Chemosensory landscape of order Vibrionales: Genome-scale phylogenomics uncovers four chemosensory architectures driven by bipartite genome organization

  1. Shravani Rajan Rawool
  2. Gaurav Sharma

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Abstract

Chemosensory systems (CSSs) are multi-protein assemblies regulating bacterial motility and cellular functions. Despite extensive study of chemotaxis in Vibrio cholerae , a comprehensive evolutionary analysis of CSSs across order Vibrionales has been lacking. Using 116 curated representative genomes across 28 Vibrio clades and ~10,000 RefSeq/metagenome-assembled genomes, we characterized the chemosensory toolkit of Vibrionales. CheA-based phylogenetics, CSS architecture, sequence similarity networks, structural comparisons, and synteny analysis identified four discrete CSS types: F6, F7, F9, and a novel lineage, F8. F6 is universally conserved on chromosome I and essential for flagellar motility, while F7, F8, and F9 show patchy, replicon-flexible distributions reflecting lineage-specific retention or horizontal acquisition. F6, F7, and F8 were vertically inherited from Gammaproteobacteria; F9 was horizontally acquired from Alphaproteobacteria. Structural analysis reveals conserved CheA folds despite sequence divergence, with lineage-specific domain insertions in F8 and F9. Collectively, this study reveals a two-tier chemosensory architecture within order Vibrionales, 1) a chromosomally stable F6 motility core under purifying selection, 2) overlaid by dynamically evolving F7, F8, and F9 accessory systems, wherein multipartite genome organization itself serves as an evolutionary substrate for sensory innovation, enabling rapid niche adaptation without compromising core chemotactic fidelity.

Importance

Bacteria actively navigate their environments using molecular sensors called chemosensory systems, moving toward nutrients and away from harm. In Vibrio bacteria, which cause cholera and serious seafood-borne infections, these same sensors help bacteria locate and colonize the human gut, making them direct contributors to disease. Yet how systems evolved across the broader Vibrio family remained unknown. By analyzing over 10,000 Vibrio genomes, this study maps CSS diversity order-wide, identifies a previously uncharacterized system (F8), and reveals how the bacteria’s distinctive two-chromosome genome enables flexible, niche-tailored sensory assembly. These findings expose key signaling proteins, CheA and MCP, as promising drug targets to disarm Vibrio pathogens by crippling their ability to sense and colonize the human host.

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    Reply to the reviewers

    Reviewer #1

    Evidence, reproducibility and clarity

    The manuscript presents a comparative genomic analysis of chemosensory systems across the order Vibrionales. By combining phylogenetic analyses, genomic context, MCP repertoires, and structural comparisons, the authors investigate the diversity and evolution of chemosensory systems within Vibrionales. The study addresses an interesting question and assembles a substantial genomic dataset. The manuscript is generally well illustrated and contains several potentially useful observations regarding the distribution and organization of chemosensory systems across Vibrionales genomes. However, there are a number of conceptual, methodological, and presentation-related issues that should be addressed before the evolutionary conclusions can be fully supported.

    The claim that F8 represents a novel chemosensory system appears to be incorrect Lines 407-424, as well as several other places throughout the manuscript, describe F8 as a "previously uncharacterized lineage" and a "newly identified" system. However, F8 chemosensory systems were previously described by Wuichet and Zhulin and have been part of the established chemosensory system classification framework for more than a decade. Furthermore, F8 systems are already annotated as such in MiST. For example, the genome GCF_003390675.1 (Vibrio anguillarum), which is included in this study, contains CheA, CheR, and CheB proteins assigned to an F8 system in MiST.

    Consequently, describing F8 as a novel, newly identified, or newly designated system appears inappropriate. This issue requires substantial revision throughout the manuscript. The authors should clearly distinguish between the previously established F8 chemosensory class and any novel observations reported here. If the novelty lies in the distribution of F8 systems within Vibrionales, their genomic organization, their evolutionary history, or some other aspect, this should be stated explicitly.

    __Response: __We thank the reviewer for appreciating our work and further commenting on the points to improve this study. As correctly mentioned by you, F8 CSS has been discovered by Wuichet and Zhulin and already annotated in the MiST database. Since this study focuses on experimentally studied CSS clusters within Vibrio organisms, where F6, F7, and F9 have already been studied very well; however, the F8 is not studied experimentally at least in *Vibrio cholerae *in any literature. We have revised the statement about F8 CSS cluster being novel throughout the manuscript. We agree that MiST database reports different Che proteins, such as CheA, CheR, and CheB, assigned to F8 class, but the information about the entire F8 gene cluster in those Vibrio species has not been provided. Following this, we have identified this entire F8 gene cluster in our study and discussed it as an ‘experimentally unexplored CSS’ in the manuscript.

    The conclusions regarding horizontal gene transfer and vertical inheritance are not sufficiently supported. Lines 429-445 contain evolutionary interpretations that appear internally inconsistent. The manuscript interprets the sporadic distribution of F7 as evidence of vertical inheritance coupled with lineage-specific adaptation, whereas several lines later the similarly patchy distribution of F8 is interpreted as evidence of horizontal gene transfer. Similar distribution patterns should not be used to support contrasting evolutionary scenarios without additional supporting evidence.

    More broadly, patchy phylogenetic distributions alone are generally insufficient evidence for horizontal gene transfer. Alternative explanations, including differential gene loss, genome reduction, incomplete sampling, or rapid sequence divergence, should also be considered. Furthermore, the conclusions regarding vertical inheritance and HGT imply reconstruction of deep evolutionary history across broad bacterial groups. Based on the methods presented, these inferences appear to rely primarily on CheA phylogenies and analyses of homologous sequences. While informative, these analyses may not be sufficient to confidently infer ancestral origins. The authors should either provide additional phylogenetic evidence supporting these conclusions or moderate the language throughout the manuscript. In their current form, the proposed evolutionary scenarios would be more appropriately presented as hypotheses rather than demonstrated conclusions.

    __Response: __We thank the reviewer for this insightful comment. We agree that the evolutionary interpretations presented in the original manuscript need additional supporting evidence. Since our inferences regarding vertical inheritance and horizontal gene transfer were primarily based on phylogenetic distribution patterns and CheA-based phylogenetic analyses. In response, we have carefully revised the relevant sections of the manuscript and moderated the language throughout. The previously presented statements as a conclusion have been reformulated as hypotheses or possible evolutionary scenarios. It must be noted that comparative analysis of protein architecture of F9 CSS, along with phylogeny, revealed a high degree of similarity between Vibrionales F9 protein architecture and their Alphaproteobacterial counterparts, putatively suggesting the HGT event-based acquisition of these CSS within order Vibrionales.

    References are frequently missing, incomplete, or potentially inappropriate. One major concern is the quality and completeness of referencing throughout the manuscript. Multiple statements either lack references altogether or appear to cite sources that do not directly support the associated claims. For example, lines 76-78 cite Ulrich et al. (2005) in support of the statement that two-component systems constitute a dominant signaling paradigm in prokaryotes, whereas the cited article is entitled "One-component systems dominate signal transduction in prokaryotes." The text and/or citation should therefore be reconsidered. Similarly, the statement in lines 100-101 that 17 classes of flagellar chemosensory systems have been designated should be accompanied by an appropriate reference. In addition, numerous ecological, physiological, and evolutionary statements throughout the Introduction and Results sections either lack citations or would benefit from more precise supporting references. I recommend that the authors carefully review all references and ensure that each citation directly supports the corresponding statement.

    __Response: __We thank the reviewer for this comment. We have added the correct reference at the place of Ulrich et al 2005. Along with this we have further checked the references throughout the manuscript and corrected them wherever it is necessary.

    The manuscript would benefit from substantial restructuring and shortening The manuscript is considerably longer than necessary, and several sections appear only loosely connected to the central biological question. In particular, the Introduction contains extensive discussions of Vibrio ecology, virulence, motility, host interactions, and general signal transduction. While these topics are relevant, the overall narrative currently reads more like a broad review article than an introduction to comparative genomics study. I recommend substantially shortening and restructuring the Introduction so that the central biological question and the specific objectives of the study become more apparent to the reader.

    Similarly, the section entitled "Multipartite genome and extensive RNA gene repertoire reflect niche adaptation in Vibrionales" (lines 302-342) contains several observations regarding genome size, tRNA counts, and rRNA copy numbers. However, it remains unclear how these analyses contribute to the primary conclusions regarding chemosensory system evolution. This section should either be shortened substantially and more explicitly connected to the central theme of the manuscript or moved to supplementary material. The manuscript would also benefit from substantial language editing. Numerous grammatical and stylistic issues are present throughout the text, including awkward phrasing, subject-verb agreement errors, and overly long sentences. A thorough language revision would improve readability and help the reader focus on the scientific content.

    __Response: __We thank the reviewer for this comment. We agree that this manuscript is considerably larger and we have shortened several parts in the introduction such as Vibrio ecology, virulence, motility, host interactions and focused more on study objectives. We have also moved the result tilted as “Multipartite genome and extensive RNA gene repertoire reflect niche adaptation in Vibrionales” in the supplementary part. We have carefully revised the entire manuscript to address grammatical errors, improve sentence structure, correct subject–verb agreement issues, and eliminate awkward phrasing. We have also streamlined several lengthy sentences and paragraphs to enhance clarity, readability, and the overall presentation of the scientific content.

    Specific comments:

    Line 20: The phrase "28 Vibrio clades" requires clarification. It is not clear what these clades represent, how they were defined, or whether "clades" is the most appropriate term. Please define these groups more clearly. Perhaps the term "genera" would be more appropriate.

    __Response: __We thank the reviewer for this comment. We agree that the terms clade and genera can be confusing. Jiang et al., 2022 have given this clade classification for Vibrionaceae family members based on phylogenetic analysis of 8 core genes. We have adopted this classification for our study and cited it wherever it is needed. We have explained this in the introduction section.

    Lines 23 and 39: F8 is described as a "novel lineage" and a "previously uncharacterized system." As discussed above, F8 systems have already been described and are annotated in MiST. Please revise these statements.

    __Response: __We thank the reviewer for this comment. We have revised this sentence throughout the manuscript.

    Lines 35-36: The statement could be interpreted as implying that the involvement of chemosensory systems in host colonization is unique to Vibrio. Since chemosensory systems are broadly distributed across bacteria and frequently contribute to host interactions, I suggest rephrasing this sentence to avoid overstatement.

    __Response: __We thank the reviewer for this comment. We rephrased this sentence.

    Lines 41-42: The conclusion that CheA and MCP proteins represent promising drug targets is not directly supported by the analyses presented in this manuscript. The study does not evaluate essentiality, druggability, inhibition, or therapeutic feasibility. I recommend removing this statement.

    __Response: __We thank the reviewer for this comment. We agree that this study does not directly evaluate the essentiality of CheA/MCP proteins as a therapeutic target. Therefore, we removed this part from the manuscript.

    Line 66: The sentence describing responses to environmental cues via "chemotaxis, quorum sensing, and response to nutrients" is awkwardly phrased, since chemotaxis itself often represents a response to nutrients. Please revise for clarity.

    __Response: __We have revised this sentence.

    Lines 69-70: The statement that signal transduction in Vibrio species is "highly precise" and senses chemical gradients "very accurately" require both a reference and a clearer explanation. Relative to what system or organism is this precision being evaluated?

    __Response: __We removed this sentence.

    Lines 76-78: Please reconsider the citation to Ulrich et al. (2005) and revise the associated statement accordingly.

    __Response: __We have corrected this part.

    Lines 100-101: Please provide a reference supporting the statement that 17 classes of flagellar chemosensory systems have been designated.

    __Response: __We have added reference for this sentence.

    Line 106: The manuscript states that our understanding of CSS architecture and function derives primarily from a limited number of model organisms, particularly Escherichia coli. However, later sections highlight the extensive literature on Vibrio cholerae chemotaxis. Since V. cholerae itself is one of the better-characterized organisms in this field, the rationale for emphasizing E. coli alone is unclear.

    __Response: __We agree with your point. We have removed the part of chemosensory system of Escherichia coli and focused on Vibrio cholerae.

    Lines 278-286: This paragraph appears to contain contradictory statements regarding the number of genera included in the analysis. Please clarify how many genera were included, which were excluded, and the criteria used for inclusion.

    __Response: __We have clarified the criteria for inclusion of genera in our study.

    Lines 289-291: Please provide a reference supporting the statement regarding the mutualistic association between Aliivibrio fischeri and squid.

    __Response: __We thank the reviewer for this comment. We have provided the necessary reference for this statement.

    Lines 291 onward: Several ecological and physiological statements in this section require appropriate references.

    __Response: __We have provided the necessary references for these statements. Also, we have shortened some parts of it.

    Lines 302-342: This section would benefit from substantial shortening or a clearer connection to the central theme of chemosensory system evolution.

    __Response: __We have moved this part into the supplementary material, since it is not directly related to the central theme of the manuscript.

    Lines 369-371: This statement is difficult to interpret. MCPs are generally much more variable in abundance than core chemotaxis proteins, and conservation of abundance alone does not demonstrate essentiality. Please clarify and revise this conclusion.

    __Response: __We agree with the reviewer. We have revised this statement in the manuscript. MCP protein shows more abundance than other che proteins.

    Lines 429-445: The contrasting interpretations of F7 and F8 distributions require additional supporting evidence or a more cautious presentation.

    __Response: __We thank the reviewer for this insightful observation. We have rephrased the statement in the manuscript.

    Lines 538-541: This sentence is difficult to follow and would benefit from reformulation. In addition, CheA domain architectures, including those associated with Vibrionales F6, F7, F8, and F9 systems, have recently been described in detail by Berry et al., 2023. The authors should discuss their observations within the context of this work.

    __Response: __We thank the reviewer for this insightful observation. We have reformulated this result part in accordance with the Berry et al., 2023 paper. We have mentioned the possible reasons behind the absence of P2 domain in Che-F6 as well as extra structured insertion between F8 and F9.

    Line 545: The phrase "additional insertion domain" is not well defined. If this region corresponds to a recognized domain, it should be identified explicitly. If it represents an insertion or a poorly structured region, more appropriate terminology should be used.

    __Response: __We thank the reviewer for this comment. We have used the terminology “an insertion” as it is not a recognizable domain.

    Figure 4: It would be helpful to include the identifiers of the proteins used in the structural comparisons.

    __Response: __We thank the reviewer for this comment. We have added the identifiers for the CheA proteins.

    __

    __

    __ __

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    This article has to be completely rewritten because it is now very confused and difficult to read, starting with the title: landscape, architecture, and bipartite organization are difficult terms to employ when describing the "chemosensory system" of bacteria. Systems, in my opinion, does not refer to assemblies, and chemosensing in bacteria refers to anything that involves quorum sensing. Nevertheless, not all bacteria use quorum sensing for flagellar motility, and if these Che-As (and MCPs) are found in "non-chemosensory" systems and are mapped in the abstract without much explanation, this should probably be thoroughly further discussed before debating the organization of the genome and the division of "chemosensory" genes.

    __Response: __We thank the reviewer for these comments regarding terminology and conceptual framing. We agree that bacterial signalling encompasses a wide range of signaling mechanisms beyond those involved in flagellar motility. In our manuscript, use of chemosensory system refers specifically to CheA/CheW/CheY/MCP-based signalling pathways as defined by Wuichet & Zhulin, 2010. We have revised the introduction to clarify the difference between chemosensory systems and other bacterial sensory systems such as quorum sensing. We also agree that not all CheA-containing pathways are necessarily associated with flagellar motility and because of this, we have used chemosensory array and chemostaxis in different contexts. Accordingly, we have moderated several statements throughout the manuscript and mentioned that some chemosensory pathways may regulate other cellular functions. We also agree that here wording “driven by bipartite genome architecture” should be changed to distribution of chemosensory systems across different replicons. Overall, following your suggestions, we have significantly changed the title, introduction, results and discussion.

    There are often few attempts to take into account the "modern" literature to describe the evolution of bacteria and vibrionales and their capacity for quorum sensing, particularly in the introduction and discussion. Such two lengthy, in-depth paragraphs about the biology and diversity of Vibrionales that span more than two pages and only include six references are rather inappropriate for a research article, particularly when the topic is chemosensing and genetics. The description of the Che family, whose abbreviation is never explained, is highly ambiguous and very confusing in comparison to very basic understanding about vibrionales. Despite their significance intracellularly, we typically gain little from reading this section of Ches. Without making a distinction between chemotaxis and quorum sensing, the third paragraph is intended to a lesson about one component systems, two component systems, and chemosensory systems. Which kind of audience do the authors hope to reach? Microbiologists? Examples of these systems that are better characterized can be found throughout the literature.

    Bacterial chemosensory systems are then abruptly introduced. "Chemosensory systems are extremely modular (?) and are typically organized as gene clusters within bacterial genomes". Which sensory genes? Which clusters? What families of bacteria? Which references? When it comes to the classification of chemosensory systems, only Gumerov et al. 2021 is cited. However, what role do other groups play in the study of bacterial Ches and their distribution among the vast diversity of bacteria, such as proteobacteria, actinomycetes, and firmicutes? In order to give a more pertinent Introduction, there are fewer acronyms to employ and undoubtedly more efforts to thoroughly analyze the literature.

    __Response: __We thank the reviewer for this detailed and constructive comment. We agree that the earlier introduction was giving more space to the general biology and diversity of Vibrionales and did not provide sufficient context on the development, diversity, and evolution of bacterial CSS. We have therefore substantially revised and shortened the introductory sections describing Vibrionales biology and diversity, while expanding and restructuring the section on bacterial signal transduction and chemosensory systems. Specifically, we have

    • reduced the number of acronyms and simplified the description of OCSs, TCSs, and CSSs;
    • explicitly distinguished chemosensory signaling from quorum sensing, emphasizing that quorum sensing is primarily a population-dependent signaling process, whereas chemosensory systems detect environmental cues and regulate cellular behaviors such as motility;
    • defined the Che (stands for Chemosensory) proteins and introduced the major components of the chemosensory machinery before discussing their organization; and
    • expanded the discussion of the diversity and evolutionary distribution of bacterial chemosensory systems beyond Vibrionales, incorporating additional literature covering their occurrence and diversification across major bacterial lineages. We have also revised the text describing chemosensory systems as “modular” to clarify that this refers to the combinatorial organization and evolutionary diversification of core signaling proteins, accessory components, and sensory receptors, rather than simply the co-occurrence of che genes within a genomic region. We have further added relevant references to provide a broader and more contemporary overview of bacterial chemosensory system diversity and evolution.

    It learns a little bit more about the chemosensory clusters (whose genes?) in V. cholera, but this information is not really helpful for the study's objective, which at the very end of this incredibly long and badly worded introduction is still ambiguous and essentially unknown. The non-chemosensory/chemotactic motile Vibrionale mutants (line 143) were built by which research team? The authors? Every sentence the authors utilize in the introduction, such as "motility and chemotaxis directly or indirectly contribute to the pathogenicity of bacteria, is somewhat disorganized without a citation (lines 152-155). "The complex (?) interaction between chemotaxis and virulence gene expression in pathogenic Vibrio species" is not better to consider. There are no references included, even while discussing the last three decades of V. cholera research. This is not really appropriate for publication. After a lengthy introduction to the many proteins that mediate chemosensing (quorum sensing) in bacteria, the study is restricted to informatics work, see material and methods, and finally limited on CheAs, which is fairly harmful. For correlation analysis, phylogeny, and structure modeling, the authors use previously available information. Therefore, what distinguishes all of these tables and figures from what is currently understood about bacterial genetics and evolution?

    Response: We thank the reviewer for this important comment. We agree that the original introduction contained excessive discussion of well-characterized chemosensory systems in Vibrio cholerae. We have therefore shortened this section, clarified the study objectives, and added appropriate references to statements concerning motility, chemotaxis, and virulence.

    Regarding the novelty of the study, our objective was not to rediscover individual chemosensory proteins, but to provide a comparative, order-wide analysis of chemosensory system evolution across Vibrionales. Using 116 representative genomes and ~10,000 additional genomes/MAGs, we resolved four distinct CSS lineages (F6-F9), characterized their contrasting genomic distributions and replicon localization, and identified F8 as an experimentally unexplored system. We further provide evolutionary evidence for distinct trajectories of these systems, including probable horizontal acquisition of F9, and reveal a conserved F6 core alongside more dynamic accessory system. We have also revised the introduction to make these objectives, findings, and the significance of the comparative analysis more explicit.

    Reviewer #2 (Significance (Required)):

    The majority of the figures are too little to make any sense. Additionally, there are some unexpected "surprises". For example, the authors' in silico data on Photobacterium toruni, E. coli, and Vibrio qinghaiensis while Introduction led us to anticipate or pick V. cholera as the primary target (see last part of introduction). Bootstrap analysis and a clear display of the clades are necessary for phylogenetic validation. A well-established protein structure (Che-A? Che-B? Other Ches?) is required as an unambiguous reference in order to validate protein structure modelling.

    I would also add that since Vibrionaceae is a family of g-proteobacteria in the order Vibrionales, it is not surprising that they are common traits in the genomes of proteobacteria and vibrionales. However, I'm not sure what the authors mean when they say that patchy and replicon-flexible groups are vertically inherited from g-bacteria. A set of genes (discrete CSS types?) that would be horizontally acquired from alphaproteobacteria are the subject of the same critical point. What is the duration of the convergence of Alphaproteobacteria and Vibrionales? Please refer to Sonnenberg and Haugen (2023) about "bipartite" genome and horizontal transfer.

    __Response: __We thank the reviewer for these suggestions. We have improved the readability of a few figures by increasing font size, clarifying clade annotations, and adding bootstrap support values to the phylogenetic trees. We have also revised the introduction to clarify that the study focuses on chemosensory systems across Vibrionales, rather than V. cholerae alone, and have highlighted Photobacterium toruni and Vibrio qinghaiensis because of their unusual CSS distribution across replicons. In order to have a well-established protein structure from AFDB for structures validation, we have used the pLDDT and pTM score for already available and predicted structures, respectively, and we have added this point in our methodology. Finally, we have revised our interpretation of vertical inheritance and horizontal acquisition, moderating the relevant claims. We have further refined the evolutionary interpretation of F6, F7, and F8 by placing their distribution within the broader Gammaproteobacteria context, while F9 is discussed separately based on its close phylogenetic association with Alphaproteobacteria and the evidence supporting its possible horizontal acquisition.

    __ Reviewer #3 (Evidence, reproducibility and clarity (Required)):__

    This manuscript by Rawool and Sharma is a comprehensive bioinformatics analysis of the chemosensory systems in Vibrionales, their chromosomal locations, likely evolution and acquisition, and differences in CheA architectures. The Abstract and Discussion sections are beautifully written, but the language in the rest of the paper, especially in the Introduction section, is difficult to follow at times (I have many comments below under minor points). An impressive amount of data was acquired and analyzed for this paper, including the identification of F8 systems in Vibrios, and I commend the authors for filling the knowledge gap with such a comprehensive data set. However, the paper overall is extremely long and very detailed, and it took me a very long time to plow through all of it.

    The Introduction section, for example, reads like a literature review from a dissertation, but without figures, and could be streamlined without losing impact. Lines 132-154, include many details on environmental sensing and virulence studies, but it reads like a long list of disconnected sentences with findings from different studies, rather than an integrated summary of current knowledge. The Methods section is also very detailed, and although I am not a bioinformatician, the level of detail often seems excessive. In the Results section, data sentences are often followed by discussion or qualification sentences, which makes the Results section even longer. So, while the science in this paper and its interpretation is sound, the paper needs reworking to make it more palatable for most readers. I think it will be difficult for most readers to remain engaged through the entire manuscript the way it is currently presented.

    Response: We thank the reviewer for their appreciation of our study. Following their suggestions, we have shortened the introduction part as well as the overall manuscript. In the methods part, we have included the detailed parameters used in our analysis, so that result can be reproducible for others. We agree about the lengthy result part, and we have shortened this part as well.

    Minor points:

    General - sometimes clades are written in capitals, sometimes not, and sometimes they are in italics and other times not. Was this intentional? Shouldn't the formatting be consistent throughout?

    __Response: __We thank the reviewer for noting down this inconsistency. There should not be variation in the formatting. We have carefully reviewed the entire manuscript and standardized the formatting of clade names throughout the text, figures, figure legends, and supplementary materials to ensure consistency.

    Line 35 - "moving towards nutrients and away from harm" is a very narrow interpretation of chemosensory systems, referring solely to chemotaxis, which only 1 of the 4 chemosensory systems in Vibrio likely supports. The statement here should be more inclusive.

    __Response: __We thank the reviewer for this important clarification. We agree that the original statement focused primarily on chemotaxis and did not adequately reflect the broader functional diversity of bacterial chemosensory systems. The sentence has been revised to emphasize that chemosensory systems mediate the detection of environmental cues and can regulate a variety of cellular behaviors, including but not limited to motility.

    Line 56 and line 702 - "V. cholerae" not "V. cholera" - a common victim of autocorrect

    __Response: __We thank the reviewer for identifying this typographical error. "V. cholera" has been corrected to "V. cholerae" at the indicated locations and throughout the manuscript.

    Line 64 - "marine sea"? Marine = of the sea. So, effectively "sea sea" = redundancy.

    __Response: __We thank the reviewer for noting this. We have corrected this part in the manuscript.

    Lines 67-68 - the English on these lines doesn't make sense to me. Perhaps "...reaching swimming speeds of 40-200 mm/sec, which requires 1-2 orders of magnitude more energy for propulsion than that required for Escherichia coli".

    __Response: __We thank the reviewer for noting this point. Since this sentence is not directly related to the chemosensory system, we have removed this line from the manuscript.

    Line 69 - Why mention V. alginolyticus here as an example of a Na+-driven flagellar motor when it's relevant for other Vibrios as well? Also, no reference is provided.

    __Response: __We thank the reviewer for noting this point. Since this sentence is not directly related to the chemosensory system, we have removed this line from the manuscript.

    Lines 79-95 - While introducing the proteins found in a chemosensory pathway, chemotaxis itself is given as the "pathway", whereas it should be indicated that it is an example pathway. Not all chemosensory pathways have CheYs that interact with FliM. And not all chemoreceptors in Vibrios have periplasmic sensing domains - some are cytoplasmic.

    __Response: __We agree with the reviewer on this point. We have revised the phrasing in the manuscript.

    Lines 91-93 - awkward sentence where the last clause reads like a non-sequitur.

    __Response: __We thank the reviewer for this observation. We have revised and streamlined the relevant paragraph to improve its clarity, organization, and overall flow.

    Lines 110-113 - The "function" of chemosensory systems are determined by their output, whereas the signals recognized determine their specificity. Please correct.

    __Response: __We thank the reviewer for this clarification. We agree that the signals recognized by chemoreceptors determine the specificity of a chemosensory system, whereas its function is defined by the downstream cellular response it regulates. Accordingly, we have revised the text to distinguish between signal specificity and system function.

    Line 111 - Aer is not an MCP as it is not a "methyl-accepting" receptor in E. coli. Change "MCP proteins" to "chemoreceptors" to be accurate.

    Response: We have changed this part.

    Line 115 = 43 MCPs; Line 149 = 45 chemoreceptors; Line 225 = 46 MCPs - there is variation in the total number of MCPS in Vibrios. Perhaps give a number range where appropriate (line 115, V. cholerae in general), and specific numbers where specific strains are mentioned.

    __Response: __We thank reviewer for noticing this variation in MCP gene numbers. We have revised this statement.

    Line 116 - "forms"

    __Response: __We have changed this part.

    Line 119 - Change "the" to "a" and what is meant by "double-layered membrane structure" since it isn't in the membrane? Please use a more accurate description.

    __Response: __We thank reviewer for this comment. We agreed that it is not a double-layered membrane, rather F9 CSS cluster form a double-layered appearance in the cytoplasm. We have rephrased this in the manuscript.

    Line 121 - Replace the comma with a semi-colon before "overall".

    __Response: __We have changed this part.

    Line 124 - You've already told us that F9 is a cytoplasmic array

    __Response: __We have changed this part.

    Line 226 - Change "during" to "via"

    __Response: __We have changed this part.

    1. Line 133 - "which is involved" and "indicating a link"

    __Response: __We have changed this part.

    Lines 137-139 - If V. cholerae shows a chemotaxis response to these chemicals, then it isn't clear why you would say that they sense the environment "through these CSS clusters" - only 1 cluster (F6) is known to be involved in chemotaxis.

    __Response: __We thank reviewer for this comment. We have corrected this statement.

    Line 140 - "for epithelial colonization"

    __Response: __We have changed this part.

    Line 151 - gene names should be in italics

    __Response: __We have changed this part throughout the manuscript.

    Line 153- "contributes"

    __Response: __We have changed this part.

    Line 156 - "are associated"

    __Response: __We have changed this part.

    Line 162 - "Studies" don't perform anything. It is an inappropriate subject. But kudos for using the word "lacuna" so eloquently!

    __Response: __We have changed this part.

    Line 179 - "from which a pie chart”!

    __Response: __We have changed this part.

    Line 187 - "using the ggsignif"

    __Response: __We have changed this part.

    Line 209 - "A total of 154"

    __Response: __We have changed this part.

    Line 219 - "using parameters the same"

    __Response: __We have changed this part.

    Line 225 - "the 46 MCP proteins were aligned"

    __Response: __We have changed this part.

    Line 240 - "was converted"

    __Response: __We have changed this part.

    Figure 1 - the labels under parts C, D and E are too small

    __Response: __We have increased the font size of the labels and revised the figures.

    Line 346 - "encodes"

    __Response: __We have changed this part.

    Line 349 - "with average values"

    __Response: __We have changed this part.

    Line 352 - defined HK and RR on lines 347-348

    __Response: __We have changed this part.

    Line 365 - replace "chemotaxis-associated" with "chemosensory-associated". Chemotaxis is not inclusive.

    __Response: __We have changed this part.

    Line 380 - shouldn't "cheA" be in italics?

    __Response: __We have changed this part.

    Line 281 - again, "chemosensory proteins" not "chemotaxis proteins"

    __Response: __We have changed this part.

    Line 382 - "in trends with" makes no sense

    __Response: __We have changed this part.

    Line 283 - "an average of 33"

    __Response: __We have changed this part.

    Lines 387-388 - I don't understand the comment in brackets - why was the protein count limited to 40?

    __Response: __We thank the reviewer for this comment. We agree that the rationale for the cutoff value was unclear. Therefore, we have revised the analysis and replaced the previously used cutoff of 40 MCP proteins with the average MCP protein count of 34 calculated from the dataset. The corresponding text has been updated in the revised manuscript.

    Lines 398-405 - It isn't clear which species have lost flagella, and did they also loose the F6 system? Did they retain the other systems? Lines 427-429 doesn't make it any clearer and I am interested to know.

    __Response: __We thank reviewer for this comment. The species from three clades namely Marinum, Rumoiensis, and Halioticoli don’t have any CSS proteins present in it and all these organisms are well-known to be non-motile, suggesting that they don’t have flagellar proteins. These species lost F6 as well as all other F classes.

    Another organism, Vibrio *qinghaiensis *Q67 does not encode F6 system, however it has F7 system.

    Line 423 - "HubP protein functions" makes no sense

    __Response: __We thank the reviewer for this comment. The text has been revised to clarify that HubP is a polar landmark protein that acts as an anchoring factor for the localization of chemotaxis arrays through its interaction with the ParC/ParP complex. We have given proper reference for this.

    Line 470 - why are your reporting 2 F7s and then explaining further on on Line 474 that this is a genome assembly artifact? The information is dislocated and should be streamlined.

    __Response: __We thank reviewer for this comment. As per our analysis, two F7 clusters are present in *Vibrio qinghaiensis *Q67 species. When we checked further, these clusters reside within nearly identical (~99% sequence identity) terminal regions spanning ~20.3 kb at both ends of the replicon. This is artifact during genome assembly process which involves duplicated terminal sequences. We have corrected the order of writing these details in the text.

    Line 499 - "with the smaller"

    __Response: __We have changed this part.

    Lines 536-538 - The F7 system in Vibrios isn't involved in chemotaxis signaling - please correct your language here.

    __Response: __We have revised the statement for clarification and added the figure number.

    Lines 550-551 - "Structural superpositions analyses similarity...." makes no sense. I don't understand what you are trying to say.

    __Response: __We have rephrased this sentence.

    Lines 612 and 630 report the same thing - sensing of bile and mucin. Are both instances necessary?

    Response: We have removed the redundant sentence.

    Figure 6B - "against" not "againts"

    __Response: __We have changed this text.

    Line 634 - "accounting for 69% of sensory inputs"

    __Response: __We have changed this part.

    Line 659 - Hiremath et al., 2015b isn't an appropriate reference for the sensory repertoire of PAS domains. Please reference a PAS domain review instead, e.g., Stuffle and Watts, 2021. PMID: 33647528; PMCID: PMC8169565.

    __Response: __We thank reviewer for this comment. We agreed that the reference cited was not completely based on PAS domain, but the authors have mentioned about PAS domain in their paper. However, we have also cited paper for PAS domain which you mentioned in the comment.

    Line 664 - "ligand" not "legend"?

    __Response: __We have changed this part.

    Line 740 = "chemosensory" instead of "chemotaxis"

    __Response: __We have changed this part.

    Line 744 - "between the extra domain..."

    __Response: __We have changed this part.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript by Rawool and Sharma is a comprehensive bioinformatics analysis of the chemosensory systems in Vibrionales, their chromosomal locations, likely evolution and acquisition, and differences in CheA architectures. The Abstract and Discussion sections are beautifully written, but the language in the rest of the paper, especially in the Introduction section, is difficult to follow at times (I have many comments below under minor points). An impressive amount of data was acquired and analyzed for this paper, including the identification of F8 systems in Vibrios, and I commend the authors for filling the knowledge gap with such a comprehensive data set. However, the paper overall is extremely long and very detailed, and it took me a very long time to plow through all of it.

    The Introduction section, for example, reads like a literature review from a dissertation, but without figures, and could be streamlined without losing impact. Lines 132-154, include many details on environmental sensing and virulence studies, but it reads like a long list of disconnected sentences with findings from different studies, rather than an integrated summary of current knowledge. The Methods section is also very detailed, and although I am not a bioinformatician, the level of detail often seems excessive. In the Results section, data sentences are often followed by discussion or qualification sentences, which makes the Results section even longer. So, while the science in this paper and its interpretation is sound, the paper needs reworking to make it more palatable for most readers. I think it will be difficult for most readers to remain engaged through the entire manuscript the way it is currently presented.

    Minor points:

    1. General - sometimes clades are written in capitals, sometimes not, and sometimes they are in italics and other times not. Was this intentional? Shouldn't the formatting be consistent throughout?
    2. Line 35 - "moving towards nutrients and away from harm" is a very narrow interpretation of chemosensory systems, referring solely to chemotaxis, which only 1 of the 4 chemosensory systems in Vibrio likely supports. The statement here should be more inclusive.
    3. Line 56 and line 702 - "V. cholerae" not "V. cholera" - a common victim of autocorrect
    4. Line 64 - "marine sea"? Marine = of the sea. So, effectively "sea sea" = redundancy.
    5. Lines 67-68 - the English on these lines doesn't make sense to me. Perhaps "...reaching swimming speeds of 40-200 m/sec, which requires 1-2 orders of magnitude more energy for propulsion than that required for Escherichia coli".
    6. Line 69 - Why mention V. alginolyticus here as an example of a Na+-driven flagellar motor when it's relevant for other Vibrios as well? Also, no reference is provided.
    7. Lines 79-95 - While introducing the proteins found in a chemosensory pathway, chemotaxis itself is given as the "pathway", whereas it should be indicated that it is an example pathway. Not all chemosensory pathways have CheYs that interact with FliM. And not all chemoreceptors in Vibrios have periplasmic sensing domains - some are cytoplasmic.
    8. Lines 91-93 - awkward sentence where the last clause reads like a non-sequitur.
    9. Lines 110-113 - The "function" of chemosensory systems are determined by their output, whereas the signals recognized determine their specificity. Please correct.
    10. Line 111 - Aer is not an MCP as it is not a "methyl-accepting" receptor in E. coli. Change "MCP proteins" to "chemoreceptors" to be accurate.
    11. Line 115 = 43 MCPs; Line 149 = 45 chemoreceptors; Line 225 = 46 MCPs - there is variation in the total number of MCPS in Vibrios. Perhaps give a number range where appropriate (line 115, V. cholerae in general), and specific numbers where specific strains are mentioned.
    12. Line 116 - "forms"
    13. Line 119 - Change "the" to "a" and what is meant by "double-layered membrane structure" since it isn't in the membrane? Please use a more accurate description.
    14. Line 121 - Replace the comma with a semi-colon before "overall".
    15. Line 124 - You've already told us that F9 is a cytoplasmic array
    16. Line 226 - Change "during" to "via"
    17. Line 133 - "which is involved" and "indicating a link"
    18. Lines 137-139 - If V. cholerae shows a chemotaxis response to these chemicals, then it isn't clear why you would say that they sense the environment "through these CSS clusters" - only 1 cluster (F6) is known to be involved in chemotaxis.
    19. Line 140 - "for epithelial colonization"
    20. Line 151 - gene names should be in italics
    21. Line 153- "contributes"
    22. Line 156 - "are associated"
    23. Line 162 - "Studies" don't perform anything. It is an inappropriate subject. But kudos for using the word "lacuna" so eloquently!
    24. Line 179 - "from which a pie chart"
    25. Line 187 - "using the ggsignif"
    26. Line 209 - "A total of 154"
    27. Line 219 - "using parameters the same"
    28. Line 225 - "the 46 MCP proteins were aligned"
    29. Line 240 - "was converted"
    30. Figure 1 - the labels under parts C, D and E are too small
    31. Line 346 - "encodes"
    32. Line 349 - "with average values"
    33. Line 352 - defined HK and RR on lines 347-348
    34. Line 365 - replace "chemotaxis-associated" with "chemosensory-associated". Chemotaxis is not inclusive.
    35. Line 380 - shouldn't "cheA" be in italics?
    36. Line 281 - again, "chemosensory proteins" not "chemotaxis proteins"
    37. Line 382 - "in trends with" makes no sense
    38. Line 283 - "an average of 33"
    39. Lines 387-388 - I don't understand the comment in brackets - why was the protein count limited to 40?
    40. Lines 398-405 - It isn't clear which species have lost flagella, and did they also loose the F6 system? Did they retain the other systems? Lines 427-429 doesn't make it any clearer and I am interested to know.
    41. Line 423 - "HubP protein functions" makes no sense
    42. Line 470 - why are your reporting 2 F7s and then explaining further on on Line 474 that this is a genome assembly artifact? The information is dislocated and should be streamlined.
    43. Line 499 - "with the smaller"
    44. Lines 536-538 - The F7 system in Vibrios isn't involved in chemotaxis signaling - please correct your language here.
    45. Lines 550-551 - "Structural superpositions analyses similarity...." makes no sense. I don't understand what you are trying to say.
    46. Lines 612 and 630 report the same thing - sensing of bile and mucin. Are both instances necessary?
    47. Figure 6B - "against" not "againts"
    48. Line 634 - "accounting for 69% of sensory inputs"
    49. Line 659 - Hiremath et al., 2015b isn't an appropriate reference for the sensory repertoire of PAS domains. Please reference a PAS domain review instead, e.g., Stuffle and Watts, 2021. PMID: 33647528; PMCID: PMC8169565.
    50. Line 664 - "ligand" not "legend"?
    51. Line 740 = "chemosensory" instead of "chemotaxis"
    52. Line 744 - "between the extra domain..."

    Referees cross-commenting

    I concur with Reviewer 1, major comment 3, and inadvertently omitted a similar comment from my review. There is absent or inappropriate referencing throughout much of the manuscript that should be addressed.

    Significance

    Strengths and limitations: Provides a comprehensive assessment of the chemosensory landscape of Vibrionales by analyzing 116 genomes and a large-scale RefSeq and MAG set of ~10,000 organisms. The major limitation of this study is in its writing, which is very detailed and long.

    Advance: Yes, this study fills the gap in our understanding of the chemosensory repertoire of Vibrionales.

    Audience: The major audience will be the bacterial chemosensing community.

    Expertise: I work in bacterial chemosensing and have worked on chemoreceptors from several Vibrio species.

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    Referee #2

    Evidence, reproducibility and clarity

    This article has to be completely rewritten because it is now very confused and difficult to read, starting with the title: landscape, architecture, and bipartite organization are difficult terms to employ when describing the "chemosensory system" of bacteria. Systems, in my opinion, does not refer to assemblies, and chemosensing in bacteria refers to anything that involves quorum sensing. Nevertheless, not all bacteria use quorum sensing for flagellar motility, and if these Che-As (and MCPs) are found in "non-chemosensory" systems and are mapped in the abstract without much explanation, this should probably be thoroughly further discussed before debating the organization of the genome and the division of "chemosensory" genes.

    There are often few attempts to take into account the "modern" literature to describe the evolution of bacteria and vibrionales and their capacity for quorum sensing, particularly in the introduction and discussion. Such two lengthy, in-depth paragraphs about the biology and diversity of Vibrionales that span more than two pages and only include six references are rather inappropriate for a research article, particularly when the topic is chemosensing and genetics. The description of the Che family, whose abbreviation is never explained, is highly ambiguous and very confusing in comparison to very basic understanding about vibrionales. Despite their significance intracellularly, we typically gain little from reading this section of Ches. Without making a distinction between chemotaxis and quorum sensing, the third paragraph is intended to a lesson about one component systems, two component systems, and chemosensory systems. Which kind of audience do the authors hope to reach? Microbiologists? Examples of these systems that are better characterized can be found throughout the literature.

    Bacterial chemosensory systems are then abruptly introduced. "Chemosensory systems are extremely modular (?) and are typically organized as gene clusters within bacterial genomes". Which sensory genes? Which clusters? What families of bacteria? Which references? When it comes to the classification of chemosensory systems, only Gumerov et al. 2021 is cited. However, what role do other groups play in the study of bacterial Ches and their distribution among the vast diversity of bacteria, such as proteobacteria, actinomycetes, and firmicutes? In order to give a more pertinent Introduction, there are fewer acronyms to employ and undoubtedly more efforts to thoroughly analyze the literature. It learns a little bit more about the chemosensory clusters (whose genes?) in V. cholera, but this information is not really helpful for the study's objective, which at the very end of this incredibly long and badly worded introduction is still ambiguous and essentially unknown. The non-chemosensory/chemotactic motile Vibrionale mutants (line 143) were built by which research team? The authors? Every sentence the authors utilize in the introduction, such as "motility and chemotaxis directly or indirectly contribute to the pathogenicity of bacteria, is somewhat disorganized without a citation (lines 152-155). "The complex (?) interaction between chemotaxis and virulence gene expression in pathogenic Vibrio species" is not better to consider. There are no references included, even while discussing the last three decades of V. cholera research. This is not really appropriate for publication. After a lengthy introduction to the many proteins that mediate chemosensing (quorum sensing) in bacteria, the study is restricted to informatics work, see material and methods, and finally limited on CheAs, which is fairly harmful. For correlation analysis, phylogeny, and structure modeling, the authors use previously available information. Therefore, what distinguishes all of these tables and figures from what is currently understood about bacterial genetics and evolution?

    Significance

    The majority of the figures are too little to make any sense. Additionally, there are some unexpected "surprises". For example, the authors' in silico data on Photobacterium oruni, E. coli, and Vibrio qinghaiensis while Introduction led us to anticipate or pick V. cholera as the primary target (see last part of introduction). Bootstrap analysis and a clear display of the clades are necessary for phylogenetic validation. A well-established protein structure (Che-A? Che-B? Other Ches?) is required as an unambiguous reference in order to validate protein structure modelling.

    I would also add that since Vibrionaceae is a family of g-proteobacteria in the order Vibrionales, it is not surprising that they are common traits in the genomes of proteobacteria and vibrionales. However, I'm not sure what the authors mean when they say that patchy and replicon-flexible groups are vertically inherited from g-bacteria. A set of genes (discrete CSS types?) that would be horizontally acquired from alphaproteobacteria are the subject of the same critical point. What is the duration of the convergence of Alphaproteobacteria and Vibrionales? Please refer to Sonnenberg and Haugen (2023) about "bipartite" genome and horizontal transfer.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript presents a comparative genomic analysis of chemosensory systems across the order Vibrionales. By combining phylogenetic analyses, genomic context, MCP repertoires, and structural comparisons, the authors investigate the diversity and evolution of chemosensory systems within Vibrionales. The study addresses an interesting question and assembles a substantial genomic dataset. The manuscript is generally well illustrated and contains several potentially useful observations regarding the distribution and organization of chemosensory systems across Vibrionales genomes. However, there are a number of conceptual, methodological, and presentation-related issues that should be addressed before the evolutionary conclusions can be fully supported.

    Major concerns

    1. The claim that F8 represents a novel chemosensory system appears to be incorrect Lines 407-424, as well as several other places throughout the manuscript, describe F8 as a "previously uncharacterized lineage" and a "newly identified" system. However, F8 chemosensory systems were previously described by Wuichet and Zhulin and have been part of the established chemosensory system classification framework for more than a decade. Furthermore, F8 systems are already annotated as such in MiST. For example, the genome GCF_003390675.1 (Vibrio anguillarum), which is included in this study, contains CheA, CheR, and CheB proteins assigned to an F8 system in MiST. Consequently, describing F8 as a novel, newly identified, or newly designated system appears inappropriate. This issue requires substantial revision throughout the manuscript. The authors should clearly distinguish between the previously established F8 chemosensory class and any novel observations reported here. If the novelty lies in the distribution of F8 systems within Vibrionales, their genomic organization, their evolutionary history, or some other aspect, this should be stated explicitly.
    2. The conclusions regarding horizontal gene transfer and vertical inheritance are not sufficiently supported Lines 429-445 contain evolutionary interpretations that appear internally inconsistent. The manuscript interprets the sporadic distribution of F7 as evidence of vertical inheritance coupled with lineage-specific adaptation, whereas several lines later the similarly patchy distribution of F8 is interpreted as evidence of horizontal gene transfer. Similar distribution patterns should not be used to support contrasting evolutionary scenarios without additional supporting evidence. More broadly, patchy phylogenetic distributions alone are generally insufficient evidence for horizontal gene transfer. Alternative explanations, including differential gene loss, genome reduction, incomplete sampling, or rapid sequence divergence, should also be considered. Furthermore, the conclusions regarding vertical inheritance and HGT imply reconstruction of deep evolutionary history across broad bacterial groups. Based on the methods presented, these inferences appear to rely primarily on CheA phylogenies and analyses of homologous sequences. While informative, these analyses may not be sufficient to confidently infer ancestral origins. The authors should either provide additional phylogenetic evidence supporting these conclusions or moderate the language throughout the manuscript. In their current form, the proposed evolutionary scenarios would be more appropriately presented as hypotheses rather than demonstrated conclusions.
    3. References are frequently missing, incomplete, or potentially inappropriate One major concern is the quality and completeness of referencing throughout the manuscript. Multiple statements either lack references altogether or appear to cite sources that do not directly support the associated claims. For example, lines 76-78 cite Ulrich et al. (2005) in support of the statement that two-component systems constitute a dominant signaling paradigm in prokaryotes, whereas the cited article is entitled "One-component systems dominate signal transduction in prokaryotes." The text and/or citation should therefore be reconsidered. Similarly, the statement in lines 100-101 that 17 classes of flagellar chemosensory systems have been designated should be accompanied by an appropriate reference. In addition, numerous ecological, physiological, and evolutionary statements throughout the Introduction and Results sections either lack citations or would benefit from more precise supporting references. I recommend that the authors carefully review all references and ensure that each citation directly supports the corresponding statement.
    4. The manuscript would benefit from substantial restructuring and shortening The manuscript is considerably longer than necessary, and several sections appear only loosely connected to the central biological question. In particular, the Introduction contains extensive discussions of Vibrio ecology, virulence, motility, host interactions, and general signal transduction. While these topics are relevant, the overall narrative currently reads more like a broad review article than an introduction to a comparative genomics study. I recommend substantially shortening and restructuring the Introduction so that the central biological question and the specific objectives of the study become more apparent to the reader. Similarly, the section entitled "Multipartite genome and extensive RNA gene repertoire reflect niche adaptation in Vibrionales" (lines 302-342) contains several observations regarding genome size, tRNA counts, and rRNA copy numbers. However, it remains unclear how these analyses contribute to the primary conclusions regarding chemosensory system evolution. This section should either be shortened substantially and more explicitly connected to the central theme of the manuscript or moved to supplementary material. The manuscript would also benefit from substantial language editing. Numerous grammatical and stylistic issues are present throughout the text, including awkward phrasing, subject-verb agreement errors, and overly long sentences. A thorough language revision would improve readability and help the reader focus on the scientific content.

    Specific comments

    Line 20: The phrase "28 Vibrio clades" requires clarification. It is not clear what these clades represent, how they were defined, or whether "clades" is the most appropriate term. Please define these groups more clearly. Perhaps the term "genera" would be more appropriate.

    Lines 23 and 39: F8 is described as a "novel lineage" and a "previously uncharacterized system." As discussed above, F8 systems have already been described and are annotated in MiST. Please revise these statements.

    Lines 35-36: The statement could be interpreted as implying that the involvement of chemosensory systems in host colonization is unique to Vibrio. Since chemosensory systems are broadly distributed across bacteria and frequently contribute to host interactions, I suggest rephrasing this sentence to avoid overstatement.

    Lines 41-42: The conclusion that CheA and MCP proteins represent promising drug targets is not directly supported by the analyses presented in this manuscript. The study does not evaluate essentiality, druggability, inhibition, or therapeutic feasibility. I recommend removing this statement.

    Line 66: The sentence describing responses to environmental cues via "chemotaxis, quorum sensing, and response to nutrients" is awkwardly phrased, since chemotaxis itself often represents a response to nutrients. Please revise for clarity.

    Lines 69-70: The statement that signal transduction in Vibrio species is "highly precise" and senses chemical gradients "very accurately" requires both a reference and a clearer explanation. Relative to what system or organism is this precision being evaluated?

    Lines 76-78: Please reconsider the citation to Ulrich et al. (2005) and revise the associated statement accordingly.

    Lines 100-101: Please provide a reference supporting the statement that 17 classes of flagellar chemosensory systems have been designated.

    Line 106: The manuscript states that our understanding of CSS architecture and function derives primarily from a limited number of model organisms, particularly Escherichia coli. However, later sections highlight the extensive literature on Vibrio cholerae chemotaxis. Since V. cholerae is itself one of the better-characterized organisms in this field, the rationale for emphasizing E. coli alone is unclear.

    Lines 278-286: This paragraph appears to contain contradictory statements regarding the number of genera included in the analysis. Please clarify how many genera were included, which were excluded, and the criteria used for inclusion.

    Lines 289-291: Please provide a reference supporting the statement regarding the mutualistic association between Aliivibrio fischeri and squid.

    Lines 291 onward: Several ecological and physiological statements in this section require appropriate references.

    Lines 302-342: This section would benefit from substantial shortening or a clearer connection to the central theme of chemosensory system evolution.

    Lines 369-371: This statement is difficult to interpret. MCPs are generally much more variable in abundance than core chemotaxis proteins, and conservation of abundance alone does not demonstrate essentiality. Please clarify and revise this conclusion.

    Lines 429-445: The contrasting interpretations of F7 and F8 distributions require additional supporting evidence or a more cautious presentation.

    Lines 538-541: This sentence is difficult to follow and would benefit from reformulation. In addition, CheA domain architectures, including those associated with Vibrionales F6, F7, F8, and F9 systems, have recently been described in detail by Berry et al., 2023. The authors should discuss their observations within the context of this work.

    Line 545: The phrase "additional insertion domain" is not well defined. If this region corresponds to a recognized domain, it should be identified explicitly. If it represents an insertion or a poorly structured region, more appropriate terminology should be used.

    Figure 4: It would be helpful to include the identifiers of the proteins used in the structural comparisons. Recommendation: Major Revision.

    Significance

    General assessment

    This manuscript presents a comparative genomic analysis of chemosensory systems across the order Vibrionales. The study addresses an interesting question and compiles a substantial genomic dataset. Its main strength lies in the large-scale comparative analysis of chemosensory system distribution, organization, and diversity across Vibrionales genomes, providing a useful resource for future studies of Vibrio signal transduction and evolution.

    The manuscript contains several potentially valuable observations regarding chemosensory-system diversity and generates hypotheses about their evolutionary history. However, some of the central evolutionary conclusions currently appear stronger than the evidence presented. In particular, the interpretation of vertical inheritance and horizontal gene transfer, as well as the treatment of F8 as a novel lineage, require clarification and revision. The manuscript would also benefit from substantial shortening and restructuring to improve focus and readability.

    Advance

    The primary advance of this study is a comprehensive comparative survey of chemosensory systems across Vibrionales. The work expands current knowledge of the distribution, genomic organization, and diversity of these systems and provides a useful synthesis of available genomic data.

    The study is primarily descriptive and comparative in nature. It generates interesting evolutionary hypotheses and provides a valuable dataset for future investigations. However, I do not believe that the manuscript currently establishes the existence of a novel chemosensory class, and several proposed evolutionary scenarios would benefit from additional supporting evidence.

    Audience

    This work will be of greatest interest to researchers studying bacterial signal transduction, chemotaxis, comparative genomics, microbial evolution, and Vibrio biology. It is primarily a basic research contribution and will likely serve as a resource for future studies of chemosensory-system evolution and function.

    Expertise

    My expertise includes bacterial signal transduction, chemosensory systems and chemotaxis, two-component systems, comparative and evolutionary genomics, and microbial genome annotation.

  5. Version published to 10.64898/2026.05.11.724241 on bioRxiv