Spatial integration of sensory input and motor output in Pseudomonas aeruginosa chemotaxis through colocalized distribution

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.

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.

(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.

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.

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?

(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.

(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.

(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? 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.

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.

(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.

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.

(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?

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.

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.