Exploring the genomic landscape of Chlamydiifrater species: Novel features include multiple truncated CDS of the major outer membrane porin (MOMP), more than 150 unique genes, and an ortholog of the chlamydial plasticity zone

  1. Martin Hölzer
  2. Charlotte Reuschel
  3. Fabien Vorimore
  4. Karine Laroucau
  5. Konrad Sachse

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Abstract

Recently discovered obligate intracellular bacteria belonging to the genus Chlamydiifrater (Cf.) with the species of Cf. phoenicopteri and Cf. volucris were studied to explore the composition of their genomes and their relatedness to Chlamydia, the other genus of the family Chlamydiaceae. We investigated four isolates of Cf. volucris, two of them newly sequenced, and one of Cf. phoenicopteri alongside 12 representatives of the Chlamydia species. Using state-of-the-art bioinformatics techniques, including a hybrid sequencing approach and advanced annotation pipelines, our study reveals previously unrecognized genomic structures within Cf. and provides insights into species-specific adaptations and evolutionary dynamics. The integration of long-read sequencing data, comprehensive re-annotation strategies, and pan-genomics enabled localization of the unique plasticity zone and identification of novel gene clusters in Chlamydiifrater strains, which improves our understanding of chlamydial genome architecture and plasticity in the family Chlamydiaceae. Our analysis revealed that 761 CDS are shared among members of both genera (approx. 80%). We further identified 158 unique genes of Cf. species but their annotation remains challenging because of the absence of functionally annotated orthologs in public databases. A full-length ompA gene encoding the major outer membrane porin (MOMP) was seen in all Cf. strains. One of the most interesting findings is the presence in Cf. of multiple truncated CDS of members of the ompA family, whose localization and structure we describe here. While genome analysis of Chlamydiifrater spp. confirmed numerous common features shared with representatives of the genus Chlamydia, many unique genomic elements were identified that underpin the distinct phenotype and separate genetic position of these new microorganisms.

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  1. Access Microbiology

    The work presented is clear and the arguments well formed. 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.

  2. Access Microbiology

    Comments to Author

    The presented paper entitled "Exploring the genomic landscape of Chlamydiifrater species: Novel features include multiple truncated CDS of the major outer membrane porin (MOMP), more than 150 unique genes, and an ortholog of the chlamydial plasticity zone" is interesting and very extensive, the methodological issues have been described in detail which makes its methodological reproducibility feasible, the data analysis that is proposed makes the comparisons between the species of the family Chlamydiaceae clear and allows elucidating the relationships between the genera Chlamydia and Chalmydiifrater. The results presentation is coherent with the methodology and logically presents the relationships being sought between the genera compared. The figures are adequately explained, and the computer tools have been well used to give context to the relationship to be determined. The paper is well explained, so it is clear that this new genus Chlamydiifrater is closely related to the genus Chlamydia and opens a world of possibilities to explore as the development of strategies to determine pathogenicity factors, adaptation patterns, and their relationship with the disease. Although the literature is still scarce regarding this new genus, the authors discuss the results adequately with the established literature. The article is fascinating since they makes an exhaustive analysis of the total sequences, genes among them ompA and plasticity zone, which has not been done before, which makes it a complete and important work for future studies.

    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

  3. Access Microbiology

    Comments to Author

    Summary: The authors report two new genomes for Chlamydiifrater volucris—a Chlamydiaceae family member—using both short- and long-read technologies. They perform comparative genomics with 2 other Cf. volucris and 1 Cf. phoenicopteri isolates as well as 12 Chlamydia spp. to determine unique genomic features between Chlamydiifrater and Chlamydia. The main differences are that Chlamydiifrater have a slightly larger genome, ~16% more (hypothetical) genes, multiple truncated ompA genes in two genetic islands, and a unique plasticity zone that lacks many canonical PZ genes found in Chlamydia spp. The underlying data is deposited on Open Science Forum. Comments: Line 85: Please add another sentence or two about the isolation to the introduction. Was there something wrong with the flamingos? From where on the flamingos was the Chlamydiifrater isolated? Line 92: Please give examples of what is meant by "typical characteristics". Line 139: Were the additional swabs also collected from flamingos? Please specify the country that Etang du Fangassier is in. Line 145: It is unclear why it is mentioned that the three genomes were re-analyzed. Were differences observed? The next sentence that the original calls were used for consistency suggests that there were differences. Line 185: Although it makes sense to have all genomes start at the same gene, how was hemB chosen as the first gene? This is typically the start for Chlamydia trachomatis serovar L2/434/Bu, but this species is not part of the alignment, so it isn't obvious as to why hemB was chosen. Table 1: C. trachomatis serovar D has a plasmid. Do you mean that there is no sequence for a plasmid available in GenBank? Please clarify for all species/strains in the table. Line 277-279 & Figure 1: Presenting the data as the percentage of hypotheticals/total CDSs may be quicker to understand your point. How expected hypotheticals are in Cf strains based on time of strain discovery is hard to say. Relatedness to genes in other organisms would be more correlative to not being annotated as hypothetical. Line 331: It looks like there are ~20 genes in each PZ in Table 2, but, as written, the text suggests that there are no intervening ORFs. Please clarify if the issue is just that the genes did not Blast to known PZ members. Figure 4: Please increase the font size and make sure that the resolution is high enough. Figures 5 and 6: It is hard to tell what the truncated ompA alleles look like in Figure 5. Figure 6 would fit better in the results section and should be moved from the discussion to immediately follow Figure 5. As is, it is unclear how extreme the truncations are and if they would have an effect when the concept is first introduced in the lead up to Figure 5. Lines 432-434: It seems quite unusual from a biological perspective that no Pmps or Incs could be identified. Missing Pmps and Incs would suggest that Cf uses very different methods from Chlamydia spp. to get nutrients and fend of immune defenses among other reasons. Are there other potential bioinformatic reasons that these would be missed? Line 513: As it sounds like the missing sequence is quite small, could Sanger sequencing be used to close the O09 genome? Is there a probable reason that this area cannot be closed, such as sequence that is highly similar/duplicated elsewhere in the genome?

    Please rate the manuscript for methodological rigour

    Very good

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

    Satisfactory

    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?

    No: I assume that these samples came from flamingos like their previous study. There is no statement of review board approval.

  4. Version published to 10.1099/acmi.0.000936.v1 on Access Microbiology