Metagenome-assembled genomes of three Hepatoplasmataceae provide insights into isopod-mollicute symbiosis

  1. Satoshi Kawato
  2. Reiko Nozaki
  3. Hidehiro Kondo
  4. Ikuo Hirono

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Abstract

The digestive organs of terrestrial isopods harbour bacteria of the recently proposed mollicute family Hepatoplasmataceae . The only complete genome available so far for Hepatoplasmataceae is that of ‘ Candidatus Hepatoplasma crinochetorum’. The scarcity of genome sequences has hampered our understanding of the symbiotic relationship between isopods and mollicutes. Here, we present four complete metagenome-assembled genomes (MAGs) of uncultured Hepatoplasmataceae members identified from shotgun sequencing data of isopods. We propose genomospecies names for three MAGs that show substantial sequence divergence from any previously known Hepatoplamsataceae members: ‘ Candidatus Tyloplasma litorale’ identified from the semiterrestrial isopod Tylos granuliferus , ‘ Candidatus Hepatoplasma vulgare’ identified from the common pill bug Armadillidium vulgare , and ‘ Candidatus Hepatoplasma scabrum’ identified from the common rough woodlouse Porcellio scaber . Phylogenomic analysis of 155 mollicutes confirmed that Hepatoplasmataceae is a sister clade of Metamycoplasmataceae in the order Mycoplasmoidales . The 16S ribosomal RNA gene sequences and phylogenomic analysis showed that ‘ Candidatus Tyloplasma litorale’ and other semiterrestrial isopod-associated mollicutes represent the placeholder genus ‘g_Bg2’ in the r214 release of the Genome Taxonomy Database, warranting their assignment to a novel genus. Our analysis also revealed that Hepatoplasmataceae lack major metabolic pathways but has a likely intact type IIA CRISPR-Cas9 machinery. Although the localization of the Hepatoplasmatacae members have not been verified microscopically in this study, these genomic characteristics are compatible with the idea that these mollicutes have an ectosymbiotic lifestyle with high nutritional dependence on their host, as has been demonstrated for other members of the family. We could not find evidence that Hepatoplasmataceae encode polysaccharide-degrading enzymes that aid host digestion. If they are to provide nutritional benefits, it may be through extra-copy nucleases, peptidases, and a patatin-like lipase. Exploration of potential host-symbiont interaction-associated genes revealed large, repetitive open reading frames harbouring beta-sandwich domains, possibly involved with host cell adhesion. Overall, genomic analyses suggest that isopod-mollicute symbiosis is not characterized by carbohydrate degradation, and we speculate on their potential role as defensive symbionts through spatial competition with pathogens to prevent infection.

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  1. Version published to 10.1099/acmi.0.000592.v3 on Access Microbiology
  2. Access Microbiology

    The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community.

  3. Access Microbiology

    Comments to Author

    All comments have been remarkably addressed by the authors. The work is rather complete, with a noteworthy methodological rigour and provides an unique overview into isopod-associated mollicutes.

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  4. Version published to 10.1099/acmi.0.000592.v2 on Access Microbiology
  5. Access Microbiology

    The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments. Please provide more detail in the Methods section and ensure that software is consistently cited and its version and parameters included.

  6. Access Microbiology

    Comments to Author

    Please find the review of the manuscript entitle: "Metagenome-assembled genomes of 1 three Hepatoplasmataceae provide insights into isopod-mollicute symbiosis" by Satoshi Kawato, Reiko Nozaki, Hidehiro Kondo, Ikuo Hirono 1. Methodological rigour, reproducibility and availability of underlying data In a global overview, the bioinformatique approaches are correct and procedures are available online. 2. Presentation of results Over all presentation is very good and figures are weel done and informative 3. How the style and organization of the paper communicates and represents key findings The style is correct and clear 4. Literature analysis or discussion The literature is well described and almost complete 5. Any other relevant comments The comparison of several genomes is really of use to better understand the potential roles of these uncultivated lineages but still of importance as they are retrieved in many isopods lineages would they be terrestrial to aquatic and deep sea, and also in some shrimps, raising the question of a continuum from land to deep sea in symbioses. The lack of many genes implied in metabolisms and the highly reduced genomes makes them dependant on their host still, their transmission is not fully understood. Key may be found in genome data. Finally the CRISPcas system retrieved raise the question of an immunity implication with a defence role for the host then playing a key role in fitness and colonisation success of these isopods. The absence of identified toxin may be due to their novelty and gene data may failed to detect them. Else, it is possible that spatial competition with pathogen would prevent infestation. Using genome data would be nice to design probes for microscopy purposes. Down below please find some comments that may help to improve the manuscript. -line 42 abstract you mention an ectosymbiotic lifestyle for all but do you have any microscopy data to mention this point? Or do you only refer to papers with precise localization? If yes should be mentioned. -introduction may be improved giving some information about the hosts. - line 113 Please indicate whether you extract DNA from the host (ie holobiont). Using illumina you have a circularized genome, which is quite rare so please check. - In several points please check the version you used which differ between (file methods.html) and those in material and methods part : -Line 131 you indicate 2.9 de Flye and in the code you indicate 2.8.3, the same for SPAdes - -line 141 you mention version 3.15.5 and in the codes its version 3.15.3. Then in the code you indicate you have done filtration on illumina sequences but not in material and methods. - line 158 How was done the phylogenenomic tree (indicate not only on the code). - line 174, Table 1 is about statistics on genomes but not on sequences obtained, please check. A table with all the sequencing done would be informative. - line 176-185 you explain very briefly the genomes obtained but would it be possible to have a more accurate estimation using phyloflash for example? - line 193 What is the tool used to determine/realise the variant calling. - line 197 Please check names you indicate « Fukuoka2020 » but in the tree you indicate « Ca. Typlasma littorales ». Line 207 you indicate «Candidatus Hepatoplasma scaber » but figure 1 indicate Ps-JP. Globally, it is a bit difficult to follow the manuscript as you use several names for the genomes. -Line 200 Would you have ANI values for genome comparisons? -line 216 IQ-TREE and not « IQ-TREE2 » -line 225 you use HHpred but it is not mentioned in material and methods. -line 339 you give the hypothesis about a physical barrier for host defence, would you have done some microscopy on the hepatopancreas to illustrate this? Or is this just speculative? -line 360-361 mind the pagination

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  7. Access Microbiology

    Comments to Author

    SYNOPSIS AND GENERAL COMMENTS The manuscript "Metagenome-assembled genomes of three Hepatoplasmataceae provide insights into isopod-mollicute symbiosis" presents a great overview of metagenome-recovered genomes from Mollicutes symbionts living in association with isopod crustaceans. The text is well-written and objective, the authors restrict their discussion based on applied methodological and analytical approaches and the data is freely available for access in accordance with the guidelines for transparency, reproducibility and accessibility of -omics derived data. All Figures and Tables are well-structured and clean, guaranteeing an easy and straightforward comprehension of the study's results. Finally, the work is original and certainly will be used as a reference for future endeavours dealing with isopod-mollicute symbiosis. Nonetheless, there are a number of issues that should be dealt with for the manuscript to be in an acceptable format for publication. Firstly, there is a need to organize the Material and Methods text regarding the genome assembly; it is not clear if the hybrid assembly mode was adeopted for the reconstruction of all MAGs. Also, other bioinformatic tools and biological databases could have been applied for genome annotation and taxonomic assignment of the MAGs. In particular, phylogenetic analyses should have been detailed in depth, both in the Material and Methods text and corresponding Figure legends. In the Results section, the authors defend that the isopod-associated hepatoblastoma should bear a defensive role instead of a nutritional one, in contrast with what has been previously observed in literature for these symbioses. This has some reasoning based on the apparent lack of "digestive" enzymes (which were solely restricted to CAZymes and should be expanded to lipolytic, proteolytic and nucleic-acid degrading-enzymes as well) in the MAGs-coding repertoire. , the MAGs should have been explored in terms of the secondary metabolism and related pathways for this hypothesis to be minimally considered viable. Other symbiotic hallmarks features, such as eukaryotic-like proteins (ELPs) and proteins involved with host interaction, could have been manually detected from the MAGs content. The Discussion is extremely short and deserves more attention, specifically taking into account the novelty and impact of the work in the long-term for the study of this host-microbe association. DETAILED REVIEW PAGE 2 Abstract Line 44 Lower the affirmative tone on ", which suggests that they may act as defensive symbionts". An alternative would be: "leaving the suggestion their potential primary role as defensive symbionts". PAGE 3 Impact Statement Lines 52 and 53 Apply a synonym for "host survival's", it has already been applied in line 49. Line 54 Add "(MAGs)" after the "metagenome-assembled genomes". General comment on the impact statement: the authors should provide some rationale (specific bioinformatic-based analyses for these inferences) for this suggestion of the potential defensive role played by these isopod-associated mollicutes. This would better ascertain that these symbionts exert this role, instead of strictly suggesting it in both abstract and impact statement texts. PAGE 5 Introduction Lines 90-94 It could be added some relevant information about these genome-based works previously performed. Have they explored these genomes with attention to potential symbiotic-related adaptations? This could even place your work on a higher standing compared to these few genomic reports. Line 98 Don't categorically affirm that these are novel species, better just to suggest it based on the genomic investigation pursued in the current work. Add a "potentially" before "novel species". PAGE 6 Line 107 Change the title of this Material and Methods (M&M) subtopic. Perhaps "Isopod origin and genome sequencing"? Line 108 Specify how these Tylos granuliferus were really purchased (exact source, company?) and the number of individuals as well. Lines 109-110 Provide the number of sampled individuals for Armadillidium vulgare and Porcellio scaber. Line 113 What particular phenol-chloroform-isoamyl alcohol extraction protocol was followed (original reference, even with minor modifications)? Lines 114-118 So, both Oxford Nanopore and Illumina sequencing were run for the same genomic DNA samples (hybrid assembly)? The authors associated second and third-generation genome sequencing for recovering of all MAGs? Is that it? If this is the case, it should be better clarified by the end of this M&M subtopic or even highlighted afterwards in the Discussion section. PAGES 7 and 8 Lines 123-150 It should be better addressed which MAGs were solely recovered using just Oxford Nanopore sequencing and which resulted from a hybrid assembly. Also, why a different approach was adopted for each recovered MAG? This would shed more light on the methodological approach used for genome assembly, in general. Lines 151-154 Why DFAST was used for genome annotation? There is a number of other widely-accepted MAG annotation tools that could be applied for this purpose (take a look at the following review to check a number of these tools: https://doi.org/10.1016/j.csbj.2021.11.028). Also, CheckM should be the program used for assessing both completeness and contamination for all recovered MAGs. Lines 155-165 Details on the bootstrap analysis and selection of best-fit evolution models for both phylogenetic trees are missing in the text. Please, specify all of that here. Crucial remark for the M&M section: These MAGs should be taxonomically classified using GTDB-Tk (https://doi.org/10.1093/bioinformatics/btac672) using the updated version of the GTDB database (https://doi.org/10.1093/nar/gkab776). That's of fundamental importance for even proposing they are likely to be new Molicutes genus and species. PAGE 9 Line 173 Correct "Metanenome" for "Metagenome". Also, write "Metagenome-assembled genomes". Line 188 Rewrite the sentence about the number of predicted CDS. A suggestion: "A range of 530 to 597 protein-coding genes were detected in the recovered MAGs". PAGE 10 Line 205 In each case, provide this alleged low nucleotide identity threshold to propose new genera and species for these MAGs. PAGE 11 Lines 215-218 Which was the taxonomic reference the authors considered for reconstructing this wide phylogenomic inference for the Mollicutes group? Again, the GTDB database should be prioritized in running these analyses. PAGE 12 Line 239 "Hepatoplasma" in "Hepatoplasma spp." should be written in italics. No "Candidatus" word is written before, so it isn't following the nomenclature accepted for candidate taxa. PAGE 13 Line 267 "Mycoplasma" should be written in italics. PAGE 15 Lines 315-327 For this inference on the lack of "digestive enzymes", the authors should have pursued the search for lipases/esterases and proteases as well, not being solely restricted to CAZymes. General comment for results: it should have searched biosynthetic gene clusters (BGCs) to get a wider view of the potential defensive role of these mollicute symbionts. antiSMASH 6.0 (https://doi.org/10.1093/nar/gkab335) and NaPdoS (https://doi.org/10.1016/j.jbc.2022.102480) could have been run to obtain these results. Lines 323-325 This paragraph is too short: either add some background information to introduce the Discussion topic or join it to the next one. Page 16 Lines 335-350 The Discussion is too short! The authors could have broadened up the inferences on the genomic survey in a more detailed way and compared them with other examples present in literature for isopod-mollicutes symbiosis. Page 18 Lines 369 and 379 "N" in "N-Acetyl-D-glucosamine, and N-Acetyl-muramic" acid should be written in italics. Figure 2 Change the orientation of the accession number and the strain taxa and identification, such as: "Ureaplasma urealyticum (NR041710.1)". In addition, provide the accession number between parentheses for the MAGs recovered in the study. Figure 4 Details on how the metabolic pathways were inferred should be presented in the Figure legend (for instance, bioinformatic tools considered in this reconstruction and additional manual curation of the data for the representation of these pathways). Moreover, how the figure was plotted (which program) should be detailed in M&M and the Figure legend. Table 3 Gene names should be italicized and written correctly (the protein products are shown instead of gene abbreviations for some of the table rows).

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  8. Version published to 10.1099/acmi.0.000592.v1 on Access Microbiology