PGENETICS-D-26-00078

Control of motility and cell shape of Haloferax volcanii is linked by a transcriptional regulator

PLOS Genetics

Dear Dr. Albers,

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PLOS Genetics

Danielle Garsin

Section Editor

PLOS Genetics

Aimée Dudley

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PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

Additional Editor Comments:

Three expert reviewers have supplied comments for your manuscript. As you can see, all reviewers expressed many positive qualities about the work, but also provided a number of comments they felt were important for improving the manuscript. Some consistent concerns involved better resolution of whether CsmR directly regulates expression of investigated motility and rod genes, along with concerns about the nature of its connection to cell morphology. There are also a number of other comments to address to improve the quality of the work. Please consider addressing many of these comments if you would like to submit a revised manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

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Reviewer #1: attachment

Reviewer #2: The manuscript titled “Control of motility and cell shape of Haloferax volcanii is linked by a transcriptional regulator” investigates the transcriptional coordination of motility and cell-shape transitions in Haloferax volcanii by identifying and characterizing a novel transcription factor, CsmR. The authors employ a combination of genetic manipulations, motility assays, microscopy, transcriptional profiling, and computational structural predictions to demonstrate CsmR is required for motility, and functions as a major transcriptional regulator linking motility and cell shape in response to environmental cues across distinct growth phases.

The authors show that deletion of csmR abolishes motility and archaellum formation while over expression results in hyper motility and maintenance of rod-shaped cells. RNA-Seq and promoter motif analysis indicate a prominent role for CsmR in transcriptional regulation of archaellum formation, cemotaxis and cell-shape- associated genes, and structural predictions are consistent with CsmR functioning as a DNA-binding protein. Moreover, proposing a hierarchical regulation of motility and cell shape- with CsmR as the central regulator and CirA as a fine-tuner -is significant, and identification of HVO_1211s as regulatory RNA is exciting.

The experimental approaches are appropriate and data analyses are done well. The manuscript is written clearly mostly.

Taken together, this study addresses an important question regarding how environmental cues are transmitted to cellular machinery to elicit coordinated changes in motility, morphology, and physiology. However, the current data provide indirect and partial support for hierarchical regulation of motility, cell shape and environmental adaptation by CsmR, CirA and RosR. Below are my comments from major to minor, addressing these points would substantially strengthen the study and its contribution to the field.

1. The central conclusion of the manuscript is that CsmR functions as a transcriptional regulator that directly controls expression of motility- and cell-shape–associated genes. While RNA-seq, promoter motif analysis, and computational structural predictions support this a direct experimental evidence that CsmR binds to DNA or target promoters is missing. Demonstrating CsmR binds DNA using available methods, such as ChIP-qPCR, ChIP-seq, electron mobility shift assay (EMSA), or promoter-reporter assays with mutated motif would significantly strengthen the conclusion that CsmR directly regulates the listed target genes.

2. Deletion of csmR leads to upregulation of motility and chemosensory genes while complete loss of motility is observed across growth phases. Despite promoter motif enrichment and structural predictions suggest that CsmR may directly regulate these genes, the manuscript does not clearly distinguish direct transcriptional effects from indirect or compensatory responses might be arising from disrupted motility or signaling. Clarify to distinguish CsmR primarily acts as a direct regulator or whether the observed transcriptional changes is a compensatory response would be significant and important for the mechanistic understanding.

3. The manuscript introduces CsmR as a novel transcriptional regulator of motility and cell shape in Haloferax volcanii, but its conservation among closely related haloarchaeal species is not explored. A basic homology analysis (e.g., BLAST-based identification of CsmR homologs), together with examination of synteny (e.g., proximity to archaellum or chemotaxis genes), would be helpful in determining if CsmR represents a conserved regulatory feature within haloarchaea or a species-specific adaptation. This analysis would provide valuable evolutionary context and contribute to the impact of the study.

4. CsmR has been implied many times as an integrator of environmental or metabolic signals, largely based on its classification within the Lrp/AsnC family and comparisons to bacterial regulators and prior work on TrmB/TbsP. However, no evidence for metabolic regulation of CsmR is presented, I suggest rephrasing such sentences (for example, lines 111-113 ) to clearly distinguish possible roles from demonstrated functions.

5. The manuscript looks at transcriptional changes at OD600 = 0.02, 0.2, and 2.0, corresponding to early, mid-exponential, and stationary phases, respectively. Additional clarification on the rationale for selecting OD600 = 2.0 as the stationary time point would be helpful since at this density cells may also be experiencing stress responses that could interfere with interpretation of physiological adaptation related specifically to motility and cell shape. Providing a growth curve and/or brief justification for these OD cutoffs would help.

6. HVO_1211s is described as a regulatory RNA and refers to it as an sRNA, while also suggesting that it stabilizes cirA mRNA through extensive sequence overlap. It would be helpful to clarify the mechanistic basis for this proposed stabilizing effect and to more clearly define how HVO_1211s should be classified.

7. Different adjusted P-value cutoffs (0.05 to 0.1) are used in RNASeq analyses but the rationale for using these different thresholds is not always stated. Justification for the choice of adjusted P-value cutoffs, especially where padj < 0.1 is used to define gene sets for downstream analyses, would be important. In addition, at line ~263 (arCOG enrichment analysis), results are described using “P-value < 0.05” without specifying whether this is raw P-value or adjusted value.

8. OD measurements of cells for northern blot analysis (Section 6.11, line ~740), are at 650nm however OD600 is used elsewhere in manuscript to define growth phases and sampling points. What is the rationale for using OD650? It would be more consistent to use a single wavelength in the manuscript, or explicitly state how OD650 values relate to OD600. This would be important for clarity and reproducibility.

Reviewer #3: This interesting manuscript reports on the identification of a regulator of motility and cell shape in archaea. Until now such a main regulator was not yet identified in haloarchaea. This regulator has connections with different other regulators involved in cell shape and motility that were discovered in recent years. Therefore, this is a very timely manuscript and also important for the community.

The introduction well written, required information given and it is coming quickly to the point. The quality of the figures in this manuscript is very high. The paper contains a very high density of good quality data. The paper has a high number of (supplementary) figures. The regulatory network seems quite complex and the data points towards a multi-layered regulatory network. This makes the manuscript at times difficult to follow.

I have several suggestions to make the manuscript easier to read for a broader audience.

Introduction/abstract

Line 20: to make it more accessible for a broader audience: add short explanation of EarA. For non-archaeal readers this might not be evident.

Line 27: archaellum gene expression ?

Line 28: Generalizing CsmR's role to all Haloarchaea lacks supporting evidence, as no comparative analysis is presented for ortholog presence, synteny near archaellin genes, or conservation in other Haloferax species or haloarchaea (e.g., Hfx. mediterranei, Hbt. salinarum, where multiple Lrp/AsnC regulators exist but vary in function). In order to justify the broad claims, add a bioinformatics survey (e.g. BLAST and synteny plots) of CsmR-like genes across haloarchaea. Otherwise, limit the survey to H. volcanii.

Results

Line 137: It is difficult to assess whether the cells lack archaella, as the entire cell cannot be seen in the micrographs. Furthermore, line 160 states that the aim was to test whether the csmR deletion leads to cell shape defects. Were the shapes not visible in the electron micrographs?

Line 165: the authors write : ‘To see if those morphological changes impact the composition of membrane lipid types the abundance of several lipid species present in….. ‘At the end of this paragraph they conclude ‘Hence, it is unlikely that the observed changes in cell morphology during growth are caused by an altered lipid composition.’. This analysis of the cell lipid composition comes a bit abrupt and it is not completely clear what the purpose of this analysis is and what the causative order is. Please make this clearer in the text.

Line 169: PG maybe write out in full first time

Fig1 : complementation with CsmR in the deletion of the CsmR strain leads to hypermotility, likely due to the strong xylose promoter. Is it possible to use less xylose and see then only partial complementation or is this promoter still quite leaky?

Figure 1A: The absence of scale bars from the motility halo images prevents quantitative size comparisons across conditions. Please add them with specified units.

Figure 1D: Graph lacks a statistical analysis to support the significance claims. Furthermore, the assertion that csmR overexpression in H26 stabilises rod shapes throughout growth phases is undermined by length distributions that resemble mixed populations during the mid-growth phase. I recommend including statistics and clarifying this.

Fig2 C: are similar figures for mid and stationary phase available or are they looking similar and are therefore not shown?

Fig 3D: I do not understand the different circles. The size circles indicate the nr of affected genes, but then at the same time they also indicate the significance or not. How to distinguish between these two features? Please add some information to the figure legend.

Figure 5B, Figure S9 D&F, Figure S14 D&E, Figure S17 D&E: Please provide statistical analysis of cell length measurements.

Figure 5C, Figure S9 A&B, Figure S14A: Please provide scale bars in motility halo images.

Line 268: two times upstream in the title. Maybe remove one

Line 275: ‘half of the genes….’ How many of these 61 genes of which the upstream region was analyzed, are functionally linked with motility/chemotaxis/cell shape ect? It is mentioned that of the 21 with the motif, half of the genes are functionally linked with motility/chemotaxis/cells shape. Is this an over or underrepresentation? Please add this information, otherwise the statement doesn’t say much.

While in silico evidence (AlphaFold3 β-sandwich core, pLDDT>90; sequence homology) supports predicted Lrp/AsnC family membership for CsmR, firm claims that it encodes a functional transcriptional regulator might overstate the data. RNA-seq reveals transcriptional changes, but lacks proof of direct DNA binding/regulation. The standard method for proving these claims would be EMSA/footprinting on target promoters, including autoregulation; ideally ChIP-seq for direct binding proof. As a reader, one might wonder why not EMSAs were attempted. I assume that this is because purification and DNA interaction studies with such a halophilic protein are not possible to perform. This would be a good location to outline the experimental challenges of this model system and justify why only in silico methods were used.

Recommended revisions is also replace definitive phrasing (e.g., "master regulator") with "putative regulator" or "modulates".

The authors use several structural prediction tools to study the possibility of Csmr DNA binding capabilities. I would further recommend to perform a structure-based multiple sequence alignment of CsmR with characterised Lrp/AsnC family members (e.g. E. coli Lrp/AsnC and B. subtilis LrpC). This should highlight the conserved residues in the N-terminal HTH DNA-binding domain and the C-terminal oligomerisation/effector pocket, providing robust support for the predicted family membership alongside AlphaFold3.

Fig 5I: can some more information be added to the legend? I find it difficult to understand the figure.

Material & Methods

Line 661: A paired t-test is appropriate only when mutant and wild-type (WT) measurements are directly paired within the same experimental unit, such as on identical plates or from matched biological replicates. However, the figure legends (Fig. 1, line 144; Fig. 5, line 364, Fig S9, Fig. S14, Fig. S17, Fig. S18) describe motility halo areas normalised to the average WT halo area across replicates, treating the groups as independent. I recommend revising to an unpaired (independent) two-tailed t-test.

Line 662: When harvesting different volumes of culture (1 L, 20 mL or 3 mL) for RNA sequencing, there is a risk of distorting gene expression profiles due to physiological artefacts such as nutrient gradients and oxygen limitation that affect transcription independently of cell density. From the M&M I cannot extract if different container types for cultivation also lead to different processing times. e.g. whether centrifugation or filtration is used, and how long pellets are left to settle.

Please provide full details on harvesting method and pellet processing times. I understand that there are practical considerations and experimental constraints. Therefore, it would be good to describe the different treatment in the M&M and also (maybe in the discussion) mention that these might also impact gene expression.

Discussion/model/ abstract: I like the division of the discussion in a summary, general discussion and model good. However, I find it still hard to follow the model. What is now the hypothesis that the authors have that CsmR knock-out leads to a non-motile phenotype, while at the same time leading to increased expression of archaellum genes?

In the model it is implied that archaellation and cell shape are necessary coupled together. Is this really the case? In different haloarchaea there are reports of round cells that can also still be archaellated. Should this not be separated in the model?

The authors discuss quorum sensing. Does the way that they sample cells also result in differences in quorum sensing between the samples, for example when the samples are concentrated in the process of harvesting them?

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics   data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: None

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: CsmR.review.pdf

PGENETICS-D-26-00078R1

CsmR controls both, motility and cell shape, in Haloferax volcanii

PLOS Genetics

Dear Dr. Albers,

Thank you for submitting your manuscript to PLOS Genetics. After careful consideration, we feel that it has merit but does not fully meet PLOS Genetics's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript within by Jun 28 2026 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 plosgenetics@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgenetics/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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PLOS Genetics

Danielle Garsin

Section Editor

PLOS Genetics

Aimée Dudley

Editor-in-Chief

PLOS Genetics

Anne Goriely

Editor-in-Chief

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Additional Editor Comments:

Two of the three original Reviewers agreed to review the revised manuscript. Both Reviewers gave very positive comments for the revised manuscript. There is one comment from Reviewer 2 that does need addressing. Therefore, the revised mansucript is being sent back to the authors for additional editing.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #2: One minor revision is needed in Supplementary figure1: the gene cooccurance plot could use a legend to aid the reader in interpreting the figure.

Other than that, the authors have adequately addressed all my prior comments, and substantially strengthened the manuscript. Particularly, the addition of ChIP-Seq data provides a significant support for direct regulation by CsmR and a critical contribution to the study. Also, the newly included phylogenetic/ conservation analyses present a valuable evolutionary context and broader relevance to the field.

Reviewer #3: I am very satisfied with how the manuscript has been revised. The chip-seq is a great addition to the paper. Moreover, all points I raised were addressed.

**********

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics   data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: No

Reviewer #3: No

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Dear Dr Albers,

We are pleased to inform you that your manuscript entitled "CsmR controls both, motility and cell shape, in Haloferax volcanii" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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