Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
The study by Wu et al presents interesting data on bacterial cell organization, a field that is progressing now, mainly due to the advances in microscopy. Based mainly on fluorescence microscopy images, the authors aim to demonstrate that the two structures that account for bacterial motility, the chemotaxis complex and the flagella, colocalize to the same pole in Pseudomonas aeruginosa cells and to expose the regulation underlying their spatial organization and functioning.
Strengths:
The subject is of importance.
Weaknesses:
The conclusions are too strong for the presented data. The lack of statistical analysis makes this paper incomplete. The novelty of the findings is not clear.
We have strengthened the data analysis by including appropriate statistical tests to support our conclusions more convincingly. Additionally, we have refined the description of the research background to better emphasize the novelty and significance of our findings. Please see the detailed responses below for further information.
Major issues:
(1) The novelty is in question since in the Abstract the authors highlight their main finding, which is that both the chemotaxis complex and the flagella localize to the same pole, as surprising. However, in the Introduction they state that "pathway-related receptors that mediate chemotaxis, as well as the flagellum are localized at the same cell pole17,18". I am not a pseudomonas researcher and from my short glance at these references, I could not tell whether they report colocalization of the two structures to the same pole. However, I trust the authors that they know the literature on the localization of the chemotaxis complex and flagella in their organism. See also major issue number 5 on the novelty regarding the involvement of c-di-GMP.
We thank the reviewer for this valuable comment and appreciate the opportunity to clarify our statements.
Kazunobu et al. (ref. 18) used scanning electron microscopy to preliminarily characterize the flagellation pattern of Pseudomonas aeruginosa during cell division, showing that existing flagella are located at the old pole. Zehra et al. (ref. 17), through fluorescence microscopy, observed that CheA and CheY proteins in dividing cells are typically also present at the old pole. Based on these observations, we inferred in the Introduction that the chemotaxis complex and flagellum may localize to the same cell pole.
However, this inference is indirect and lacks direct live-cell evidence of colocalization, leaving its validity to be confirmed. This uncertainty was indeed the starting point and motivation for our study.
In our work, we simultaneously visualized flagellar filaments and core chemoreceptor proteins at the single-cell level in P. aeruginosa. We characterized the assembly and spatial coordination of the chemotaxis network and flagellar motor throughout the cell cycle, providing direct evidence of their colocalization and coordinated assembly. This represents a significant advance beyond prior indirect observations and supports the novelty of our study.
Accordingly, we have revised the relevant statements in lines 71-75 of the manuscript to better reflect the current state of the literature and emphasize the novelty of our direct observations.
(2) Statistics for the microscopy images, on which most conclusions in this manuscript are based, are completely missing. Given that most micrographs present one or very few cells, together with the fact that almost all conclusions depend on whether certain macromolecules are at one or two poles and whether different complexes are in the same pole, proper statistics, based on hundreds of cells in several fields, are absolutely required. Without this information, the results are anecdotal and do not support the conclusions. Due to the importance of statistics for this manuscript, strict statistical tests should be used and reported. Moreover, representative large fields with many cells should be added as supportive information.
We thank the reviewer for this important comment, which significantly improves the rigor and persuasiveness of our manuscript.
For the colocalization analyses presented in Fig. 1D and Fig. 2B, we quantified 145 and 101 cells with fluorescently labeled flagella, respectively, and observed consistent colocalization of the chemoreceptor complexes and flagella in all examined cells (now added in the figure legends). Regarding the distribution patterns of chemoreceptors shown in Fig. 3A, we have now included comprehensive statistical analyses for both wild-type and mutant strains. For each strain, more than 300 cells were analyzed across at least three independent microscopic fields, providing robust statistical power (detailed data are presented in Fig. 3C).
To further strengthen the evidence, statistical tests were applied to confirm the significance and reproducibility of our findings (Fig. 3C). In addition, representative large-field fluorescence images containing numerous cells have been added to the supplementary materials (Fig. S1 and Fig. S3).
The problem is more pronounced when the authors make strong statements, as in lines 157-158: "The results revealed that the chemoreceptor arrays no longer grow robustly at the cell pole (Figure 2A)". Looking at the seven cells shown in Figure 2A, five of them show polar localization of the chemoreceptors. The question is then: what is the percentage of cells that show precise polar, near-polar, or mid cell localization (the three patterns shown here) in the mutant and in the wild type? Since I know that these three patterns can also be observed in WT cells, what counts is the difference, and whether it is statistically significant.
We thank the reviewer for raising this important point. Following the reviewer's suggestion, we have now analyzed and categorized the distribution of the chemotaxis complex in both wild-type and flhF mutant strains into three patterns: precise-polar, near-polar, and mid-cell localization. For each strain, more than 200 cells across three independent fields of view were quantified.
Our statistical analysis shows that in the wild-type strain, approximately 98% of cells exhibit precise polar localization of the chemotaxis complex. In contrast, the ΔflhF mutant displays a clear shift in distribution, with about 5% of cells showing mid-cell localization and 9.5% showing near-polar localization. These differences demonstrate a significant alteration in the spatial pattern upon flhF deletion.
We have revised the relevant text in lines 166-170 accordingly and included the detailed statistical data in the newly added Fig. S4.
Even for the graphs shown in Figures 3C and 3D, where the proportion of cells with obvious chemoreceptor arrays and absolute fluorescence brightness of the chemosensory array are shown, respectively, the questions that arise are: for how many individual cells these values hold and what is the significance of the difference between each two strains?
The number of cells analyzed for each strain is indicated in the original manuscript: 372 wild-type cells (line 123), 221 ΔflhF cells (line 172), 234 ΔfliG cells (line 197), 323 ΔfliF cells (line 200), 672 ΔflhFΔfliF cells (line 202), and 242 ΔmotAΔmotCD cells (line 207). For each strain, data were collected from three independent fields of view. We have now also provided the number of cells in Fig. 3 legend.
We have now performed statistical comparisons using t-tests between strains. Notably, the measured values in Fig. 3C exhibit a clear, monotonic decrease with successive gene knockouts, supporting the robustness of the observed trend.
Regarding the absolute fluorescence intensity shown in the original Fig. 3D, the mutants did not display consistent directional changes compared to the wild type. Reliable comparison of absolute fluorescence intensity requires consistent fluorescent protein maturation levels across strains. Given the likely variability in maturation levels between strains, we concluded that this data may not accurately reflect true differences in protein concentrations. Therefore, we have removed the fluorescence intensity graph from the revised manuscript to avoid potential misinterpretation.
(3) The authors conclude that "Motor structural integrity is a prerequisite for chemoreceptor self-assembly" based on the reduction in cells with chemoreceptor clusters in mutants deleted for flagellar genes, despite the proper polar localization of the chemotaxis protein CheY. They show that the level of CheY in the WT and the mutant strains is similar, based on Western blot, which in my opinion is over-exposed. "To ascertain whether it is motor integrity rather than functionality that influences the efficiency of chemosensory array assembly", they constructed a mutant deleted for the flagella stator and found that the motor is stalled while CheY behaves like in WT cells. The authors further "quantified the proportion of cells with receptor clusters and the absolute fluorescence intensity of individual clusters (Figures 3C-D)". While Figure 3DC suggests that, indeed, the flagella mutants show fewer cells with a chemotaxis complex, Figure 3D suggests that the differences in fluorescence intensity are not statistically significant. Since it is obvious that the regulation of both structures' production and localization is codependent, I think that it takes more than a Western blot to make such a decision.
We thank the reviewer for the suggestions. To further clarify that the assembly of flagellar motors and chemoreceptor clusters occurs in an orderly manner rather than being merely codependent, we performed additional experiments. Specifically, we constructed a ΔcheA mutant strain, in which chemoreceptor clusters fail to assemble. Using in vivo fluorescent labeling of flagellar filaments, we observed that the proportion of cells with flagellar filaments in the ΔcheA strain was comparable to that of the wild type (Fig. S5).
In contrast, mutants lacking complete motor structures, such as ΔfliF and ΔfliG, showed a significant reduction in the proportion of cells with obvious receptor clusters (Fig. 3C). Based on these results, we conclude that the structural integrity of the flagellar motor is, to a certain extent, a prerequisite for the self-assembly of chemoreceptor clusters.
Accordingly, we have revised the relevant statement in lines 213-217 of the manuscript to reflect this clarification.
(4) I wonder why the authors chose to label CheY, which is the only component of the chemotaxis complex that shuttles back and forth to the base of the flagella. In any case, I think that they should strengthen their results by repeating some key experiments with labeled CheW or CheA.
We thank the reviewer for this valuable suggestion. In our study, we initially focused on the positional relationship between chemoreceptor clusters and flagella, then investigated factors influencing cluster distribution and assembly efficiency. The physiological significance of motor and cluster co-localization was ultimately proposed with CheY as the starting point.
Previous work by Harwood's group demonstrated that both CheY-YFP and CheA-GFP localize to the old poles of dividing Pseudomonas aeruginosa cells. Since our physiological hypothesis centers on CheY, we chose to label CheY-EYFP in our experiments.
To further strengthen our conclusions, we constructed a plasmid expressing CheA-CFP and introduced it into the cheY-eyfp strain via electroporation. Fluorescence imaging revealed a high degree of spatial overlap between CheA-CFP and CheY-EYFP (Fig. S2), confirming that CheY-EYFP accurately marks the location of the chemoreceptor complex.
We have revised the manuscript accordingly (lines 119-123) and added these data as Fig. S2.
(5) The last section of the results is very problematic, regarding the rationale, the conclusions, and the novelty. As far as the rationale is concerned, I do not understand why the authors assume that "a spatial separation between the chemoreceptors and flagellar motors should not significantly impact the temporal comparison in bacterial chemotaxis". Is there any proof for that?
We apologize for the lack of clarity in our original explanation. The rationale behind the statement was initially supported by comparing the timescales of CheY-P diffusion and temporal comparison in chemotaxis. Specifically, the diffusion time for CheY-P to traverse the entire length of a bacterial cell is approximately 100 ms (refs 39&40), whereas the timescale for bacterial chemotaxis temporal comparison is on the order of seconds (ref 41).
To clarify and strengthen this argument, we have expanded the discussion as follows:
The diffusion coefficient of CheY in bacterial cells is about 10 µm2/s, which corresponds to an estimated end-to-end diffusion time on the order of 100 ms (refs 40&41). If the chemotaxis complexes were randomly distributed rather than localized, diffusion times would be even shorter. In contrast, the timescale for the chemotaxis temporal comparison is on the order of seconds (ref. 42). Additionally, a study by Fukuoka and colleagues reported that intracellular chemotaxis signal transduction requires approximately 240 ms beyond CheY or CheY-P diffusion time (ref. 41). Moreover, the intervals of counterclockwise (CCW) and clockwise (CW) rotation of the P. aeruginosa flagellar motor under normal conditions are 1-2 seconds, as determined by tethered cell or bead assays (refs. 30&43).
Taken together, these indicate that for P. aeruginosa, which moves via a run-reverse mode, the potential 100 ms reduction in response time due to co-localization of the chemotaxis complex and motor has a limited effect on overall chemotaxis timing.
We have revised the corresponding text accordingly (lines 238-245) to better explain this rationale.
More surprising for me was to read that "The signal transduction pathways in E. coli are relatively simple, and the chemotaxis response regulator CheY-P affects only the regulation of motor switching". There are degrees of complexity among signal transduction pathways in E. coli, but the chemotaxis seems to be ranked at the top. CheY is part of the adaptation. Perfect adaptation, as many other issues related to the chemotaxis pathway, which include the wide dynamic range, the robustness, the sensitivity, and the signal amplification (gain), are still largely unexplained. Hence, such assumptions are not justified.
We apologize for the confusion and imprecision in our original statements. Our intention was to convey that the chemotaxis pathway in E. coli is relatively simple compared to the more complex chemosensory systems in P. aeruginosa. We did not mean to generalize this simplicity to all signal transduction pathways in E. coli.
We acknowledge that E. coli chemotaxis is a highly sophisticated system, involving processes such as perfect adaptation, wide dynamic range, robustness, sensitivity, and signal amplification, many aspects of which remain incompletely understood. CheY indeed plays a crucial role in adaptation and motor switching regulation.
Accordingly, we have revised the original text (lines 249-255) to avoid any misunderstanding.
More perplexing is the novelty of the authors' documentation of the effect of the chemotaxis proteins on the c-di-GMP level. In 2013, Kulasekara et al. published a paper in eLife entitled "c-di-GMP heterogeneity is generated by the chemotaxis machinery to regulate flagellar motility". In the same year, Kulasekara published a paper entitled "Insight into a Mechanism Generating Cyclic di-GMP Heterogeneity in Pseudomonas aeruginosa". The authors did not cite these works and I wonder why.
We apologize for having been unaware of these important references and thank the reviewer for bringing them to our attention. We have now cited the eLife paper and the PhD thesis titled "Insight into a Mechanism Generating Cyclic di-GMP Heterogeneity in Pseudomonas aeruginosa" by Kulasekara et al.
Regarding novelty, there are key differences between our findings and those reported by Kulasekara et al. While they proposed that CheA influences c-di-GMP heterogeneity through interaction with a specific phosphodiesterase (PDE), our results demonstrate that overexpression of CheY leads to an increase in intracellular c-di-GMP levels.
We have revised the original text accordingly (lines 358-362) to clarify these distinctions.
(6) Throughout the manuscript, the authors refer to foci of fluorescent CheY as "chemoreceptor arrays". If anything, these foci signify the chemotaxis complex, not the membrane-traversing chemoreceptors.
We thank the reviewer for this clarification. We have revised the manuscript accordingly to refer to the fluorescent CheY foci as representing the chemotaxis complex rather than the chemoreceptor arrays.
Conclusions:
The manuscript addresses an interesting subject and contains interesting, but incomplete, data.
Reviewer #2 (Public Review):
Summary:
Here, the authors studied the molecular mechanisms by which the chemoreceptor cluster and flagella motor of Pseudomonas aeruginosa (PA) are spatially organized in the cell. They argue that FlhF is involved in localizing the receptors-motor to the cell pole, and even without FlhF, the two are colocalized. FlhF is known to cause the motor to localize to the pole in a different bacterial species, Vibrio cholera, but it is not involved in receptor localization in that bacterium. Finally, the authors argue that the functional reason for this colocalization is to insulate chemotactic signaling from other signaling pathways, such as cyclic-di-GMP signaling.
Strengths:
The experiments and data look to be high-quality.
Weaknesses:
However, the interpretations and conclusions drawn from the experimental observations are not fully justified in my opinion.
I see two main issues with the evidence provided for the authors' claims.
(1) Assumptions about receptor localization:
The authors rely on YFP-tagged CheY to identify the location of the receptor cluster, but CheY is a diffusible cytoplasmic protein. In E. coli, CheY has been shown to localize at the receptor cluster, but the evidence for this in PA is less strong. The authors refer to a paper by Guvener et al 2006, which showed that CheY localizes to a cell pole, and CheA (a receptor cluster protein) also localizes to a pole, but my understanding is that colocalization of CheY and CheA was not shown. My concern is that CheY could instead localize to the motor in PA, say by binding FliM. This "null model" would explain the authors' observations, without colocalization of the receptors and motor. Verifying that CheY and CheA are colocalized in PA would be a very helpful experiment to address this weakness.
We thank the reviewer for this valuable suggestion. We agree that verifying the colocalization of CheY and CheA would strengthen our conclusions. To address this, we constructed a plasmid expressing CheA-CFP and introduced it into the CheY-EYFP strain by electroporation. Fluorescence imaging revealed a high degree of spatial overlap between CheA-CFP and CheY-EYFP signals, indicating that CheY-EYFP indeed marks the location of the chemoreceptor complex rather than the flagellar motor.
We have revised the manuscript accordingly (lines 118-123) and included these results in the new Fig. S2.
(2) Argument for the functional importance of receptor-motor colocalization at the pole:
The authors argue that colocalization of the receptors and motors at the pole is important because it could keep phosphorylated CheY, CheY-p, restricted to a small region of the cell, preventing crosstalk with other signaling pathways. Their evidence for this is that overexpressing CheY leads to higher intracellular cdG levels and cell aggregation. Say that the receptors and motors are colocalized at the pole. In E. coli, CheY-p rapidly diffuses through the cell. What would prevent this from occurring in PA, even with colocalization?
We appreciate the reviewer's insightful question. The colocalization of both the signaling source (the kinase) and sink (the phosphatase) at the chemoreceptor complex at the cell pole results in a rapid decay of CheY-P concentration within approximately 0.2 µm from the cell pole, leading to a nearly uniform distribution elsewhere in the cell, as demonstrated by Vaknin and Berg (ref. 46). This spatial arrangement effectively confines high CheY-P levels to the pole region. When the motor is also localized at the cell pole, this reduces the need for elevated CheY-P concentrations throughout the cytoplasm, thereby minimizing potential crosstalk with other signaling pathways.
We have revised the manuscript accordingly (lines 280-286) to clarify this point.
Elevating CheY concentration may increase the concentration of CheY-p in the cell, but might also stress the cells in other unexpected ways. It is not so clear from this experiment that elevated CheY-p throughout the cell is the reason that they aggregate, or that this outcome is avoided by colocalizing the receptors and motor at the same pole. If localization of the receptor array and motor at one pole were important for keeping CheY-p levels low at the opposite pole, then we should expect cells in which the receptors and motor are not at the pole to have higher CheY-p at the opposite pole. According to the authors' argument, it seems like this should cause elevated cdG levels and aggregation in the delta flhF mutants with wild-type levels of CheY. But it does not look like this happened. Instead of varying CheY expression, the authors could test their hypothesis that receptor-motor colocalization at the pole is important for preventing crosstalk by measuring cdG levels in the flhF mutant, in which the motor (and maybe the receptor cluster) are no longer localized in the cell pole.
We thank the reviewer for raising the important point regarding potential cellular stress caused by elevated CheY concentrations, as well as for the suggestion to test the hypothesis using ΔflhF mutants.
First, as noted above, CheY-P concentration rapidly decreases away from the receptor complex. While deletion of flhF alters the position of the receptor complex, thereby shifting the region of high CheY-P concentration, it does not increase CheY-P levels elsewhere in the cell. Importantly, in the ΔflhF strain, the receptor complex and the motor still colocalize, so this mutant may not effectively test the role of receptor-motor colocalization in preventing crosstalk as suggested.
Regarding the possibility that elevated CheY levels stress the cells independently of CheY-P signaling, prior work in <i.E. coli by Cluzel et al. (ref. 11) showed that overexpressing CheY several-fold did not cause phenotypic changes, indicating that simple CheY overexpression alone may not be generally stressful. Furthermore, our data indicate that the increase in c-di-GMP levels and subsequent cell aggregation upon CheY overexpression is not an all-or-none switch but occurs progressively as CheY concentration rises.
To further confirm that CheY overexpression promotes aggregation through increased c-di-GMP levels, we performed additional experiments co-overexpressing CheY and a phosphodiesterase (PDE) from E. coli to reduce intracellular c-di-GMP. These experiments showed that PDE expression mitigates cell aggregation caused by CheY overexpression (Fig. S8).
We have revised the manuscript accordingly (lines 290-294) and added these new results in Fig. S8.
Reviewer #3 (Public Review):
Summary:
The authors investigated the assembly and polar localization of the chemosensory cluster in P. aeruginosa. They discovered that a certain protein (FlhF) is required for the polar localization of the chemosensory cluster while a fully-assembled motor is necessary for the assembly of the cluster. They found that flagella and chemosensory clusters always co-localize in the cell; either at the cell pole in wild-type cells or randomly-located in the cell in FlhF mutant cells. They hypothesize that this co-localization is required to keep the level of another protein (CheY-P), which controls motor switching, at low levels as the presence of high levels of this protein (if the flagella and chemosensory clusters were not co-localized) is associated with high-levels of c-di-GMP and cell aggregations.
Strengths:
The manuscript is clearly written and straightforward. The authors applied multiple techniques to study the bacterial motility system including fluorescence light microscopy and gene editing. In general, the work enhances our understanding of the subtlety of interaction between the chemosensory cluster and the flagellar motor to regulate cell motility.
Weaknesses:
The major weakness in this paper is that the authors never discussed how the flagellar gene expression is controlled in P. aeruginosa. For example, in E. coli there is a transcriptional hierarchy for the flagellar genes (early, middle, and late genes, see Chilcott and Hughes, 2000). Similarly, Campylobacter and Helicobacter have a different regulatory cascade for their flagellar genes (See Lertsethtakarn, Ottemann, and Hendrixson, 2011). How does the expression of flagellar genes in P. aeruginosa compare to other species? How many classes are there for these genes? Is there a hierarchy in their expression and how does this affect the results of the FliF and FliG mutants? In other words, if FliF and FliG are in class I (as in E. coli) then their absence might affect the expression of other later flagellar genes in subsequent classes (i.e., chemosensory genes). Also, in both FliF and FliG mutants no assembly intermediates of the flagellar motor are present in the cell as FliG is required for the assembly of FliF (see Hiroyuki Terashima et al. 2020, Kaplan et al. 2019, Kaplan et al. 2022). It could be argued that when the motor is not assembled then this will affect the expression of the other genes (e.g., those of the chemosensory cluster) which might play a role in the decreased level of chemosensory clusters the authors find in these mutants.
We thank the reviewer for the insightful comments. P. aeruginosa possesses a four-tiered transcriptional regulatory hierarchy controlling flagellar biogenesis. Within this system, fliF and fliG belong to class II genes and are regulated by the master regulator FleQ. In contrast, chemotaxis-related genes such as cheA and cheW are regulated by intracellular free FliA, and currently, there is no evidence that FliA activity is influenced by proteins like FliG.
To verify that the expression of core chemotaxis proteins was not affected by deletion of fliG, we performed Western blot analyses to compare CheY levels in wild-type, ΔfliF, and ΔfliG strains. We observed no significant differences, indicating that the reduced presence of receptor clusters in these mutants is not due to altered expression of chemotaxis proteins.
Accordingly, we have revised the manuscript (lines 341-348) and updated Fig. 3B to reflect these findings.
Recommendations for the authors:
Reviewing Editor (Recommendations For The Authors):
The reviewers comment on several important aspects that should be addressed, namely: the lack of statistical analysis; the need for clarifications regarding assumptions made regarding receptor localization; the functional importance of receptor-motor colocalization; and the need for an elaborate discussion of flagellar gene expression. Also, two reviewers pointed out the need to prove the co-localization of CheY and CheA; This is important since CheY is dynamic, shuttling back and forth from the chemotaxis complex to the base of the flagella, whereas CheA (or cheW or, even better, the receptors) is considered less dynamic and an integral part of the chemotaxis complex.
Reviewer #1 (Recommendations For The Authors):
Minor points:
Line 43: "ubiquitous" - I would choose another word.
We changed "ubiquitous" to "widespread".
Line 49: "order" - change to organize.
We changed "order" to "organize".
Line 52: "To grow and colonize within the host, bacteria have evolved a mechanism for migrating...". Motility "towards more favorable environments" is an important survival strategy of bacteria in various ecological niches, not only within the host.
We revised it to "grow and colonize in various ecological niches".
Line 72: Define F6 in "F6 pathway-related receptors".
The proteins encoded by chemotaxis-related genes collectively constitute the F6 pathway, which we have now explained in the manuscript text.
Line 72-73: Do references 17 &18 really report colocalization of the chemotaxis receptor and flagella to the same pole? If these or other reports document such colocalization, then the sentence in the Abstract "Surprisingly, we found that both are located at the same cell pole..." is not correct.
Kazunobu et al. (ref. 18) used scanning electron microscopy to preliminarily characterize the flagellation pattern of Pseudomonas aeruginosa during cell division, showing that existing flagella are located at the old pole. Zehra et al. (ref. 17), through fluorescence microscopy, observed that CheA and CheY proteins in dividing cells are typically also present at the old pole. Based on these observations, we inferred in the Introduction that the chemotaxis complex and flagellum may localize to the same cell pole.
However, this inference is indirect and lacks direct live-cell evidence of colocalization, leaving its validity to be confirmed. This uncertainty was indeed the starting point and motivation for our study.
In our work, we simultaneously visualized flagellar filaments and core chemoreceptor proteins at the single-cell level in P. aeruginosa. We characterized the assembly and spatial coordination of the chemotaxis network and flagellar motor throughout the cell cycle, providing direct evidence of their colocalization and coordinated assembly. This represents a significant advance beyond prior indirect observations and supports the novelty of our study.
Accordingly, we have revised the relevant statements in lines 71-75 of the manuscript to better reflect the current state of the literature and emphasize the novelty of our direct observations.
Line 108: "CheY has been shown to colocalize with chemoreceptors". The authors rely here (reference 29) and in other places on findings in E. coli. However, in the Introduction, they describe the many differences between the motility systems of P. aeruginosa and E. coli, e.g., the number of chemosensory systems and their spatial distribution (E. coli is a peritrichous bacterium, as opposed to the monotrichous bacterium P. aeruginosa). There seem to be proofs for colocalization of the Che and MCP proteins in P. aeruginosa, which should be cited here.
Thank you for pointing this out. Harwood's group reported that a cheY-YFP fusion strain exhibited bright fluorescent spots at the cell pole, which disappeared upon knockout of cheA or cheW-genes encoding structural proteins of the chemotaxis complex. This strongly suggests colocalization of CheY with MCP proteins in P. aeruginosa. We have now cited this study as reference 17 in the manuscript.
Figure 1B: Please replace the order of the schematic presentations, so that the cheY-egfp fusion, which is described first in the text, is at the top.
We have modified the order of related images in Fig. 1B.
Line 127: "by introducing cysteine mutations". Replace either by "by introducing cysteines" or by "by substituting several residues with cysteines".
We changed the relevant statement to "by introducing cysteines".
Line 144-145: "Given that the physiological and physical environments of both cell poles are nearly identical.". I think that also the physical, but certainly the physiological environment of the two poles is not identical. First, one is an old pole, and the other a new pole. Second, many proteins and RNAs were detected mainly or only in one of the poles of rod-shaped Gram-negative bacteria that are regarded as symmetrically dividing. Although my intuition is that the authors are correct in assuming that "it is unlikely that the unipolar distribution of the chemoreceptor array can be attributed to passive regulatory factors", relating it to the (false) identity between the poles is incorrect.
We thank the reviewer for this important correction. We agree that the physiological environments of the two poles are not identical, given that one is the old pole and the other the new pole, and that many proteins and RNAs show polar localization in rod-shaped Gram-negative bacteria. Accordingly, we have revised the original text (lines 150-152) to read:
“Despite potential differences in the physical and especially physiological environments at the two cell poles, it is unlikely that the unipolar distribution of the chemotaxis complex can be attributed to passive regulatory factors.”
Lines 151-154: "Considering the consistent colocalization pattern between chemosensory arrays and flagellar motors in P. aeruginosa". Does the word consistent relate to different reports on such colocalization or to the results in Figure 1D? In case it is the latter, then what is the word consistent based on? All together only 7 cells are presented in the 5 micrographs that compose Figure 1D (back to statistics...).
We thank the reviewer for raising this point. To clarify, the word "consistent" refers to the observation of colocalization shown in Figure 1D & Figure S3. As noted in the revised figure legend for Figure 1D, a total of 145 cells with labeled flagella were analyzed, all exhibiting consistent colocalization between flagella and chemosensory arrays. Additionally, we have included a new image showing a large field of co-localization in the wild-type strain as Figure S3 to better illustrate this consistency.
Figure 2A: Omit "Subcellular localization of" from the beginning of the caption.
We removed the relevant expression from the caption.
Reviewer #2 (Recommendations For The Authors):
I strongly recommend checking that CheY localizes to the receptor cluster in PA. This could be done by tagging cheA with a different fluorophore and demonstrating their colocalization. It would also be helpful to check that they are colocalized in the delta flhF mutant.
We thank the reviewer for this valuable suggestion. We constructed a plasmid expressing CheA-CFP and introduced it into the CheY-EYFP strain by electroporation. Fluorescence imaging revealed a high degree of spatial overlap between CheA-CFP and CheY-EYFP signals, indicating that CheY-EYFP indeed marks the location of the chemoreceptor complex.
We have revised the manuscript accordingly (lines 118-123) and included these results in the new Fig. S2.
The experiments under- and over-expressing CheY part seemed too unrelated to receptor-motor colocalization. I think the authors should think about a more direct way of testing whether colocalization of the motor and receptors is important for preventing signaling crosstalk. One way would be to measure cdG levels in WT and in delta flhF mutants and see if there is a significant difference.
We thank the reviewer for raising the important point regarding potential cellular stress caused by elevated CheY concentrations, as well as for the suggestion to test the hypothesis using flhF mutants.
First, as noted in the response to your 2nd comment in Public Review, CheY-P concentration rapidly decreases away from the receptor complex. While deletion of flhF alters the position of the receptor complex, thereby shifting the region of high CheY-P concentration, it does not increase CheY-P levels elsewhere in the cell. Importantly, in the ΔflhF strain, the receptor complex and the motor still colocalize, so this mutant may not effectively test the role of receptor-motor colocalization in preventing crosstalk as suggested.
Regarding the possibility that elevated CheY levels stress the cells independently of CheY-P signaling, prior work in E. coli by Cluzel et al. (ref. 11) showed that overexpressing CheY several-fold did not cause phenotypic changes, indicating that simple CheY overexpression alone may not be generally stressful. Furthermore, our data indicate that the increase in c-di-GMP levels and subsequent cell aggregation upon CheY overexpression is not an all-or-none switch but occurs progressively as CheY concentration rises.
To further confirm that CheY overexpression promotes aggregation through increased c-di-GMP levels, we performed additional experiments co-overexpressing CheY and a phosphodiesterase (PDE) from E. coli to reduce intracellular c-di-GMP. These experiments showed that PDE expression mitigates cell aggregation caused by CheY overexpression (Fig. S8).
We have revised the manuscript accordingly (lines 290-294) and added these new results in Fig. S8.
Reviewer #3 (Recommendations For The Authors):
(1) Can the authors elaborate more on the hierarchy of flagellar gene expression in P. aeruginosa and how this relates to their work?
We thank the reviewer for the suggestion. We have now described the hierarchy of flagellar gene expression in P. aeruginosa in lines 341-348.
(2) I would suggest that the authors check other flagellar mutants (than FliF and FliG) where the motor is partially assembled (e.g., any of the rod proteins or the P-ring protein), together with FlhF mutant, to see how a partially assembled motor affects the assembly of the chemosensory cluster.
We thank the reviewer for this valuable suggestion. The P ring, primarily composed of FlgI, acts as a bushing for the peptidoglycan layer, and its absence leads to partial motor assembly. We constructed a ΔflgI mutant and observed that the proportion of cells exhibiting distinct chemotactic complexes was similar to that of the wild-type strain, suggesting that the assembly of the receptor complex is likely influenced mainly by the C-ring and MS-ring structures rather than by the P ring. We have revised the original text accordingly (lines 217-220) and added the corresponding data as Figure S6.
(3) I would suggest that the authors check the levels of CheY in cells induced with different concentrations of arabinose (i.e., using western blotting just like they did in Figure 3B).
We have assessed the levels of CheY in cells induced with different concentrations of arabinose using western blotting, as suggested. The results have been incorporated into the manuscript (lines 274-275) and are presented in Figure S7.
(4) To my eyes, most of the foci in FliF-FlhF mutant in Figure 3A are located at the pole (which is unlike the FlhF mutant in Figure 2). Is this correct? I would suggest that the authors also investigate this to see where the chemosensory cluster is located.
We thank the reviewer for pointing this out. The distribution of the chemotaxis complex in the ΔflhFΔfliF strain was investigated and showed in Fig. S4. Indeed, most of the chemoreceptor foci in this mutant are located at the pole. This probably suggests that, in the absence of both FlhF and an assembled motor, the position of the receptor complex may be largely influenced by passive factors such as membrane curvature. This interesting possibility warrants further investigation in future studies.
