EFS073 cells harboring plasmids pPA114 (wild-type Tsr) and pFSRB-GFP (CheR and CheB-GFP) are shown (n = 22 cells). Gray and blue represent before and after the addition of isoleucine, respectively. The error bar shows the SE. **P < 0.01 and ***P < 0.001 by Welch’s t test.

Identity of the CheB-binding site in the receptor array after repellent stimulation

CheB is known to interact with both the NWETF pentapeptide at the C terminus of Tsr and Tar and with the P2 domain of CheA. To elucidate which interaction is important for the increase in the localization of CheB-GFP after stimulation with isoleucine, we examined different mutant receptor arrays. First, we compared localization of CheB in cells expressing wild-type Tsr and TsrΔNWETF. We defined the increased FI (ΔFI) at the cell pole as the difference between the average intensity before stimulation and the maximum intensity after stimulation. The ΔFI at the pole of wild-type Tsr cells was 621 ± 225 (means ± SD), whereas that of TsrΔNWETF cells was 540 ± 235. There is no significant difference in these results (Figs. 2, C and F, and 5A).

Next, we investigated the localization of CheB in cells having the ΔP2 variant of the chromosomal cheA gene, which encodes CheA lacking the P2 domain (CheAΔP2; see table S1). These cells showed transient and weak localization of CheB-GFP immediately after addition of isoleucine (Fig. 5B, top, and movie S3). There was also a small positive ΔFI of ~1.1-fold at the cell pole (Fig. 5C and fig. S6). No increased localization of CheB-GFP was observed in the cells containing both CheAΔP2 and TsrΔNWETF (Fig. 5B, middle, and movie S4), and there was no notable change in ΔFI at the cell pole in these cells (Fig. 5C and fig. S7). These results indicate that the main target for localization of CheB to ON state receptor array is the P2 domain of CheA, with the NWETF pentapeptide of Tsr also contributing slightly to the increased localization of CheB.

Fig. 5. CheB localization in cells expressing mutant variants of Tsr or CheA.

A) Comparison of the increase in the CheB localization (ΔFI) between EFS073 cells producing wild-type Tsr and EFS073 cells producing TsrΔNWETF. The light blue and orange show the results with cells expressing wild-type Tsr (n = 20 cells) or TsrΔNWETF (n = 22 cells). The data are taken from Fig. 2 (C and F). Each data point represents the ΔFI of an individual cell. Bar graphs and error bars indicate the means and SDs, respectively. P > 0.05 (ns) by Welch’s t test. (B) Sequential 10-s fluorescence images (the average images every 10 s) of cells producing CheB-GFP and different CheA mutant proteins before and after stimulation with 25 mM isoleucine. Top: An EFS119 (CheAΔP2) cell producing wild-type Tsr showed weak localization (arrowhead). Middle: An EFS119 (CheAΔP2) cell producing TsrΔNWETF showed no localization. Bottom: An EFS121 cell (CheA-H48Q) cell producing wild-type Tsr showed no localization. Scale bars, 1 μm. See figs. S6 to S8 for time traces of fluorescence intensities at the cell pole and in the cytoplasm and the rotational velocity of the cells shown in (B). (C) Comparison of the FI at the cell pole before and after the addition of 25 mM isoleucine. The intensity at each cell pole was normalized by the average FI immediately before the addition of isoleucine (20 s at most), shown by the black dashed line. The average FI values at the poles of individual cells during the first 10 s after the addition of isoleucine are plotted. P > 0.05 (ns) and *P < 0.05 by Welch’s t test.

Autophosphorylation of CheA is essential for CheB localization

We next asked whether phosphorylation of the P1 domain of CheA is essential for increased localization of CheB. To investigate this question, we used a strain with a chromosomal cheA gene with the mutation that confers the H48Q substitution (see table S1). The His48 residue of CheA becomes phosphorylated upon receptor activation, and the substitution from His to Gln abolishes this process (39). No increase in CheB-GFP at the cell pole was observed in this mutant (Fig. 5B, bottom, and movie S5), and there was no notable ΔFI at the cell pole after the addition of isoleucine (fig. S8B). All 12 cells that we observed behaved in this way (Fig. 5C and fig. S8D). Therefore, phosphorylation of His48 in the P1 domain of CheA is essential for binding of CheB to the P2 domain.

CheR localization to the receptor array does not change with repellent stimulation

CheR catalyzes methylation of specific glutamate residues in MCPs, and CheR interacts with the same target residues as CheB (NWETF pentapeptide) during the adaptation process. To investigate whether the localization of CheR is affected by repellent stimulation, we used the EFS073 strain, which produces plasmid-encoded wild-type CheB and GFP-fused CheR at the N terminus (GFP-CheR), pFSGFPRB1, and plasmid-encoded wild-type Tsr, pPA114, (see table S1). We note that the GFP-CheR fusion and wild-type CheB function comparably as adaptation enzymes under our experimental condition (fig. S1). We confirmed that GFP-CheR localizes at the cell pole as shown previously (Fig. 6A) (19, 21). Cells of this strain gave a repellent behavioral response to 25 mM isoleucine essentially identical to CheB-GFP cells, with a period of CW flagellar rotation followed by a period of adaptation and an overshoot to CCW rotation (Fig. 6B, green). However, there was no obvious change in localization of GFP-CheR after the addition of isoleucine (Fig. 6, A and B, and movie S6). This was true for all eight cells that we observed (Fig. 6, C and D). Localization of CheR also did not change upon addition of the attractant (1 μM l-serine). Cells of this strain gave a typical attractant behavioral response to 1 μM l-serine, with a period of CCW flagellar rotation followed by adaptation (fig. S9 and movie S7). Thus, localization of CheR to the receptor array, presumably via the NWETF pentapeptide, does not change with either repellent or attractant stimulation.

Fig. 6. Response to isoleucine in cells producing GFP-CheR.

(A) Sequential fluorescence images (the average images every 10 s) of an EFS073 cell carrying plasmids pPA114 (wild-type Tsr) and pFSGFPRB1 (GFP-CheR and CheB) before and after the addition of 25 mM isoleucine. The arrowhead points to GFP-CheR localized at a cell pole. Scale bar, 1 μm. (B) The time traces of fluorescence intensities at the cell pole (red) and cytoplasm (blue) and the rotational velocity (green) of the cell shown in (A). The vertical dashed line indicates the time of isoleucine stimulation. (C) Gray lines indicate the normalized FI at the cell pole of eight individual cells. In these traces, the values on the x axis were adjusted so that the addition of isoleucine was at 0 s. Time traces of the FI at the cell pole were normalized to the average value for 20 s immediately before stimulation. The red line shows the average of the traces for individual cells. (D) FI at the cell pole after the addition of isoleucine. The average intensities of the first 10 s immediately after stimulation of cells producing GFP-CheR (blue; n = 8 cells) and CheB-GFP (gray; n = 20 cells). The black dashed line represents the average intensity before the addition of isoleucine.

DISCUSSION

Two proteins, CheR and CheB, interact with the MCP-CheA-CheW receptor array to mediate sensory adaptation in the E. coli chemotaxis system. CheR methylates receptors in the kinase-OFF state to shift their output toward the kinase-ON state. CheB demethylates receptors in the kinase-ON state to shift their output toward the OFF state. Thus, the methylation level of MCPs is feedback controlled by CheR and CheB in response to their signaling activity (16–18). CheR and CheB localize to the receptor array at the cell pole (19–21), and they undergo turnover under stable conditions (27). In the current study, we asked how these adaptation enzymes behave in response to repellent stimulation by observing changes in the intracellular distribution of CheB and CheR fused to GFP in single cells. These changes were correlated with the output activity of the receptor array by observing the CW/CCW rotation of a bead attached to a sticky flagellar filament of the same cell.

After the addition of the repellent l-isoleucine, there was a transient increase in the polar localization of CheB-GFP during the time the flagellum exhibited exclusively CW rotation. The duration of the CheB localization and CW flagellar rotation increased with isoleucine concentration up to 25 mM and then plateaued. Both increased localization and CW rotation lasted about 15 s at and above 25 mM isoleucine. Thus, polar localization of CheB and flagellar rotation track the ON state of the receptor array in unison. The subsequent decrease in polar localization of CheB and the return of CCW flagellar rotation represent the decrease in the activity of the receptor array. We know that the return to prestimulus conditions represents adaptation because cells containing TsrΔNWETF cannot adapt. They respond to isoleucine with polar localization of CheB and CW flagellar rotation but never decrease CheB-GFP localization nor return to CCW flagellar rotation.

We estimate that about 22 molecules of CheB-GFP localize to the cell pole before exposure to isoleucine. That number increased by about 100 molecules to a total of 123 molecules after the addition of 25 mM isoleucine (Fig. 4A). Correspondingly, we estimate that the number of CheB-GFP molecules in the cytoplasm decreased from 375 molecules before the addition of isoleucine to about 237 after the addition of isoleucine, a difference of 138. Given the uncertainty in the determination of the precise number of CheB-GFP molecules, it seems reasonable to conclude that the same number of CheB-GFP molecules left the cytoplasm and became associated with the polar receptor array. Thus, CheB moved from the cytoplasm to the receptor array in response to addition of the repellent isoleucine.

The receptor array typically contains 4000 to 5000 CheA molecules (37). As we confirmed here, CheB primarily binds to the P2 domain of CheA (20). Because about 123 molecules of CheB-GFP localize to the receptor array after a saturating repellent stimulus, there is about a 40-fold excess of potential binding sites for CheB in the ON state array. Because about 237 CheB molecules remain in the cytoplasm, there is the possibility of turnover of CheB between the receptor array and the cytoplasm even when the receptor array is in the ON state.

Step-down stimulation by an attractant also activates the receptor array (6, 34, 40). The response to step-down l-aspartate stimuli by a single swimming cell, which is trapped by two optical tweezers at cell poles, showed little variation over two orders of magnitude change in the size of the step-down concentration jumps; the adaptation time to step-down l-aspartate stimuli (from 5, 100, 500 to 0 μM) was constant (~15 s) regardless of the magnitude in step-down size (34). In contrast, both the time for increased localization of CheB at the receptor array and the duration of CW rotation exhibit a very clear dependence of the strength of the repellent stimulus (Fig. 3A).

One clear difference in these two CW responses is that an attractant-bound receptor is in a more-highly methylated state than a ligand-free receptor. When it returns to its unbound state, adaptation of the CW response occurs by the removal of additional methyl groups that were added to the receptor during adaptation to the attractant. When a repellent is added, the receptor assumes a new repellent-bound conformation that sends a CW signal. Adaptation occurs via removal of a smaller number of methyl groups that were on the receptor in its ligand-free state. Thus, both the conformation of the receptor and the number and identity of the methyl groups that must be removed are different in these two cases. Therefore, it is perhaps expected that the adaptation processes after repellent addition and after attractant removal show very different kinetics.

The increase in FI (ΔFI) at the cell pole after the addition of saturating isoleucine (25 mM) was the same in cells expressing wild-type Tsr and Tsr∆NWETF (Fig. 5A). Thus, the C-terminal NWETF pentapeptide plays no significant role in the initial recruitment of CheB to the receptor array. In cells containing CheA lacking the P2 domain (CheAΔP2), there was only very weak localization of CheB after the addition of isoleucine. Even this weak localization was abolished in cells having both CheAΔP2 and TsrΔNWETF. These results confirm that the main target for localization of CheB to the ON state receptor array is the P2 domain of CheA (20). The minor role played by the NWETF pentapeptide is consistent with its low affinity (Kd ~ 150 μM) for CheB (33). Cells producing CheA(H48Q), in which the His residue in P1 that gets phosphorylated is converted to Gln, cannot autophosphorylate (39). CheB-GFP did not localize to the cell pole in cells whose receptor array contained CheA(H48Q). Thus, autophosphorylation of CheA is essential for the stable binding of CheB to the P2 domain of CheA. During the repellent response to isoleucine, Tsr signals CheA through its kinase control domain and promotes CheA autophosphorylation, which then stabilizes binding of CheB to the P2 domain of CheA.

The interactions between chemotaxis proteins to attractant stimulus is reported by detecting total Förster resonance energy transfer from cell population (41); CheB dissociates from CheA and Tar during the transition from steady state to the kinase-OFF state by attractant stimulus. On the other hand, our single-cell analyses showed recruitment of CheB to CheA and Tsr during the transition from steady state to kinase-ON state array due to the direct sensing of repellent and dissociation from the array by adaptation. These results indicate that CheB binding to the receptor array is sensitive to the active level of chemoreceptor array. The quantitative comparison of these two responses will be necessary to further understand bacterial signal transduction.

We also tested whether the localization of CheR to the receptor array is affected by the signaling state of the receptor array using GFP-fused CheR. There was no change in the intracellular distribution of CheR in response to addition of either the repellent l-isoleucine or the attractant l-serine. We conclude that CheR localization, which is known to occur primarily through the high affinity of CheR for the NWETF pentapeptide (16, 17, 22), is independent of the signaling state of the receptor. The turnover of CheR on the receptor array occurs over the time on the order of tens of seconds (27). Therefore, it appears that CheB association with the receptor array is dynamic and depends on the signaling state of the array and that CheR is relatively stably localized and exchanges at a rate that is independent of the signaling state of the array.

In the steady state before the addition of isoleucine, the kinase activity of the receptor array blinks between the OFF and ON states by the interplay of CheR and CheB action (Fig. 7, top) (19). In the kinase-OFF state of the receptor array, CheA has a low affinity for binding CheB. When Tsr binds isoleucine, the receptor array is strongly biased toward the kinase-ON state, and the His48 residue in CheA in the array is phosphorylated by adenosine 5′-triphosphate (ATP) (Fig. 7, middle, the step 1). This phosphorylation would induce a conformational change in CheA (42, 43). This conformational change allows CheB to bind to the CheA-P2 domain, and CheB receives a phosphoryl group by transfer from His48 in the P1 domain of CheA (Fig. 7, middle, the step 2). The affinity of CheB for the phosphorylated form of CheA is about 3.4-fold higher than that for nonphosphorylated CheA (fig. S4). CheB-P is released from CheA (Fig. 7, middle, the step 3). The local concentration of CheB-P around the receptor array would increase from steps 2 to 3. Released CheB-P interacts with the NWETF pentapeptide of Tsr. While CheB-P bound to the NWETF pentapeptide at the end of the flexible C-terminal tail of Tsr, it can remove the methyl group from Em residues of Tsr (Fig. 7, middle, the step 4). The interaction of CheB-P with the NWETF pentapeptide is essential for demethylation; however, this interaction may be transient, and CheB is immediately released into the cytoplasm. This transient interaction may be due to the fast autodephosphorylation of CheB with its half-life less than 2 s (44) and the difference in the NWETF pentapeptide binding affinity to CheB-P and non–phosophorylated CheB (24). Steps 2 to 4 would be repeated during the adaptation process. Progressive demethylation of MCP gradually reduces the activity of the receptor array; thereby, CheB localization decreases (Fig. 7, middle, the step 5). When adaptation is complete, the receptor array resumes the blinking of activity even in the presence of isoleucine (Fig. 7, bottom). In the receptor array blinking between the OFF and ON states, weak localization is observed because CheB binds only to CheA in the ON state array. The binding of the CheR methyltransferase with the NWETF pentapeptide is unaffected by the activity of the receptor array. Adaptive methylation activity is determined by the accessibility of target glutamate residues, which changes with the conformation of the receptor in different signaling states.

Fig. 7. CheB localization depends on the activity of the receptor array.

(Top) Steady state before the addition of isoleucine. The kinase activity of receptor array blinks between the OFF and ON states due to the oscillations at the methylation level (magenta circles in cytoplasmic domain of Tsr) by the interplay of CheB (green circle marked B) and CheR (orange circle marked R) action. Weak localization is observed because CheB binds only to CheA in the ON state array. (Middle) Active state. (1) When the repellent isoleucine binds to Tsr (red diamond), the receptor array is strongly biased toward the kinase-ON state. His48 in CheA is phosphorylated by ATP. (2) Phosphorylation induces a conformational change in CheA, which allows CheB to bind to CheA-P2 domain, and CheB is phosphorylated by CheA. This step is observed as majority of the increase in the polar localization of CheB after the addition of isoleucine. (3) CheB-P is released from CheA. (4) CheB-P is then free to interact with the NWETF pentapeptide of Tsr to perform demethylation of Tsr. The cycle from (1) to (4) would be repeated during the demethylation process. (5) Progressive demethylation of the MCP reduces the activity of the receptor array; therefore, the localization of CheB decreases. (Bottom) Steady state after adaptation. When adaptation is complete, the receptor array resumes the blinking of the array’s activity even in the presence of isoleucine. Because of demethylation by CheB, the methylation level of the receptor array is low compared to the top panel. CheR binds to the C-terminal NWETF pentapeptide of Tsr with a constant affinity regardless of the activity state of the receptor array.

The basic mechanisms of signal transduction networks through phosphotransfer are not only present in bacterial chemotactic systems but also shared by many organisms, including eukaryotes (45–47). Our findings provide useful insights into the mechanism of signal transduction in a prokaryotic cell, but we believe that they also provide a different perspective on the dynamic behavior of more complex signal transduction networks.

MATERIALS AND METHODS

E. coli strains, plasmids, and cell growth conditions

The strains and plasmids used in this study are listed in table S1. All strains were derived from the K12 strain RP437, which is wild type for chemotaxis (48). Replacements of the wild-type fliC gene with the fliC-sticky gene (31), of the wild-type cheA gene with cheA encoding the H48Q substitution, and of the wild-type cheA gene with cheA deleted for the P2 domain were carried out using the λ red recombinase and the tetracycline sensitivity selection method (49, 50).

LB broth [1% bactotryptone (BD, Sparks, MD), 0.5% yeast extract (BD, Sparks, MD), and 0.5% NaCl (Nakarai, Kyoto, Japan)] was used for culture growth, transformations, and plasmid isolation. Tryptone broth (1% bactotryptone and 0.5% NaCl) was used to grow cells for measurements of motor rotation. For all measurements, the cells were suspended in 10NaMB [10 mM potassium phosphate buffer (Wako, Osaka, Japan) (pH 7.0), 0.1 mM EDTA-2 K (Wako, Osaka, Japan) (pH 7.0), 10 mM NaCl, and 75 mM KCl (Nakarai, Kyoto, Japan)]. Plasmid-bearing derivatives of strains EFS073, EFS119, and EFS121 were grown at 30°C for 5.25 hours in tryptone broth containing chloramphenicol (25 μg/ml) and ampicillin (50 μg/ml). Production of Tsr and TsrΔNWETF was induced with 0.5 mM salicylate, which confers optimal chemotaxis (19, 38). The production of CheB, CheB-GFP, CheR, and GFP-CheR was induced with 0.005% arabinose, which confers chemotaxis like that of a wild-type cell (fig. S1) (19).

Microscope system for the simultaneous observation of fluorescence and bright-field images of cells expressing GFP-fusion proteins

To measure both 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. 1B). For the excitation of GFP-fusion proteins, a blue laser beam (λ = 488 nm) (sapphire 488-20-SV, Coherent, Germany) was inserted into the microscope (IX71, Olympus, Tokyo, Japan) from the right-side port. The blue laser beam was reflected by a primary dichroic mirror (FF495-Di03, Semrock, Lake Forest, IL) and focused on the back focal plane of the first objective lens (APON 60XOTIRF, numerical aperture 1.49, Olympus).

Fluorescence from the GFP-fusion protein was collimated by the first objective lens to form the primary image at the focal point of the microscope imaging lens. The fluorescence was collimated by the second objective lens (UPLFLN4X, numerical aperture 0.13, Olympus) and reflected by a secondary dichroic mirror (FF580-FDi01, Semrock). The fluorescence was then passed through an emission filter (FF01-520/35, Semrock) and focused on an EMCCD camera (DU860D-CS0-BV, Andor Technology, UK) using a tube lens with a focal length of 180 mm (TTL180-A, Thorlabs, Newton, NJ). The final magnification of the fluorescence image was ×240. The fluorescence images were recorded at 20 frames/s, and EM gain was set to 1000.

Long-pass (LP1) (R66, Hoya, Tokyo, Japan) and cold (SC751, Asahi Spectra, Tokyo, Japan) filters were set in front of the halogen lamp to observe the bright-field image. The light for bright-field imaging of the cell was collimated by the first objective lens to form the primary image at the focal point of the microscope imaging lens. The light for bright-field imaging was collimated by a second objective lens and passed through a secondary dichroic mirror and recorded by a high-speed CCD camera (ICL- B0610M-KC, Imperx, Boca Raton, FL, USA) using a tube lens with a focal length of 100 mm (TTL100-A, Thorlabs). Therefore, the final magnification of the fluorescence image was ×133. A long-pass filter (LP2) (BLP01-488R-25, Semrock) was set in front of the high-speed CCD camera to prevent light from the excitation laser from leaking back through the dichroic mirrors. The bright-field images were recorded at 1000 frames/s. Each captured image was transferred via a frame-grabber card (PIXCI-EB1, EPIX, Buffalo Grove, IL) to a computer for image analysis. This high-speed CCD camera was controlled by real-time video nanometry software (51), which we developed using LabVIEW 2009 (National Instruments, Austin, TX).

Preparation of cells for observation

Cells were prepared essentially as described in a previous report (30). One milliliter of cell culture was centrifuged, and the pellet was suspended in 1 ml of 10NaMB. The cell suspension was centrifuged, and the pellet was suspended in ~750 μl of 10NaMB. The cell suspension was loaded into a sample chamber made from 18 mm by 18 mm and 24 mm by 50 mm coverslips with a 0.3-μm silicon spacer (Fig. 1B) and incubated for 15 min to allow the cells to attach to the coverslip. The inside of the sample chamber was gently perfused with additional 10NaMB to remove unattached cells. A suspension of 1.0-μm polystyrene beads (Polysciences, Warrington, PA) was diluted 50-fold with 10NaMB immediately before injection into the chamber. Incubation for 20 min allowed the beads to attach to the sticky flagellar filaments. Gentle perfusion with 30 μl of 10NaMB was used to remove unattached beads. A 30-μl aliquot of isoleucine or serine dissolved in 10NaMB was perfused into the chamber using a peristaltic pump (SJ-1220, ATTO, Tokyo, Japan) on the microscope stage during observation.

Estimation for the time trace of FI and localization duration

We applied two regions of interest (ROIs) surrounding the fluorescent spot at the cell pole (ROIpole) and cytoplasm (ROIcyto); the size of each ROI was 3 pixels by 3 pixels (0.3 μm by 0.3 μm). The localized FI was estimated as followsFIloc=FIpole−C×FIcyto(1)

where FIloc is the FI attributed to the polar-localized GFP-fusion and FIpole and FIcyto are the average fluorescence intensities of ROIpole and ROIcyto, respectively. C corrects for the background fluorescence from cytoplasm at the cell pole (see Supplementary Methods). We used 0.81 as C.

The time trace from Eq. 1 was filtered by a moving average window of 20 data points (1 s). We defined the duration of localization as the time from the addition of isoleucine until FIloc was less than half of its maximum intensity (Fig. 3A, top).

Estimation of rotational velocity and CW duration

Bright-field images of the beads were fitted by a two-dimensional Gaussian function for every sampling frame. The position of a bead was expressed as X and Y coordinates (see the Supplementary Materials) (14, 19, 30). The rotation angle was calculated for every two sampling frames. The time trace of the rotational velocity was filtered by the Chug-Kennedy filtering algorithm (52), using an analytical window of 20 data points and a weight of 10. Rotational velocities of more than +10 Hz, between +10 and −10 Hz, and less than −10 Hz were assigned as CCW rotation (+1), pause (0), and CW rotation (−1), respectively. CW bias was the fraction of time spent in CW rotation per 1 s, and the CW duration was the time from the addition of isoleucine until the CW bias was less than half of its maximum value (Fig. 3A, bottom).

Normalization of FI before and after stimulation

To compare changes in FI, time traces of the intensity at the cell pole were normalized to the average value 20 s before stimulation. The fluorescence in the cell-free area and the autofluorescence of an E. coli cell were subtracted from the time trace of total intensity at the cell pole. These values were determined using EFS073 cells that did not express a GFP fusion. The normalized FI after stimulation was estimated from the normalized trace for the first 10 s after stimulation.

Estimation of the number of GFP molecules localized at the cell pole and the cytoplasm

Details for the calculation of the number of GFP molecules are described in Supplementary Methods and figs. S10 and S11.

Acknowledgments

We thank M. Manson and S. Parkinson for critically reading the manuscript, for useful comments, and for grammatical and stylistic editing. We thank T. Nagai for the gift of the plasmid encoding monomeric segfp.

Funding: This work was supported by Japan Society for the Promotion of Science JP19H05797 (to A.I. and H.F.) and JP21K06097 (to H.F.).

Author contributions: Conceptualization: H.F. and A.I. Data curation: H.F. and A.I. Formal analysis: H.F. and A.I. Methodology: H.F. and A.I. Investigation: H.F., A.I., Y.U., K.N., T.D., T.H., and T.O. Project administration: H.F. and A.I. Resources: H.F. and A.I. Software: H.F. and A.I. Supervision: H.F. and A.I. Validation: H.F. and A.I. Writing—original draft: H.F. Writing—review and editing: H.F., A.I., Y.U., Y.-S.C.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Supplementary Materials

The PDF file includes:

Supplementary Text

Table S1

Figs. S1 to S11

Legends for movies S1 to S7

References

Other Supplementary Material for this manuscript includes the following:

Movies S1 to S7
==== Refs
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).15573139
2 R. Macnab, “Flagela and motility” in Eschericia coli and Salmonella, F. C. Neidhardt, Ed. (American Society for Microbiology, 1996), pp.123–145.
3 J. S. Parkinson, G. L. Hazelbauer, J. J. Falke, Signaling and sensory adaptation in Escherichia coli chemoreceptors: 2015 update. Trends Microbiol. 23 , 257–266 (2015).25834953
4 L. Turner, W. S. Ryu, H. C. Berg, Real-time imaging of fluorescent flagellar filaments. J. Bacteriol. 182 , 2793–2801 (2000).10781548
5 J. Liu, B. Hu, D. R. Morado, S. Jani, M. D. Manson, W. Margolin, Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells. Proc. Natl. Acad. Sci. U.S.A. 109 , E1481–E1488 (2012).22556268
6 A. Burt, C. K. Cassidy, P. Ames, M. Bacia-Verloop, M. Baulard, K. Huard, Z. Luthey-Schulten, A. Desfosses, P. J. Stansfeld, W. Margolin, J. S. Parkinson, I. Gutsche, Complete structure of the chemosensory array core signalling unit in an E. coli minicell strain. Nat. Commun. 11 , 743 (2020).32029744
7 G. E. Pinas, M. D. DeSantis, C. K. Cassidy, J. S. Parkinson, Hexameric rings of the scaffolding protein CheW enhance response sensitivity and cooperativity in Escherichia coli chemoreceptor arrays. Sci. Signal 15 , eabj1737 (2022).35077199
8 H. Fukuoka, T. Sagawa, Y. Inoue, H. Takahashi, A. Ishijima, Direct imaging of intracellular signaling components that regulate bacterial chemotaxis. Sci. Signal 7 , ra32 (2014).24692593
9 R. C. Stewart, Kinetic characterization of phosphotransfer between CheA and CheY in the bacterial chemotaxis signal transduction pathway. Biochemistry 36 , 2030–2040 (1997).9047301
10 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).12232047
11 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).9600834
12 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).8415608
13 O. Afanzar, D. Di Paolo, M. Eisenstein, K. Levi, A. Plochowietz, A. N. Kapanidis, R. M. Berry, M. Eisenbach, The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility. EMBO J. 40 , e104683 (2021).33620739
14 Y. S. Che, T. Sagawa, Y. Inoue, H. Takahashi, T. Hamamoto, A. Ishijima, H. Fukuoka, Fluctuations in intracellular CheY-P concentration coordinate reversals of flagellar motors in E. coli. Biomolecules 10 , 1544–1564 (2020).33198296
15 G. L. Hazelbauer, W. C. Lai, Bacterial chemoreceptors: Providing enhanced features to two-component signaling. Curr. Opin. Microbiol. 13 , 124–132 (2010).20122866
16 U. Alon, M. G. Surette, N. Barkai, S. Leibler, Robustness in bacterial chemotaxis. Nature 397 , 168–171 (1999).9923680
17 N. Barkai, S. Leibler, Robustness in simple biochemical networks. Nature 387 , 913–917 (1997).9202124
18 T. M. Yi, Y. Huang, M. I. Simon, J. Doyle, Robust perfect adaptation in bacterial chemotaxis through integral feedback control. Proc. Natl. Acad. Sci. U.S.A. 97 , 4649–4653 (2000).10781070
19 Y. Uchida, T. Hamamoto, Y. S. Che, H. Takahashi, J. S. Parkinson, A. Ishijima, H. Fukuoka, The chemoreceptor sensory adaptation system produces coordinated reversals of the flagellar motors on an Escherichia coli cell. J. Bacteriol. 204 , e0027822 (2022).36448786
20 S. Banno, D. Shiomi, M. Homma, I. Kawagishi, Targeting of the chemotaxis methylesterase/deamidase CheB to the polar receptor-kinase cluster in an Escherichia coli cell. Mol. Microbiol. 53 , 1051–1063 (2004).15306010
21 D. Shiomi, I. B. Zhulin, M. Homma, I. Kawagishi, Dual recognition of the bacterial chemoreceptor by chemotaxis-specific domains of the CheR methyltransferase. J. Biol. Chem. 277 , 42325–42333 (2002).12101179
22 J. Wu, J. Li, G. Li, D. G. Long, R. M. Weis, The receptor binding site for the methyltransferase of bacterial chemotaxis is distinct from the sites of methylation. Biochemistry 35 , 4984–4993 (1996).8664291
23 A. N. Barnakov, L. A. Barnakova, G. L. Hazelbauer, Efficient adaptational demethylation of chemoreceptors requires the same enzyme-docking site as efficient methylation. Proc. Natl. Acad. Sci. U.S.A. 96 , 10667–10672 (1999).10485883
24 M. Li, X. Xu, X. Zou, G. L. Hazelbauer, A selective tether recruits activated response regulator CheB to its chemoreceptor substrate. mBio 12 , e0310621 (2021).34809457
25 X. Feng, A. A. Lilly, G. L. Hazelbauer, Enhanced function conferred on low-abundance chemoreceptor Trg by a methyltransferase-docking site. J. Bacteriol. 181 , 3164–3171 (1999).10322018
26 J. Li, R. V. Swanson, M. I. Simon, R. M. Weis, The response regulators CheB and CheY exhibit competitive binding to the kinase CheA. Biochemistry 34 , 14626–14636 (1995).7578071
27 S. Schulmeister, M. Ruttorf, S. Thiem, D. Kentner, D. Lebiedz, V. Sourjik, Protein exchange dynamics at chemoreceptor clusters in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 105 , 6403–6408 (2008).18427119
28 W. W. Tso, J. Adler, Negative chemotaxis in Escherichia coli. J. Bacteriol. 118 , 560–576 (1974).4597449
29 X. Chen, S. Bi, X. Ma, V. Sourjik, L. Lai, Discovery of a new chemoeffector for Escherichia coli chemoreceptor Tsr and identification of a molecular mechanism of repellent sensing. ACS Bio. Med. Chem. Au 2 , 386–394 (2022).
30 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).21539787
31 W. S. Ryu, R. M. Berry, H. C. Berg, Torque-generating units of the flagellar motor of Escherichia coli have a high duty ratio. Nature 403 , 444–447 (2000).10667798
32 W. C. Lai, L. A. Barnakova, A. N. Barnakov, G. L. Hazelbauer, Similarities and differences in interactions of the activity-enhancing chemoreceptor pentapeptide with the two enzymes of adaptational modification. J. Bacteriol. 188 , 5646–5649 (2006).16855257
33 A. N. Barnakov, L. A. Barnakova, G. L. Hazelbauer, Allosteric enhancement of adaptational demethylation by a carboxyl-terminal sequence on chemoreceptors. J. Biol. Chem. 277 , 42151–42156 (2002).12196531
34 T. L. Min, P. J. Mears, I. Golding, Y. R. Chemla, Chemotactic adaptation kinetics of individual Escherichia coli cells. Proc. Natl. Acad. Sci. U.S.A. 109 , 9869–9874 (2012).22679285
35 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).9419353
36 P. Cluzel, M. Surette, S. Leibler, An ultrasensitive bacterial motor revealed by monitoring signaling proteins in single cells. Science 287 , 1652–1655 (2000).10698740
37 M. Li, G. L. Hazelbauer, Cellular stoichiometry of the components of the chemotaxis signaling complex. J. Bacteriol. 186 , 3687–3694 (2004).15175281
38 P. Ames, C. A. Studdert, R. H. Reiser, J. S. Parkinson, Collaborative signaling by mixed chemoreceptor teams in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 99 , 7060–7065 (2002).11983857
39 J. F. Hess, R. B. Bourret, M. I. Simon, Histidine phosphorylation and phosphoryl group transfer in bacterial chemotaxis. Nature 336 , 139–143 (1988).3185734
40 J. M. Keegstra, K. Kamino, F. Anquez, M. D. Lazova, T. Emonet, T. S. Shimizu, Phenotypic diversity and temporal variability in a bacterial signaling network revealed by single-cell FRET. eLife 6 , e27455 (2017).29231170
41 D. Kentner, V. Sourjik, Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway. Mol. Syst. Biol. 5 , 238 (2009).19156130
42 C. K. Cassidy, B. A. Himes, D. Sun, J. Ma, G. Zhao, J. S. Parkinson, P. J. Stansfeld, Z. Luthey-Schulten, P. Zhang, Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations. Commun. Biol. 3 , 24 (2020).31925330
43 T. Tran, A. Karunanayake Mudiyanselage, S. J. Eyles, L. K. Thompson, Bacterial chemoreceptor signaling complexes control kinase activity by stabilizing the catalytic domain of CheA. Proc. Natl. Acad. Sci. U.S.A. 120 , e2218467120 (2023).37523532
44 R. C. Stewart, Activating and inhibitory mutations in the regulatory domain of CheB, the methylesterase in bacterial chemotaxis. J. Biol. Chem. 268 , 1921–1930 (1993).8420965
45 T. Maeda, S. M. Wurgler-Murphy, H. Saito, A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369 , 242–245 (1994).8183345
46 S. C. Schuster, A. A. Noegel, F. Oehme, G. Gerisch, M. I. Simon, The hybrid histidine kinase DokA is part of the osmotic response system of Dictyostelium. EMBO J. 15 , 3880–3889 (1996).8670893
47 J. Q. Wilkinson, M. B. Lanahan, H. C. Yen, J. J. Giovannoni, H. J. Klee, An ethylene-inducible component of signal transduction encoded by never-ripe. Science 270 , 1807–1809 (1995).8525371
48 J. S. Parkinson, S. E. Houts, Isolation and behavior of Escherichia coli deletion mutants lacking chemotaxis functions. J. Bacteriol. 151 , 106–113 (1982).7045071
49 K. A. Datsenko, B. L. Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97 , 6640–6645 (2000).10829079
50 S. R. Maloy, W. D. Nunn, Selection for loss of tetracycline resistance by Escherichia coli. J. Bacteriol. 145 , 1110–1111 (1981).7007341
51 T. Sagawa, Y. Kikuchi, Y. Inoue, H. Takahashi, T. Muraoka, K. Kinbara, A. Ishijima, H. Fukuoka, Single-cell E. coli response to an instantaneously applied chemotactic signal. Biophys J. 107 , 730–739 (2014).25099812
52 S. H. Chung, R. A. Kennedy, Forward-backward non-linear filtering technique for extracting small biological signals from noise. J. Neurosci. Methods 40 , 71–86 (1991).1795554
53 Q. Zhou, P. Ames, J. S. Parkinson, Biphasic control logic of HAMP domain signalling in the Escherichia coli serine chemoreceptor. Mol. Microbiol. 80 , 596–611 (2011).21306449
54 K. K. Gosink, M. C. Buron-Barral, J. S. Parkinson, Signaling interactions between the aerotaxis transducer Aer and heterologous chemoreceptors in Escherichia coli. J. Bacteriol. 188 , 3487–3493 (2006).16672602
55 L. M. Guzman, D. Belin, M. J. Carson, J. Beckwith, Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177 , 4121–4130 (1995).7608087
