Dear Dr Thomas, dear Sam,

Thank you for submitting your manuscript entitled "Quantifying Multi-Signal Quorum Sensing Defines the Mapping from Environment to Bacterial Regulatory Responses" for consideration as a Research Article by PLOS Biology.

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Melissa Vazquez Hernandez, Ph.D.

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PLOS Biology

mvazquezhernandez@plos.org

Dear Dr Thomas,

Thank you for your patience while your manuscript "Quantifying Multi-Signal Quorum Sensing Defines the Mapping from Environment to Bacterial Regulatory Responses" was peer-reviewed at PLOS Biology. It has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by two independent reviewers.

In light of the reviews, which you will find at the end of this email, we would like to invite you to revise the work to thoroughly address the reviewers' reports. As you will see below, the reviewers are really positive about the study, yet some concerns have raised during revision. Reviewer #1 mentions that the mechanism of reciprocal activation is not directly tested and suggests to repeat some experiments including a deletion mutant of one of the regulators. Reviewer 2 raises a major concern over the use of a lasI/rhlI null mutant which eliminates regulatory feedbacks and perhaps may not apply to strains where feedback loops are intact, so the reviewer requests some validation on this point using a WT strain and seeing if it also mimics the model presented. S/He also requests that the authors decrease their concentration of 3-oxo-C12 to simulate more physiological concentrations. After discussion with the Academic Editor, we think these requests will indeed strengthen the study, and encourage you to address them.

IMPORTANT: we agree that repeating the experiments including a WT strain will help assessing biological relevance, but we also understand that the results may not be easy to interpret. Also, there was a concern regarding novelty, especially regarding the initial comment of Reviewer 1, so please convey your novelty more clearly.

Given the extent of revision needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is likely to be sent for further evaluation by all or a subset of the reviewers.

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Sincerely,

Melissa

Melissa Vazquez Hernandez, Ph.D.

Associate Editor

PLOS Biology

mvazquezhernandez@plos.org

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REVIEWERS' COMMENTS:

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Reviewer #1:

This manuscript asks what are the impacts of having different architectural arrangements of quorum sensing systems in species that have more than one of these systems. The results convincingly show that architectural arrangement impacts the range and sensitivity of signal responses. The results also challenge the dogma of how the architectural arrangement is understood in P. aeruginosa.

The manusciript is very nicely done with rigorous experiments, appropriate controls and valid interpretations. The work is also clearly conveyed. The figures illustrating the equations were especially appreciated.

The results are very interesting and are a significant contribution both to the field and to understanding the impacts of different network interactions more generally.

There were some larger and smaller suggestions to be addressed.

Lines 98-102 (Introduction): the wording here suggests that there is a difference in hierarchical vs. parallel systems that has not really been examined. But that difference has been examined and is primarily attributed to environmental or genetic factors (e.g. strain-to-strain variation) - which isn't really a justification for your studies. It may be better to frame this as - in standard lab conditions and in the strain you are using (PAO1), the activation of the Las system by the Rhl system has not been carefully evaluated (?), and also there are completely different hierarchical configurations in other environments and conditions, suggesting that these architectures are somewhat fluid.

Throughout the manuscript there is an assumption about the mechanism of reciprocal activation of each QS system (results in Fig. 2, model in Fig. 3E) that is not directly tested. The assumption is that the Rhl system is inducing the Las system (and vice versa) through direct effects on transcription. However, C4-HSL could induce lasI through cross-activation of LasR. This could be tested by repeating some of the experiments in Fig. 2 in the absence of LasR or RhlR (e.g. using a ∆lasI-∆rhlI-∆lasR mutant or a ∆lasI-∆rhlI-∆rhlI mutant). It is important these two mechanistic possibilities are sorted out if the mechanism remains central to the model and the conceptualization of the system. With either mechanism, the final conclusions should still be the same.

Fig. 3A-D: Is the Y-axis on a linear or log scale? This should be shown. If linear, then there does not appear to be a 30-fold difference between single signal activation and dual signal activation, as stated in the results. Perhaps this could be shown more clearly with a bar graph? Also, are the curves shown in panels A and B included in the graphs in panels C and D? if so, those lines could be color-matched to the panels A and B so they are clearer to see.

Fig. 4C is uninterpretable.

Lines 225-226: is there evidence from the literature that lasB is more sensitive to C4-HSL than to 3OC12-HSL? This is surprising, right?

Line 356 (discussion): the results don't reject population density and diffusion sensing models, it rejects the idea that quorum sensing is all one or the other (correct?). Also, isn't the model designed to reject the all one or the other idea, because it incorporates both population density and diffusion sensing?

Line 369 (discussion): the "combinatorial QS analyses" should be defined here.

Figs. 6 and 7 should be described in the results section, not the discussion. Fig. 6 could probably be moved to the supplement. Fig. 7 needs more context and explanation if it is included - it seems like it may be the basis of a whole different study.

Is the difference between Fig. 6 and Fig. 5 that in Fig. 5 both signals are incorporated into the model and in Fig. 6 it is one or the other? This is not clear in the description.

Lines 401-407: Are there any other explanations for the multiplicative effects of the signals on lasB expression other than cooperative promoter binding? And how would cooperative promoter binding be conceptualized to happen given what we know about LasR and RhlR promoter activation?

Minor:

Line 416: what is __pqs__ referring to?

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Reviewer #2:

This manuscript by Thomas et. al. challenges the dogma of the Pseudomonas aeruginosa quorum sensing field that the two acyl-homoserine lactone (AHL) based quorum sensing systems are hierarchical. Rather, they conclude that the Las system can induce the Rhl system, as is generally known, but the Rhl system also influences the Las system. They primarily derive this conclusion by measuring gene expression reporters of the two synthases, LasI and RhlI, in a lasI/rhlI null mutant by adding back specific concentrations of autoinducers. Both systems clearly seem to influence each other. They then extend these results to the downstream effector elastase (lasB). For each of these processes, they develop mathematical models to explain their results, which largely fit with their experimental observations. Finally, they use these mathematical models to demonstrate that having a reciprocal system (i.e. each node impacts the other) increases the overall response and robustness. I found the work interesting, and the models very well explained. I do accept the authors conclusions that this is a reciprocal circuit, which should be of significant interest to the quorum sensing community. My biggest concern is that all studies were done in a lasI/rhlI null mutant, which eliminates any regulatory feedbacks on the circuit, be it positive or negative. I therefore question how applicable their conclusions are to the actual QS circuit of P. aeruginosa in which these feedback loops are intact. Would they see the same results for lasB in a WT cell? I appreciate these experiments are less controllable given the bacteria are making their own autoinducer, but some validation and exploration is required in my mind for these results to be centered on the actual biology of these circuits. I also felt that there is significant work asking similar questions that merits discussion. Overall, though, this was an interesting well written manuscript.

1. The authors miss a lot of theoretical/experimental work from the Bassler/Wingreen groups on similar AI input/phenotypic output topics studying quorum sensing in Vibrios including PMIDS: 21613980, 19920810, and 19320539 that merit discussion and comparison to their work.

2. Many of the studies utilize a lasI/rhlI mutant that cannot make its own autoinducer and add either acyl-homoserine lactone (AHL) at different concentrations. The output is a transcriptional reporter fusion for lasI and rhlI. Analysis of this mutant does not allow for feedback loops in AI production (either positive or negative). How would such feedback loops alter induction of downstream products like elastase? I think the authors should compare their results from Fig. 4 in a wild type strain that has functional lasI and rhlI to see how closely it mimics the mathematical model because some of the conclusions drawn about this signaling system and its relative impacts on downstream gene expression might be different with feedback loops in place. For example, would the conclusions stated in lines 224-226 be the same if RhlI and LasI were functional and expressed by their native promoters. If not, then how biologically meaningful are these results?

3. Line 118-Can you please specify if these fusions are transcriptional or translational? It would also be useful to describe if these are single copy in the genome or multi-copy on plasmids.

4. Fig. 2A, 2D. The response to 3-oxo-C12 is quite sharp from 0 to the first point measured. This increase seems to capture most of the regulation, especially for Fig. 2D. Such results suggest to me that the concentrations of AHL used here may be above the physiological relevant concentrations as they do not capture the induction at lower concentrations. The authors should decrease the concentrations of AHL in these experiments to really determine the dose response to lower concentrations of 3-oxo-C12.

Dear Dr Thomas,

Thank you for your patience while we considered your revised manuscript "Quantifying Multi-Signal Quorum Sensing Defines the Mapping from Environment to Bacterial Regulatory Responses" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and the original reviewers.

Based on the reviews, we are likely to accept this manuscript for publication, provided you satisfactorily address the remaining points raised by the Reviewer 1. Please also make sure to address the following data and other policy-related requests.

a) We routinely suggest changes to titles to ensure maximum accessibility for a broad, non-specialist readership, and to ensure they reflect the contents of the paper. In this case, we would suggest a minor edit to the title, as follows. Please ensure you change both the manuscript file and the online submission system, as they need to match for final acceptance:

"Bacteria respond to environmental and regulatory cues through reciprocal and nonlinear quorum sensing signal integration"

b) Please add to your Competing Interests the following statement “SB is a member of PLOS Biology’s Editorial Board. The other authors declare that no competing interests exist."

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Please supply the numerical values either in the a supplementary file or as a permanent DOI’d deposition for the following figures:

Figure 2A-F, 3CD, 4BC, 5B-H, S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12

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Sincerely,

Melissa

Melissa Vazquez Hernandez, Ph.D.

Associate Editor

mvazquezhernandez@plos.org

PLOS Biology

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REVIEWERS' COMMENTS

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Reviewer #1:

The manuscript is much improved and the clear and attentive responses to feedback are much appreciated.

Just a few minor suggestions for clarity:

Fig. 1 could be improved. In 1A, Consider changing the arrows so they are not pointing from the receptor but pointing from the entire system, to take into account all mechanisms of interaction of these systems. Also consider using brackets using brackets, or something similar, to group the Las and Rhl systems, rather than circles. The circles around each system are confusing because it suggests different bacteria that are interacting.

The new model in Fig. 2E is also confusing. What are the arrows representing? What exactly do the beakers in Figs. 2 and 4 represent? They are also in Fig. 4C but not mentioned in the Fig. 4 legend (panel C).

For the 3D graphs with two X axes (Fig. 3, 4), the labels on the X axis are not clear because the text is horizontal whereas the axes are at a slant. Consider slanting the text and putting it right under each axis.

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Reviewer #2:

This revised manuscript by Thomas et. al. has addressed my concerns.

Dear Stephen, dear Sam,

Thank you for the submission of your revised Research Article "Quantitative modeling of multi-signal quorum sensing maps environment to bacterial regulatory responses" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Sara Mitri, I am pleased to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

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Melissa

Melissa Vazquez Hernandez, Ph.D., Ph.D.

Associate Editor

PLOS Biology

mvazquezhernandez@plos.org