Promoter selectivity of the RhlR quorum-sensing transcription factor receptor in Pseudomonas aeruginosa is coordinated by distinct and overlapping dependencies on C4-homoserine lactone and PqsE

Quorum sensing is a mechanism of bacterial cell-cell communication that relies on the production and detection of small molecule autoinducers, which facilitate the synchronous expression of genes involved in group behaviors, such as virulence factor production and biofilm formation. The Pseudomonas aeruginosa quorum sensing network consists of multiple interconnected transcriptional regulators, with the transcription factor, RhlR, acting as one of the main drivers of quorum sensing behaviors. RhlR is a LuxR-type transcription factor that regulates its target genes when bound to its cognate autoinducer, C4-homoserine lactone, which is synthesized by RhlI. RhlR function is also regulated by the metallo-β-hydrolase enzyme, PqsE. We recently showed that PqsE binds RhlR to alter its affinity for promoter DNA, a new mechanism of quorum-sensing receptor activation. Here, we perform ChIP-seq analyses of RhlR to map the binding of RhlR across the P. aeruginosa genome, and to determine the impact of C4-homoserine lactone and PqsE on RhlR binding to different sites across the P. aeruginosa genome. We identify 40 RhlR binding sites, all but three of which are associated with genes known to be regulated by RhlR. C4-homoserine lactone is required for maximal binding of RhlR to many of its DNA sites. Moreover, C4-homoserine lactone is required for maximal RhlR-dependent transcription activation from all sites, regardless of whether it impacts RhlR binding to DNA. PqsE is required for maximal binding of RhlR to many DNA sites, with similar effects on RhlR-dependent transcription activation from those sites. However, the effects of PqsE on RhlR specificity are distinct from those of C4-homoserine lactone, and PqsE is sufficient for RhlR binding to some DNA sites in the absence of C4-homoserine lactone. Together, C4-homoserine lactone and PqsE are required for RhlR binding at the large majority of its DNA sites. Thus, our work reveals three distinct modes of activation by RhlR: i) when RhlR is unbound by autoinducer but bound by PqsE, ii) when RhlR is bound by autoinducer but not bound by PqsE, and iii) when RhlR is bound by both autoinducer and PqsE, establishing a stepwise mechanism for the progression of the RhlR-RhlI-PqsE quorum sensing pathway in P. aeruginosa.

See response to previous comment.
3) Line 137 -It would be of interest for the authors to briefly explain how the RhlR binding motif compares to lux-box sequences for other receptors.
We have added text to address this issue.On lines 156-158, we now state: "This sequence motif matches the known RhlR binding consensus, which was previously determined using promoter sequences from the core regulon; this motif is similar to those of other LuxR-type family proteins" and provide a citation.4) Line 197 -It would be clearer for the authors to state they compared expression of direct RhlR target genes +/-rhlI to identify C4-dependent changes.
We replaced "+/-C4HSL" with "+/-rhlI" in the text on line 227 to improve clarity.5) Figure 4C -It would be useful to provide a quantitative metric of correlation in PqsE dependence that could be compared to C4-dependencies shown in Fig 3C .For Figures 3C and 4C, we performed a simple linear regression model to determine a best-fit line.We now include the R 2 and p-values on the graph, which indicate statistical significance.
6) Line 271 -The authors state the ChIP-Seq analysis of RhlR in a ∆rhlI∆pqsE strain results in nearly same decrease in binding occupancy as ∆rhlR and reference Fig 5B, which shows a comparison of ∆rhlI∆pqsE and WT enrichment.It would be helpful to include a comparison of ∆rhlI∆pqsE and ∆rhlR to justify the claim that they have "nearly the same decrease in binding occupancy".This could be a supplemental figure .We have included a new supplemental figure (Figure S3) to depict the combined contributions of rhlI and pqsE to RhlR binding across all sites.
7) It would be helpful for the authors to include statistical analyses of key comparisons, particularly in Fig 5C where some differences in expression level seem minor or have large errors.
The important conclusion from this figure is the relative difference between the effects of the loss pqsE on different genes.8) In figure 6, the authors use 1 uM C4-HSL to stimulate the activity of RhlR heterologously expressed in E. coli.Is this a sufficient concentration to stimulate maximal activity for each of the reporters?It seems unlikely as published EC50 values for RhlR expressed in E. coli range from 9 -120 uM (PMID 30114353, 26460240, 30837333).The authors should justify their choice of 1 uM C4-HSL in this experiment and consider repeating the experiment with a higher concentration of C4-HSL.
We have addressed this concern in the manuscript on lines 343-345 and included a citation of previous work done at this concentration to examine the effects of PqsE on RhlR activity.Relatedly, is it possible "C4-independent" genes simply require a higher concentration of C4-HSL than provided, or produced in these experiments?This may be true in both the E. coli studies and in P. aeruginosa where C4 production in planktonic culture could be less than that in biofilms or in other environments.Perhaps PqsE stabilizes RhlR at low concentrations of C4-HSL but at higher concentrations, the impact of PqsE is lower or even negligible?
Our data and data from earlier studies support this model.Over the course of growth, we expect that cells will be exposed to different concentrations of C4HSL.We consider the implications of this in the discussion.9) Line 518 -What was the OD of the cultures when collected for qPCR?The authors state "until the optimal cell density was achieved" without giving a specific number.
We apologize for this oversight.We have included this information in the material and methods.
Reviewer #2: This study describes a comprehensive Chip-Seq analysis of the regulon of P. aeruginosa quorum sensing regulator RhlR.The study also looks at how the RhlR-binding protein PqsE influence RhlR promoter occupancy from in vivo CHIP-seq studies.The results map the set of gene promoters that RhlR occupies in the presence and absence of its ligand C4-HSL and a regulator protein PqsE.
Global studies of RhlR binding have not been previously carried out.Thus, the studies provide important new insight into RhlR and P. aeruginosa quorum sensing biology.The finding that RhlR promoter occupancy is altered by interacting with PqsE also represents a newly described mechanism of transcription regulation and is very interesting.
The paper is very clearly written and has a very appealing systems-level approach.However, there are some issues with clarity of writing and presentation that need to be addressed before the manuscript is ready for publication.

Major criticisms
1.The Chip-seq results using natively expressed RhlR show that PqsE alters the promoter occupancy of RhlR but a limitation of this approach is that there are a variety of mechanisms that could alter RhlR promoter occupancy.Care should be taken in drawing conclusions from these results, for example by replacing terms such as "PqsE alters RhlR promoter selection" with "PqsE alters promoter occupancy." We have decided to use the terminology "selective RhlR promoter occupancy" because it addresses the reviewer's comment, and highlights that the effect of PqsE is different at different promoters.
2. There are some problems with the figures that need addressed.The figure headings are not informative and in some cases do not reflect what is seen as the main point of the figure.In some cases the figures are also difficult to interpret or lack critical information.See below for specific examples.
3. The discussion, particularly the first paragraph, needs revision.In particular, many of the statements are not clear and there are some issues with the conclusions being drawn -see below for specific examples.
We address the specific criticisms below.Specific criticisms 1. Fig. 2 needs clarification.First, the heading is not accurately describing the data.The data are more related to which genes from the Chip-seq study correlate with the RhlR-regulated genes from RNAseq.Also, it seems you used some sort of cutoff (log2 of 2?) to decide which genes were transcriptionally induced by RhlR and which weren't, where this cutoff is should be indicated on the graph or at least described in the legend.
We have changed the title of Figure 2. We have included the cut off (q-value) for defining RhlRdependent regulation in the figure legend.We also now indicate using different colors which of the genes are direct RhlR targets.
2. Fig. 3C -this figure is intuitively difficult to understand.Also, the text on lines 200-202 states that there is a positive correlation between C4-dependent changes in gene expression and RhlR binding, but this is not clear from the data, and there is no positive correlation line or statistics to support this statement.Also, based on the rest of description in the results, and looking at the data, it seems that the point is actually that RhlR does not necessarily need C4-HSL for binding but it does need it to activate.That's not really reflective of a positive correlation.
For Figures 3C and 4C, we performed a simple linear regression model to determine a best-fit line and included the R 2 and p-values on the graph, which indicate statistical significance.
3. Fig. 4 -no correlation line or statistics to support the conclusion that there is a correlation.In addition, the discussion of this data in lines 246-255 seems to be highlighting the variability (although the example on lines 245-247 was difficult to follow), and not the correlation.
We agree with the reviewer that the text was confusing around the interpretation of these data.We have clarified our assertions in the text that while the data are largely positively correlated, some genes deviate from this trend stating on line 292 "Despite the positive correlation between the dependence on PqsE for RhlR binding and activation, the expression of some genes was largely independent of PqsE notwithstanding a strong dependence of RhlR binding on PqsE." 4. Fig. 6A -it would be helpful to see these data in their raw form (+ and -C4-HSL, so that an evaluation of the relative levels of induction can be taken into consideration.In addition, statistical comparisons are needed to support the conclusions. We agree that these data are best visualized by displaying the data in their raw form.We have modified Figure 6A to accommodate these changes.5. Fig. 6B -because EMSA experiments cannot be conducted in +/-C4-HSL conditions due to technical limitations, it is unclear whether PqsE is altering the ability of RhlR to respond to C4 or simply increasing its promoter affinity.The data in Fig. 6A do seem to support the idea that it is the former and not the latter (although visualizing the data in its raw form would help with that interpretation).Also, in absence of promoter binding affinity calculations, it is difficult to determine if the promoter affinity is altered in the same way for each of the genes (in which case PqsE would be altering the promoter selectivity and not just affinity).Without the EMSA +/-C4 data and a rigorous study of promoter binding affinities with several different promoters, care should be taken in describing the interpretation of the Chip-Seq data (see major point #1 above and point #6 below).
RhlR is purified in the presence of the agonist and PqsE is added to RhlR in the EMSA reaction.Thus, PqsE cannot alter the ability of RhlR to respond to C4HSL.We agree with the reviewer that the affinity for different sites is likely to be different, and we note this in the manuscript on line 359.
6. Lines 154-155.The wording here suggests the RNAseq study used for this analysis was published previously, but upon closer examination of the methods it appears that those experiments were done for this study.This section needs to be rewritten to clarify that where the RNAseq results came from.Also, it appears that at least one other RNAseq study focused on PqsE is in the published literature (ref. 42).A comparison of the approach and differences in results of each study should be discussed either here or in the discussion.
We have clarified in the methods section that the RNA-seq data are from our earlier paper, but we note that our analysis of those data in our current study is new.We believe that lines 376-377 address this sufficiently.
7. Line 288 -the heterologous E. coli reporter system is not equivalent to an in vitro system because it is not in vitro.
We agree that it is not an "in vitro" system, but since E. coli does not produce RhlR, PqsE, or C4HSL, it can be considered as a proxy for in vitro assays.There might be other RhlR co-factors present in E. coli that we are unaware of, so we have qualified the statement with "somewhat equivalent." 8. How were the DNA probes in the EMSA experiments detected?It appears they were detected by fluorescence.This needs to be clarified in the methods.
We modified the methods to include the wavelength for the detection of SYBR Gold.
Relatively minor: Line 191: it doesn't seem like there should be a dash between "C4-HSL" and "dependent" We are not sure how to address this comment because it depends on the editorial style of the journal and believe it is a question best left to the editorial staff.
The paragraph starting at line 222 is not clear.I think what it is trying to say is that several new genes have been identified that have robust effects of PqsE, but that is not clear.
We have edited the second sentence of this paragraph on line 244 to make the text clearer.
Lines 258-260: please remind the reader where the data are to support this statement -("ChIPseq and RNA-seq data showed that RhlR binding and RhlR-dependent transcription activation of rhlA and lasB are more dependent on C4HSL than PqsE, whereas the opposite is true for hcnA and lecB.") We now refer to Table 1 to remind the reader of the location of the data.Specific examples of problems in the discussion that needs addressed (just from the first paragraph) are below --Line 306: "the direct RhlR regulon" is an unclear term Addressed (see response to comment below about lines 311-312).
-Line 307: "RhlR-dependent changes in RNA levels" is also unclear -do you mean RhlRdependent changes in gene transcription?Addressed.
-Line 308: This sentence could be softened -perhaps "provides insight into direct vs. indirect regulation" Addressed.
-Line 310: This section is unclear.Having RhlR binding sites without evidence of regulation does not necessarily support condition-specific regulation.Perhaps what was meant was that some genes are RhlR regulated only under some conditions?
We do not understand this comment, as we think the reviewer's suggestion is already described in the discussion.
-Line 311-312: what is "direct RhlR target genes" -is this meant to be "genes directly regulated by RhlR"?
We clarify this statement in the discussion and on lines 374-377, we state: "This is the first comprehensive assessment of the direct RhlR regulon (i.e., genes where regulation by RhlR is due to RhlR binding upstream)." -Line 314 -it is not clear how the RNAseq study in ref. 42 is different or how the results compared with those in this study.Also, why do the prior RNAseq studies suggest that most of the regulon is indirectly controlled by RhlR?Don't your results also suggest this?
We agree, and now make a general statement about direct vs indirect regulation by RhlR, rather than referring specifically to reference 42.
Reviewer #3: The manuscript entitled "Promoter selectivity of the RhlR quorum-sensing transcription factor receptor in Pseudomonas aeruginosa is coordinated by distinct and overlapping dependencies on C4-homoserine lactone and PqsE" by Keegan and collaborators aims to enlighten the current comprehension of the quorum sensing (QS) network in the bacterium P. aeruginosa.The authors performed ChIP-Seq analyses of the transcriptional regulator RhlR to decipher the role of the cognate autoinducer C4-HSL and PqsE in DNA binding.The significance of this work lies in its ability to unravel the direct contributions of RhlR to the QS network, thereby advancing our understanding of this crucial communication system.Despite the intriguing nature of the provided data, the manuscript suffers from a lack of overall cohesion in its structural organization, hampering the potential of the findings.

Overall remarks:
The manuscript contains crucial data for individuals interested in studying gene regulation and quorum sensing of the opportunistic pathogen P. aeruginosa.However, to enhance its impact and clarity, some structural revisions are essential.It is imperative for the authors to establish a primary focus for the manuscript and then reorganize its content accordingly.The current arrangement lacks coherence, leading to confusion even among experts in the field.