Genome-Wide Analysis Reveals that PhoP Regulates Pathogenicity in Riemerella anatipestifer

ABSTRACT Duck infectious serositis, also known as Riemerella anatipestifer disease, infects domestic ducks, geese, and turkeys and wild birds. However, the regulatory mechanism of its pathogenicity remains unclear. The PhoPR two-component system (TCS) was first reported in Gram-negative bacteria in our previous research and was demonstrated to be involved in virulence and gene expression. Here, DNA affinity purification sequencing (DAP-seq) was applied to further explore the regulation of PhoPR in relation to pathogenicity in R. anatipestifer. A conserved motif was identified upstream of 583 candidate target genes which were directly regulated by PhoP. To further confirm the genes which are regulated by PhoR and PhoP, single-gene-deletion strains were constructed. The results of transcriptome analysis using next-generation RNA sequencing showed 136 differentially expressed genes (DEGs) between the ΔphoP strain and the wild type (WT) and 183 DEGs between the ΔphoR strain and the WT. The candidate target genes of PhoP were further identified by combining transcriptome analysis and DAP-seq, which revealed that the main direct regulons of PhoP are located on the membrane and PhoP is involved in regulating aerotolerance. Using the in vivo duck model, the pathogenicity of ΔphoP and ΔphoR mutants was found to be significantly lower than that of the WT. Together, our findings provide insight into the direct regulation of PhoP and suggest that phoPR is essential for the pathogenicity of R. anatipestifer. The gene deletion strains are expected to be candidate live vaccine strains of R. anatipestifer which can be used as ideal genetic engineering vector strains for the expression of foreign antigens. IMPORTANCE Riemerella anatipestifer is a significant pathogen with high mortality in the poultry industry that causes acute septicemia and infectious polyserositis in ducks, chickens, geese, and other avian species. Previously, we characterized the two-component system encoded by phoPR and found that R. anatipestifer almost completely lost its pathogenicity for ducklings when phoPR was deleted. However, the mechanism of PhoPR regulation of virulence in R. anatipestifer had not been deeply explored. In this study, we utilized DAP-seq to explore the DNA-binding sites of PhoP as a response regulator in the global genome. Furthermore, phoP and phoR were deleted separately, and transcriptomics analysis of the corresponding gene deletion strains was performed. We identified a series of directly regulated genes of the PhoPR two-component system. The duckling model showed that both PhoP and PhoR are essential virulence-related factors in R. anatipestifer.

subsequent statement that PhoP/PhoR in R anatipestiferis first described for Gram negative. 6. Line 94. This is confusing to include the genomic loci. They are not defined as such. Perhaps just the gene names and the reference to the prior study make this more clear for the reader? 7. Line 98? What is meant by speculated? The data cited establishes a role in pathogenesis. 8. Line 99. It would help the reader to include more details as to just how it was concluded that these loci are functionally PhoP/PhoR? And how this is differentiated from PhoP/PhoQ or the aerotolerance TCS systems that comparisons will be made to. 9. Line 104. What is RR? Define abbreviations. 10. Line 107. Drawbacks is meant, not defects? 11. Line 108. What is a ChIP-level antibody? It will help the reader to avoid jargon. 12. Line 114. This would be a good place to define the abbreviation DAP-seq. 13. Line 121. Provides a foundation is better English than laid. 14. Line 121 Understanding is better English than clarifying15. Line 123 Insights is better English than sight 16. Line 124. Provides (present tense) vs provided (past tense) is better English. Conclusions are most appropriately made in present tense, while reference to data are most appropriate in past tense. 17. Line 125. See line 124 18. Line 127. No context is provide to make sense of this conclusion that these data are important for understanding cross-talk in other pathogens? More detail would help to provide context. 19. Line 140. What is meant by "self-made"? 20. Line 143. This is expansive. The presentation of negative data here does not add to the presentation. There could be many reasons for the failure to produce a suitable antibody and none are important for understanding the DAP-seq experiment. 21. Line 143-144. A stronger presentation is not to focus on the negatives and the failure to produce an antibody, but instead, to focus on the advantages of the DAP-seq approach. 22. Line 147. Was this protein phosphorylated or not? Is not clear. 23. Line 147. some details on the DAP-seq experiment are also required here, including he specific strain and negative controls to establish specificity of the His6-PhoP to DNA. 24. Line 147. The quality of these results using this method is completely dependent on the quality of the purified protein. Thus, it will be essential to include details as to the quality of this protein preparation, including it purity, its ability to bind specifically to DNA as compared to an appropriate negative control, that it is of the appropriate size and that it actually is the PhoR protein and not some fortuitously purified protein. 25. Similar to above. Characterization of the truncated DBD protein is also important for evaluation of these results. 26. Line 147. Details are required in order to evaluate the quality of these data. Including some specific details of the method, the specific strain that was analyzed, the stage of growth that were sampled and the number of technical and biological replicates 27. Line 148. What is meant by random? At best you can conclude that no pattern or order was apparent in the distribution? 28. Line 149. Reference for the MEME tools should be included. 29. Line 163. How is it concluded that all binding to the 764 genes was specific? What negative controls establish the specificity of binding? 30. Line 167. It is not possible to conclude from these data that the domain is not involved. Only that it is not sufficient for binding. It still could be necessary. 31. Line 176. These data are not sufficient to support a conclusion that phosphorylation is not important for binding DNA. The paradigm is that phosphorylation increases binding affinity, which in an in vitro assay would be sensitive to the concentration of the respective proteins. And also their stability in vitro. Thus, comparison of stability and calculation of Kd is required to support this conclusion. 32. Line 194. What is a "confident correlation"? 33. Line 197. This statement is not clear. What is meant by "changed in the same pattern"? Was this different media or stress imposed on the cultures? 34. Line 199. What is meant by "inconsistent regulation"? 35. Line 203. The rationale for this section requires more detail. Is this concordance between the two datasets? If so, whatLine 203 is the rationale for this vs possible technical differences in sensitivity that could account for non-concordance? 36. Line 203. It also appears that many more genes were found to be regulated by DAP-seq than by RNA-seq? What could explain the discrepancy? It is likely that there are many explanations that involve the strengths and weaknesses of the methods? 37. Line 203. Is the approach here that valid regulated genes are only those that appear in both data sets? That seems limited and requires more justification, as there could be many technical reasons for this. And it overlooks the result above finding the common motif in the 764 genes identified by DAP-Seq. Do the RNA-seq dataset genes share this motif? 38. Line 214. What is "membrane compromised TBDR"? 39. Line 219. This section is more a discussion rather than a presentation of data. 40. Line 234. Do the mutants have a defect in aerotolerance? That seems like it would be easy to test and these data would significantly test this hypothesis. -41. Line 264. What is a gradient dose? Is meant "a range of doses"? 42. Line 256. This conclusion of "mainly regulate..." is based on the assumptions that the only valid regulation are those in the concordance of the DAP-seq and RNA-seq datasets. The strength of this conclusion remains to be established by more information, as indicated above.
43. Line 268. What is "retarded behavior"? 44. Line 269. What is "favorable mental conditions"? 45. Line 270. huddling is a mental state? Generally, this sort of behavior is usually described as morribund or lethargic, as it does not imply that there is any direct effect on brain function, only it can be concluded that the animals are experiencing an overall state of malaise and lethargy because they are sick. 46. Line 299. "Mock infected" is more precise that "infected by PBS" 47. Line 350. As discussed above, there is no increase in clarity from the inclusion of negative data. There are many reasons why an attempt to make a highly specific antibody was not successful. None of which are important for interpretation of the actual data presented. 48. Line 395. As discussed above, additional data are required to support this conclusion. 49. Line 396. Assuming the conclusion is correct, that phosphorylation has no affect, then what is a phosphorylationindependent model that can explain regulation of these genes?
Reviewer #2 (Comments for the Author): Zhang et al. have examined the regulon of a two-component system in Riemerella anatipestifer. They already studied this TCS, that they then called PhoP-PhoR, in a previous studied and found that it was important for pathogenicity of this duck pathogen. In this work, they go in depth in the determination of the regulon by using RNA-seq and DAP-seq to delineate the full direct regulon and confirm relevance to virulence. While the technical aspects seem sound and some of the high-throughput findings are confirmed with targeted approaches, I have few concerns that would need to be addressed for this study to be considered for publication.
Major comments : -The authors have already published a study on the regulon of this two-component system (https://doi.org/10.3389/fmicb.2017.00688). In the present manuscript, they re-perform transcriptomics and perform additional DAP-seq and confirmation experiments. Both studies also present virulence data on mutants of this system. They should thus make clearer what was the rational for the need of these additional approaches and what does this new study actually brings in terms of new knowledge and biological understanding. Several of the main conclusions (i.e. the PhoPR regulon has been identified & the PhoPR system is essential for virulence in duck) are redundant between the two papers, what is actually new should be highlighted better if the authors want to claim this study as bringing novel insights.
-The authors describe this TCS as a PhoPR system that senses environmental phosphate. This claim is based solely on computational prediction and no experimental proof was made in the current or previous study. Now that they have mutants for both the response regulator and histidine kinase, and primers for RT-qPCR of their targets, it should be relatively easy to test that claim in low or high phosphate growth conditions. If it is found that this TCS does not respond to phosphate availability, renaming or at least discussion of these findings should be considered.
-The authors naming of this TCS as a PhoPR system was notably based on a KEGG / GO pathway enrichment approach from their previous RNA-seq results. In the current, much more well defined version of the regulon, what would be the result of such analysis ?
-The authors state that in their previous study they found the PhoPR system to regulate one third of genes using RNA-seq on mutants. In the present study and using a similar approach, they find a much lower number of dysregulated genes (even when combining results from both mutants), even though they use a more complete version of the genome. This difference should be discussed.
-The main method used in this study is DAP-seq, however the authors do not show anywhere how the data actually looks like. I would suggest adding a representation of the actual peaks, at least for some of the examples studied in depth (those with EMSA for instance) in order for the reader to appreciate quality and intensity of the signal, as is usually done in studies reporting DAPor ChIP-seq data.
-The authors have resequenced their bacterium to provide a more complete assembly of the genome. As this new assembly is used as a reason for the need of reanalyzing the TCS regulon, the differences brought by this new assembly should be briefly discussed.
Minor comments : -Concerning the virulence phenotype, can the authors comment on what is the reason for the decreased virulence in the PhoPR mutants ? i.e. which target genes could be responsible ? -Statistical analysis should be performed in figure 8 -DAP-seq is an emerging method for the study of TF binding sites that is not yet well known, especially in studies on prokaryotes, attention should thus be paid to properly cite the method and previous works. Particularly, the authors do not cite papers from the O'Malley group, which developed DAP-seq, but only older versions of the DAP-chip approach. At least the Nature protocol DAP-seq paper should be cited (https://doi.org/10.1038/nprot.2017.055). Additionally, the authors use a version of the protocol that is made for response regulators and uses acetyl phosphate, which has already been used in other bacterial studies (ex: https://doi.org/10.1093/nar/gkab928 ; https://doi.org/10.1111/mmi.13909) which should also be considered as references.
-Line 462: "The present study has reported the first application of the combination of DAP-seq with RNA-seq ...": that claim is not true (see https://doi.org/10.1111/mmi.13909 for an example of bacterial TCSs, and https://doi.org/10.1128/mSystems.00753-20 for bacterial OCSs). Citing previous similar studies should thus be considered.
- Figure  -Generally, the manuscript should be thoroughly proofread again for typos and confusing phrasings Staff Comments:

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Dear editors and reviewers:
Thank you for your decision and constructive comments on my manuscript.
We have carefully considered the suggestion of Reviewer and make some changes.
We have tried our best to improve and made some changes in the manuscript. We submitted the revised version with 'Marked Up Manuscript -For Review Only' that indicates the changes from the original submission and a clean and new 'Manuscript' without changes marked.
We marked the modified statement in red and provide two types of Line number according to the comments, the yellow Line number in brackets is for Manuscript. Revision notes, point-to-point, are given as follows: To Reviewer #1: 1. Introduction. I think this has too much detail and is more like a literature review.
Much of the detail could be removed without compromising clarity.
Answer: Thank you for the suggestion. We have removed some content about the researches about R. anatipestifer, and modified some contents according to the comment.
2. Line 84. What is a "reaction protein"? Is meant response regulator?
Answer: Thank you for the comment. Yes, we meant 'response regulator'. We have modified it as 'response regulator' throughout the text.
3. Line 85. "causes the expression" is poor English. What is meant is regulates the expression...? And this can be positive or negative.

Answer:
We apologize for the poor language of our manuscript. We really appreciate the suggestion. We have modified the sentence as 'The 5. Line 90. It would be useful to discuss how these are different from PhoP/PhoR.
It will help bring out the significance of the subsequent statement that PhoP/PhoR in R anatipestiferis first described for Gram negative.
Answer: Thank you for the suggestion. We have rewritten this part as 'PhoPR and PhoPQ mainly exist in Gram-positive bacteria and Gram-negative bacteria respectively, and PhoR and PhoQ showed a high similarity in their C-terminal portion. Although many research reported PhoPR and PhoPQ are required for the pathogenicity in their respective species, they are not the physiological equivalent.
PhoPR in Gram-positive bacteria is mainly activated in low phosphate conditions, regulating gene expression to cope with low phosphate environments, while the PhoPR in M. tuberculosis does not respond to the phosphate. In Gram-negative bacteria, PhoPQ mainly responds to the magnesium limitation and antibacterial peptides, and phosphate starvation is sensed by PhoBR.' in Line 88-97(75-84).
6. Line 94. This is confusing to include the genomic loci. They are not defined as such. Perhaps just the gene names and the reference to the prior study make this more clear for the reader?
Answer: Thank you for the suggestion, we modified as 'phoPR double gene deletion strain of R. anatipestifer was constructed in our previous research' in Line 104-105 (84-85).
7. Line 98? What is meant by speculated? The data cited establishes a role in pathogenesis.
Answer: Thank you for the suggestion. It's inappropriate description here, and we have modified and added the data of virulence reduction by deletion of PhoPR as 'the double gene deletion strain completely lost its pathogenicity to ducklings (LD50 > 10 11 CFU)' in Line 115(91-92).
8. Line 99. It would help the reader to include more details as to just how it was concluded that these loci are functionally PhoP/PhoR? And how this is differentiated from PhoP/PhoQ or the aerotolerance TCS systems that comparisons will be made to.
Answer: Thank you for the suggestion. This conclusion is from the previous study that PhoP/PhoR TCS was predicted by KEGG pathway and we added the detailed and reference. We added the detail of gene expression in high (1 mM 11. Line 108. What is a ChIP-level antibody? It will help the reader to avoid jargon.
Answer: Thank you for the suggestion. We have removed this part in Introduction according to Comment 1.
12. Line 114. This would be a good place to define the abbreviation DAP-seq.
Answer: Thank you for the suggestion, we added the abbreviation in Line 128 (98) according to the comment.
13. Line 121. Provides a foundation is better English than laid.
Answer: Thank you for the suggestion, and we have modified the sentence as 'Our data provides a foundation' in Line 135 (105). 14. Line 121 Understanding is better English than clarifying Answer: Sincerely thank you for this suggestion. We modified the sentence as 'Our data provides a foundation for understanding the role of the phoP/phoR two-component system in the pathogenic process of R. anatipestifer' in Line 135-137(105-106) according to this comment.

Line 123 Insights is better English than sight
Answer: Thank you for the suggestion. We modified the sentence as 'a new insight into the regulation of PhoP' in Line 137 (107). 16. Line 124. Provides (present tense) vs provided (past tense) is better English.
Conclusions are most appropriately made in present tense, while reference to data are most appropriate in past tense.
Answer: Thank you for the suggestion, and we modified as 'Our data provides a foundation for understanding the role of the phoP/phoR two 24. Line 147. The quality of these results using this method is completely dependent on the quality of the purified protein. Thus, it will be essential to include details as to the quality of this protein preparation, including it purity, its ability to bind specifically to DNA as compared to an appropriate negative control, that it is of the appropriate size and that it actually is the PhoR protein and not some fortuitously purified protein.
Answer: Thank you for the suggestion. We added the information about the purity of PhoP in supplement. In Figure S1 we provided the SDS-PAGE result and added chromatography of purified PhoP to show the purity and size, and we mentioned the detail of purification in Materia and methods. Negative control of DAP-seq we performed is that the sheared genome directly binding with resin, and the eluted DNA is the non-PhoP binding. We added the information of purity in Fig. S1, 'Chromatography of His6-PhoP by gel-filtration. The number above the peaks is the retention time. The purity of His6-PhoP is 94.4232%'.
25. Similar to above. Characterization of the truncated DBD protein is also important for evaluation of these results.
Answer: Thank you for the suggestion. We have updated the information of the purity of PhoP-DBD in supplement according to the comment. We added the information of purity in Fig. S1, 'Chromatography of His6-PhoP-DBD by gelfiltration. The number above the peaks is the retention time. The purity of His6-PhoP-DBD is 91.4392%.'.
26. Line 147. Details are required in order to evaluate the quality of these data.
Including some specific details of the method, the specific strain that was analyzed, the stage of growth that were sampled and the number of technical and biological 28. Line 149. Reference for the MEME tools should be included.
Answer: Thank you for the suggestion. We added the reference in Line 182(143).
29. Line 163. How is it concluded that all binding to the 764 genes was specific?
What negative controls establish the specificity of binding? Answer: Thank you for the suggestion. Several statements that we made were more ambiguous than intended. We added the statements that the negative control of DAP-seq was performed without PhoP, which means, the highthroughput data of negative control was generated from the unspecific bound DNA eluted from the resin. The 764 genes were enriched based on the nonspecific binding. And we validated the results by perform EMSA with the upstream region of 8 genes.
30. Line 167. It is not possible to conclude from these data that the domain is not involved. Only that it is not sufficient for binding. It still could be necessary.

Line 194. What is a "confident correlation"?
Answer: Thank you for the suggestion. We have modified this sentence as 'confirmed the accuracy of the high-throughput results' in Line 235-236(194), and added the correlation between the RT-qPCR and RNA-seq results of 8 candidate genes in Fig. S4. 33. Line 197. This statement is not clear. What is meant by "changed in the same pattern"? Was this different media or stress imposed on the cultures? Answer: Thank you for pointing out this indistinctness. We mean that the 35 genes downregulated in both ΔphoP and ΔphoR, and the 22 genes upregulated in both ΔphoP and ΔphoR. We modified the sentence as 'Further analysis of these 59 genes showed that the expression of 57 genes changed in the same trend including 35 genes downregulated and 22 genes upregulated in both ΔphoP and ΔphoR' in Line 239-241(196-199).

Line 199. What is meant by "inconsistent regulation"?
Answer: Thank you for pointing out this indistinctness. We meant that the 2 genes displayed different trend of regulation in ΔphoP and ΔphoR, and we have modified the sentence as 'only two genes displayed different trend of regulation' in .
35. Line 203. The rationale for this section requires more detail. Is this concordance between the two datasets? If so, whatLine 203 is the rationale for this vs possible technical differences in sensitivity that could account for nonconcordance?
Answer: Thank you for the suggestion. We added the information to make the section clear. 'From the RNA-seq of ΔphoP, we can know the genes directly or indirectly regulated by PhoP, and the DAP-seq of PhoP provides information of PhoP-binding sites on the genome' in Line 249-251(205-208). We generated 183 differentially expressed genes (|log2FoldChange| > 1, Padj < 0.05) from the RNAseq of ΔphoP, and 764 genes from DAP-seq. We focused on the DEGs which the PhoP binds in the upstream region, and validated 5 genes via EMSA and RT-qPCR.
We think the difference of sequencing depth and intrinsic decreased accuracy of high-throughput sequencing leads to the inconsistency between of the peakenrichment and gene expression.
36. Line 203. It also appears that many more genes were found to be regulated by DAP-seq than by RNA-seq? What could explain the discrepancy? It is likely that there are many explanations that involve the strengths and weaknesses of the methods?
Answer: Thank you for the comment. Due to the principle of DAP-seq, the data generated from DAP-seq is the sites of PhoP-binding motif on the genome. RNAseq mainly showed transcription level of all genes in RA-YM, and there are direct and indirect regulation caused by gene disruption. Strict statistical screening of DEGs (|log2FoldChange| > 1, Padj < 0.05) also lead to some regulated genes not participating in the following analysis.
37. Line 203. Is the approach here that valid regulated genes are only those that appear in both data sets? That seems limited and requires more justification, as there could be many technical reasons for this. And it overlooks the result above finding the common motif in the 764 genes identified by DAP-Seq. Do the RNAseq dataset genes share this motif? Answer: Yes, the candidate target genes are those appear in both data sets. And we admit that there are some limitations, and we can't announce that we have screened all the direct regulatory genes. We performed EMSA and RT-qPCR on selected 5 of candidate target genes to verify the confidence of data, and showed the binding region of each gene. MEME analysis of all 583 peaks (located at the upstream of 764 target genes), showed the conserved motif appeared in 579 peaks, and absent in 4 peaks (located at upstream of 4 target genes). Not all the RNA-seq dataset genes were shared the motif, but the 50 candidate target genes we screened share the binding motif.

Line 214. What is "membrane compromised TBDR"?
Answer: Thank you for the suggestion, here is our writing mistake. We have modified it as 'membrane composed of TBDR' in Line 497(392).
39. Line 219. This section is more a discussion rather than a presentation of data.
Answer: Thank you for the suggestion. We moved some content to the Discussion.
40. Line 234. Do the mutants have a defect in aerotolerance? That seems like it would be easy to test and these data would significantly test this hypothesis.
Answer: Thank you for the constructive suggestion. We did perform a preliminary study on the difference of aerotolerance of mutants. We tested the change of expression level of Bat operon after H2O2 treatment. We added the survival rate in Discussion and Figure S5, 'the survival rate of ΔphoP and ΔphoR decreased compared with WT after exposure of 10mM H2O2'. We have added the content as 'The genes batA and batC were upregulated when H2O2 was added, and downregulated either when treated anaerobic, or when phoP or phoR was deleted, the survival rate of ΔphoP and ΔphoR decreased compared with WT after exposure of 10 mM H2O2' in Line 259-264(218-221). We also are conducting future work on Bat operon and constructing gene knockout strains in our future project. 41. Line 264. What is a gradient dose? Is meant "a range of doses"?
42. Line 256. This conclusion of "mainly regulate..." is based on the assumptions that the only valid regulation are those in the concordance of the DAP-seq and RNA-seq datasets. The strength of this conclusion remains to be established by more information, as indicated above.
Answer: Thank you for the significant suggestion. Due to the difference of sequencing depth and principle between DAP-seq and RNA-seq, there is no significant concordance in the enrichment of peaks from DAP-seq and the expression change from RNA-seq. We verified seven genes by EMSA and RT-qPCR.
We removed the conclusion according to the comment. 45. Line 270. huddling is a mental state? Generally, this sort of behavior is usually described as morribund or lethargic, as it does not imply that there is any direct effect on brain function, only it can be concluded that the animals are experiencing an overall state of malaise and lethargy because they are sick.
Answer: Thank you for the suggestion. We have modified the description of symptoms as 'The ducklings infected with the ΔphoP strain showed lethargic after injection, preferred huddling together than moving' in Line 329-331(248-249).
46. Line 299. "Mock infected" is more precise that "infected by PBS" Answer: Thank you for the suggestion. We have modified the sentence as 'The tissues did not show obvious pathological changes in mock infected, . 47. Line 350. As discussed above, there is no increase in clarity from the inclusion of negative data. There are many reasons why an attempt to make a highly specific antibody was not successful. None of which are important for interpretation of the actual data presented.
Answer: Thank you for the constructive comment. We have removed the part of negative data, and rewritten this part as 'DAP-seq allows us to use phosphoryl group donor to mimic phosphorylation of RR, avoid the limitation of unknow activating signals of PhoP. DAP-seq does not require a specific antibody or tagged transgenic lines comparing with ChIP-seq, and only requires an affinity method for the tag-labeled response regulator.' in Line 405-409(323-327)  Answer: Thank you for the constructive suggestions. As you said, in our previous research, we initially identified the phoPR two-component system, and performed RNA-seq and animal models tests of ΔphoPR. We confirmed that phoPR had a great influence on the pathogenicity of RA-YM. The immunogenicity of ΔphoPR to ducklings was tested (not shown), and it was found that the pathogenicity of Δ phoPR decreased after its deletion, and its immunogenicity also decreased significantly. And we wanted to further dig the specific model of phoPR regulation on virulence and found an appropriate live vaccine candidate strain that retained the immunogenicity, so our work focuses on the direct regulatory pathway of PhoP in RA-YM this time. We updated the complete genome of RA-YM via Pacbio platform, and constructed phoP and phoR single-gene deletion strain, so as to more specifically study the effects of each component in the phoPR. We found that both ΔphoP and ΔphoR can retain immunogenicity (another article being submitted). We also obtained a further understanding of the regulation mechanism of RA-YM through the data of DAP-seq and RNA-seq. In this research, we found that the motif of PhoP-binding in RA-YM, and is mainly involved in the direct regulation of which genes, providing a foundation for the further study on the virulence of RA.
-The authors describe this TCS as a PhoPR system that senses environmental phosphate. This claim is based solely on computational prediction and no experimental proof was made in the current or previous study. Now that they have mutants for both the response regulator and histidine kinase, and primers for RT-qPCR of their targets, it should be relatively easy to test that claim in low or high phosphate growth conditions. If it is found that this TCS does not respond to phosphate availability, renaming or at least discussion of these findings should be considered.
Answer: Thank you for the suggestion. We tested the expression of phoP and phoR in low or high phosphate growth conditions. Using RT-qPCR, we found that phoP and phoR was upregulated in low Pi medium (1μM), while downregulated in high Pi medium (1mM).
RT-qPCR analysis of phoR and phoP in low (1 μM) and high (1 mM) phosphate growth condition.
-The authors naming of this TCS as a PhoPR system was notably based on a KEGG / GO pathway enrichment approach from their previous RNA-seq results. In the current, much more well defined version of the regulon, what would be the result of such analysis?
Answer: Thank you for the suggestion. We annotated the new version genome by KEGG and GO. From the predicted results, the TCS is still annotated as PhoP and PhoR in KEGG. We tested gene expression in high-or low-phosphate, and displayed the root neighbor-joining tree of PhoP and PhoR respectively. -The authors state that in their previous study they found the PhoPR system to regulate one third of genes using RNA-seq on mutants. In the present study and using a similar approach, they find a much lower number of dysregulated genes (even when combining results from both mutants), even though they use a more complete version of the genome. This difference should be discussed.
Answer: Thank you for the suggestion, we added the discussion according to the comment. 'From the perspective of gene or operon function, there should exist divergence in the DEGs produced by the deletion of phoP, phoR or phoPR due to the different regulatory pathway in cell. As a HK, PhoR not only phosphorylated the cognate RR, but also possible transferred the phosphoryl group to other RRs, while PhoP as the RR, which may also get phosphorylated by other non-cognate HKs. Therefore, we believe that there is difference in the global transcriptome between the double gene deletion strain and single-component deletion strains.
Due to the limitation in previous research, the RNA-seq of ∆phoPR was not duplicated and the reference genome was not appropriate, resulting in an inability to conduct statistical analysis.' In Line 482-491(376-385). We updated a complete genome of RA-YM by PacBio platform, and conducted three biological duplicates in RNA-seq in this study. Because of the statistical analysis of RNA-seq this time, the DEGs we obtained had a higher credibility, but reduced in number compared with previous data.
-The main method used in this study is DAP-seq, however the authors do not show anywhere how the data actually looks like. I would suggest adding a representation of the actual peaks, at least for some of the examples studied in depth (those with EMSA for instance) in order for the reader to appreciate quality and intensity of the signal, as is usually done in studies reporting DAP-or ChIPseq data.