Repression of ctrA and chpT by a transcriptional regulator of the Xre family that is expressed by RpoN3 and its cognate activator protein in Cereibacter sphaeroides

Benjamín Vega-Baray; José Hernández-Valle; Sebastián Poggio; Laura Camarena

doi:10.1371/journal.pone.0321186

Peer Review History

Original SubmissionDecember 17, 2024
17 Dec 2024 Author Response https://doi.org/10.1371/journal.pone.0321186.r001
10 Jan 2025 Decision Letter - MD MOTALEB, Editor PONE-D-24-58434Repression of ctrA and chpT by a transcriptional regulator of the Xre family that is expressed by RpoN3 and its cognate activator protein in CereibactersphaeroidesPLOS ONE Dear Dr. Camarena, Thank you for submitting your manuscript to PLOS ONE. Two expert reviewers evaluated the merits of the manuscript. As noted below, both reviewers are positive but have a few additional concerns and suggestions that I would like you to address in a revised manuscript. Please submit your revised manuscript by Feb 24 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors focused on the regulation mechanism of flagellar expression in Cereibacter sphaeroides. They found several new signaling components involved in the expression of Fla2 protein and proposed a gene-regulation model for assembling multiple polar flagella. The experiments are well-designed and proceed carefully based on the detailed discussion of the results. I suggest several modifications to the manuscript as follows. (Figure 2) Seeing the photos under the photoheterotrophic condition, I cannot understand the difference in the expansion of swimming rings between AM1 and SP13; both strains’ rings seem to be expanded similarly by MgSO4. How were the ring diameters measured? In addition, as in the other figures, please add indications of each panel by A, B, C, and D. (lines 280-283) The authors state “Conversely, in plates incubated under the photoheterotrophic condition, the inclusion of MgSO₄ only slightly promoted the swimming of BOTH strains ( ~2.5-fold) (Fig. 2).” The right panels of Fig 2 show the swimming diameter of BV11under photoheterotrophic condition is larger than that under heterotrophic condition. It would be better to revise the sentence to “Conversely, in plates incubated under photoheterotrophic conditions, the inclusion of MgSO₄ only slightly promoted the swimming of AM1.” Or, please explain the enhancement of BV11 motility under the photoheterotrophic condition. (Figure 3) It is difficult to see the scale bars in the EM photos. Please use a larger font size. (lines 389-390) The authors state “the OVEREXPRESSION of XrpA leads to the inhibition of Fla2-dependent motility even under these conditions, in which BV11 (xrpA+) is able to swim.” Fig. 6A shows that the expression levels of XrpA from BV11 and AM1/pRK_xrpA appear to be comparable. What is the rationale for the overexpression of XrpA in AM1/pRK_xrpA? (Figure 12) It would be better to include flaA2 in Figure 12, because the control of flaA2 expression is the goal of the presented signaling network. Reviewer #2: The authors report here on factors that control the expression of one of the two sets of flagellar genes (fla2 genes) in the alpha-proteobacterium Cereibacter sphaeroides. Expression of the fla2 genes is controlled by the CckA/ChpT/CtrA two-component system. The authors identify here a new transcriptional regulator, XrpA, that negatively regulates expression of the fla2 genes by reducing expression of CtrA. They further show that XrpA binds to the promoter regions of ctrA and chpT, and presumably represses transcription of these genes. They also identify one of the four paralogs of the alternative sigma factor RpoN (RpoN3) and its cognate activator (a protein that they designate as AprX) are required for transcription xrpA. The results are well documented and of broad interest. The manuscript is well written. Specific comments: 1. The authors mention that four bEBPs with a DNA-binding domain have been identified in the C. sphaeroides genome (lines 489-490). This seems to imply that there are bEBPs in C. sphaeroides that lack a DNA-binding domain. Is this correct? If it is true, it would be informative to briefly discuss these bEBPs. 2. Was expression of xrpA examined under nitrogen-fixing or limiting nitrogen conditions? The authors report that under heterotrophic growth conditions, xrpA expression was higher when succinate was included as the carbon source versus casamino acids. When using casamino acids as a carbon source, the bacteria presumably are excreting ammonia since the demand of carbon would be greater than the demand for nitrogen. This might explain the lower expression of xrpA with casamino acids as the carbon source. 3. Bacteria that have two flagellar systems typically elaborate a polar flagellum that is used for swimming and multiple lateral flagella that are used for swarming. That is not the case here, and instead both sets of flagella are used for swimming. It would be helpful if the authors pointed out this distinction. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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Revision 1
22 Feb 2025 Author Response RESPONSE TO REVIEWERS Reviewer #1: …..I suggest several modifications to the manuscript as follows. (Figure 2) Seeing the photos under the photoheterotrophic condition, I cannot understand the difference in the expansion of swimming rings between AM1 and SP13; both strains’ rings seem to be expanded similarly by MgSO4. How were the ring diameters measured? In addition, as in the other figures, please add indications of each panel by A, B, C, and D. (Figure 2) Swimming plates are inoculated with a 2 µl drop of each strain, if the drop slides on the surface of the culture medium, we observe a lopsided denser growth zone and if the strain can swim a less dense swimming ring appears. In Fig 2C in the presence of MgSO4, SP13 only shows the lopsided growth, while the AM1 strain exhibits the lopsided growth and the swimming ring. When this occurs, we measure the shortest diameter of the ring. This information was included in Material and Methods (lines 160-163). As suggested, we have added indications of each panel by A, B, C and D. (lines 280-283) The authors state “Conversely, in plates incubated under the photoheterotrophic condition, the inclusion of MgSO₄ only slightly promoted the swimming of BOTH strains ( ~2.5-fold) (Fig. 2).” The right panels of Fig 2 show the swimming diameter of BV11under photoheterotrophic condition is larger than that under heterotrophic condition. It would be better to revise the sentence to “Conversely, in plates incubated under photoheterotrophic conditions, the inclusion of MgSO₄ only slightly promoted the swimming of AM1.” Or, please explain the enhancement of BV11 motility under the photoheterotrophic condition. The purpose of Figure 2A is to explain why heterotrophic growth conditions utilizing 15 mM succinic acid as a carbon source were chosen for the subsequent experiments in this work. As it can be seen, the difference in the response of the AM1 and BV11 strains to the presence of MgSO4 is stronger under this condition, making it more suitable to study the effect of XrpA. We have slightly modified the original text to clarify this point (lines 290-293). (Figure 3) It is difficult to see the scale bars in the EM photos. Please use a larger font size. (Figure 3) As suggested, we have increased the font size and the scale bar. (lines 389-390) The authors state “the OVEREXPRESSION of XrpA leads to the inhibition of Fla2-dependent motility even under these conditions, in which BV11 (xrpA+) is able to swim.” Fig. 6A shows that the expression levels of XrpA from BV11 and AM1/pRK_xrpA appear to be comparable. What is the rationale for the overexpression of XrpA in AM1/pRK_xrpA? We have modified the text to indicate that XrpA is expressed in an unregulated manner from the pRK_xrpA plasmid (not overexpressed), and this significantly reduces the swimming ability of AM1/pRK_xrpA when grown in casamino acids (line 399). To further support this idea, we have included a supplementary figure (Fig. S5) to show the differences in XrpA levels under different growth conditions for the BV11 and AM1/pRK_xrpA strains (lines 401-405). (Figure 12) It would be better to include flaA2 in Figure 12, because the control of flaA2 expression is the goal of the presented signaling network. (Figure 12) We have included fla2 in Figure 12 as suggested. Reviewer #2 Specific comments: 1. The authors mention that four bEBPs with a DNA-binding domain have been identified in the C. sphaeroides genome (lines 489-490). This seems to imply that there are bEBPs in C. sphaeroides that lack a DNA-binding domain. Is this correct? If it is true, it would be informative to briefly discuss these bEBPs. 1. C. sphaeroides has five EBPs with the conserved motif GA(F/S)TGA which is necessary to interact with RpoN. The EBP FleT lacks the DNA-binding domain, and its transcriptional action is exerted through FleQ, another EBP. As suggested by the reviewer, we have included information about FleT in the Introduction section (lines 79-84). We believe that this information fits well in this section and clarifies the role of this protein. 2. Was expression of xrpA examined under nitrogen-fixing or limiting nitrogen conditions? The authors report that under heterotrophic growth conditions, xrpA expression was higher when succinate was included as the carbon source versus casamino acids. When using casamino acids as a carbon source, the bacteria presumably are excreting ammonia since the demand of carbon would be greater than the demand for nitrogen. This might explain the lower expression of xrpA with casamino acids as the carbon source. 2. The expression of xrpA has not been examined under nitrogen-fixing or nitrogen-limiting conditions, and the reviewer's suggestion is plausible in explaining the effect of casamino acids. We explored this matter by using the usual amount of (NH4)2SO4 in the culture medium, which corresponds to 3.7 mM, and reducing it to 1.85 mM, 0.74 mM, and 0.37 mM. Total cell extracts of BV11 grown under these conditions did not reveal any notable differences in XrpA levels between the samples. This figure has been uploaded for review. 3. Bacteria that have two flagellar systems typically elaborate a polar flagellum that is used for swimming and multiple lateral flagella that are used for swarming. That is not the case here, and instead both sets of flagella are used for swimming. It would be helpful if the authors pointed out this distinction. 3. We have included this information as suggested (lines 93-95). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0321186.r003
4 Mar 2025 Decision Letter - MD MOTALEB, Editor Repression of ctrA and chpT by a transcriptional regulator of the Xre family that is expressed by RpoN3 and its cognate activator protein in Cereibactersphaeroides PONE-D-24-58434R1 Dear Dr. Camarena, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, MD A MOTALEB, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0321186.r004
Formally Accepted
Acceptance Letter - MD MOTALEB, Editor PONE-D-24-58434R1 PLOS ONE Dear Dr. Camarena, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. MD A MOTALEB Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0321186.r005

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