Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition

Abstract

The transition between cell envelopes with one membrane (Gram-positive or monoderm) and those with two membranes (Gram-negative or diderm) is a fundamental open question in the evolution of Bacteria. Evidence of the presence of two independent diderm lineages, the Halanaerobiales and the Negativicutes, within the classically monoderm Firmicutes has blurred the monoderm/diderm divide and specifically anticipated that other members with an outer membrane (OM) might exist in this phylum. Here, by screening 1,639 genomes of uncultured Firmicutes for signatures of an OM, we highlight a third and deep branching diderm clade, the Limnochordia, strengthening the hypothesis of a diderm ancestor and the occurrence of independent transitions leading to the monoderm phenotype. Phyletic patterns of over 176,000 protein families constituting the Firmicutes pan-proteome identify those that strongly correlate with the diderm phenotype and suggest the existence of new potential players in OM biogenesis. In contrast, we find practically no largely conserved core of monoderms, a fact possibly linked to different ways of adapting to repeated OM losses. Phylogenetic analysis of a concatenation of main OM components totalling nearly 2,000 amino acid positions illustrates the common origin and vertical evolution of most diderm bacterial envelopes. Finally, mapping the presence/absence of OM markers onto the tree of Bacteria shows the overwhelming presence of diderm phyla and the non-monophyly of monoderm ones, pointing to an early origin of two-membraned cells and the derived nature of the Gram-positive envelope following multiple OM losses.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: An updated reference phylogeny of the Firmicutes reveals a third diderm clade.
Fig. 2: Phyletic patterns of protein families highlight the functional core of the diderm Firmicutes OM.
Fig. 3: Distribution and functional annotation of the protein families and PFAM domains highly correlated with the diderm phenotype and the monoderm phenotype.
Fig. 4: A large OM gene cluster is a distinguishing feature of all diderm Firmicutes.
Fig. 5: Phylogenomic analysis does not support the acquisition of the OM by HGT and supports multiple and independent losses of the OM.
Fig. 6: Distribution of monoderm and diderm cell envelopes across Bacteria and two potential evolutionary scenarios for their origin.

Data availability

All results and raw data relative to this analysis (databanks, sequence accession numbers, sequence datasets and corresponding trees, and protein families) are provided as supporting data at Mendeley Data repository14 (http://dx.doi.org/10.17632/3pcn9779gc.1).

References

  1. 1.

    Gupta, R. S. Origin of diderm (Gram-negative) bacteria: antibiotic selection pressure rather than endosymbiosis likely led to the evolution of bacterial cells with two membranes. Antonie van Leeuwenhoek 100, 171–182 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Cavalier-Smith, T. The neomuran origin of archaebacteria, the negibacterial root of the universal tree and bacterial megaclassification. Int. J. Syst. Evol. Microbiol. 52, 7–76 (2002).

    CAS  PubMed  Google Scholar 

  3. 3.

    Tocheva, E. I., Ortega, D. R. & Jensen, G. J. Sporulation, bacterial cell envelopes and the origin of life. Nat. Rev. Microbiol. 14, 535–542 (2016).

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Errington, J. L-form bacteria, cell walls and the origins of life. Open Biol. 3, 120143 (2013).

    PubMed  PubMed Central  Google Scholar 

  5. 5.

    Megrian, D., Taib, N., Witwinowski, J., Beloin, C. & Gribaldo, S. One or two membranes? Diderm Firmicutes challenge the Gram-positive/Gram-negative divide. Mol. Microbiol. 113, 659–671 (2020).

    CAS  PubMed  Google Scholar 

  6. 6.

    Mavromatis, K. et al. Genome analysis of the anaerobic thermohalophilic bacterium Halothermothrix orenii. PLoS ONE 4, e4192 (2009).

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Tocheva, E. I. et al. Peptidoglycan remodeling and conversion of an inner membrane into an outer membrane during sporulation elitza. Cell 146, 799–812 (2012).

    Google Scholar 

  8. 8.

    Campbell, C., Sutcliffe, I. C. & Gupta, R. S. Comparative proteome analysis of Acidaminococcus intestini supports a relationship between outer membrane biogenesis in Negativicutes and Proteobacteria. Arch. Microbiol. 196, 307–310 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Helander, I. M., Hurme, R., Haikara, A. & Moran, A. P. Separation and characterization of two chemically distinct lipopolysaccharides in two Pectinatus species. J. Bacteriol. 174, 3348–3354 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Antunes, L. C. et al. Phylogenomic analysis supports the ancestral presence of LPS-outer membranes in the firmicutes. eLife 5, e14589 (2016).

    PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Kojima, S. et al. Cadaverine covalently linked to peptidoglycan is required for interaction between the peptidoglycan and the periplasm-exposed S-layer-homologous domain of major outer membrane protein Mep45 in Selenomonas ruminantium. J. Bacteriol. 192, 5953–5961 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Poppleton, D. I. et al. Outer membrane proteome of Veillonella parvula: a diderm firmicute of the human microbiome. Front. Microbiol. 8, 1215 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Parks, D. H. et al. Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life. Nat. Microbiol. 2, 1533–1542 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Taib, N. et al. Data from: Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition. v.1 Mendeley Data http://dx.doi.org/10.17632/3pcn9779gc.1 (2020).

  15. 15.

    Yutin, N. & Galperin, M. Y. A genomic update on clostridial phylogeny: Gram-negative spore formers and other misplaced clostridia. Environ. Microbiol. 15, 2631–2641 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Watanabe, M., Kojima, H. & Fukui, M. Limnochorda pilosa gen. nov., sp. nov., a moderately thermophilic, facultatively anaerobic, pleomorphic bacterium and proposal of Limnochordaceae fam. nov., Limnochordales ord. nov. and Limnochordia classis nov. in the phylum Firmicutes. Int. J. Syst. Evol. Microbiol. 65, 2378–2384 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  17. 17.

    Watanabe, M., Kojima, H. & Fukui, M. Complete genome sequence and cell structure of Limnochorda pilosa, a Gram-negative spore-former within the phylum Firmicutes. Int. J. Syst. Evol. Microbiol. 66, 1330–1339 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    Silhavy, T. J., Kahne, D. & Walker, S. The bacterial cell envelope. Cold Spring Harb. Perspect. Biol. 2, a000414 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  19. 19.

    Bos, M. P., Robert, V. & Tommassen, J. Biogenesis of the Gram-negative bacterial outer membrane. Annu. Rev. Microbiol. 61, 191–214 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Sperandeo, P., Martorana, A. M. & Polissi, A. The Lpt ABC transporter for lipopolysaccharide export to the cell surface. Res. Microbiol. https://doi.org/10.1016/j.resmic.2019.07.005 (2019).

  21. 21.

    Heinz, E., Selkrig, J., Belousoff, M. J. & Lithgow, T. Evolution of the translocation and assembly module (TAM). Genome Biol. Evol. 7, 1628–1643 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Noinaj, N., Guillier, M., Barnard, T. J. & Buchanan, S. K. TonB-Dependent transporters: regulation, structure, and function. Annu. Rev. Microbiol. 64, 43–60 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Hughes, G. W. et al. Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system. Nat. Microbiol. https://doi.org/10.1038/s41564-019-0481-y (2019).

  24. 24.

    Malinverni, J. C. & Silhavy, T. J. An ABC transport system that maintains lipid asymmetry in the Gram-negative outer membrane. Proc. Natl Acad. Sci. USA 106, 8009–8014 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. 25.

    Mukherjee, S. & Kearns, D. B. The structure and regulation of flagella in Bacillus subtilis. Annu. Rev. Genet. 48, 319–340 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Jacquier, N., Yadav, A. K., Pillonel, T., Viollier, P. H. & Greub, G. A. SpoIID homolog cleaves glycan strands at the chlamydial division septum. Mol. Biol. Physiol. 10, e01128–19 (2019).

    CAS  Google Scholar 

  27. 27.

    Delsuc, F., Brinkmann, H. & Philippe, H. Phylogenomics and the reconstruction of the tree of life. Nat. Rev. Genet. 6, 361–375 (2005).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    Cavalier-Smith, T. Rooting the tree of life by transition analyses. Biol. Direct 1, 19 (2006).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  29. 29.

    Battistuzzi, F. U. & Hedges, S. B. A major clade of prokaryotes with ancient adaptations to life on land. Mol. Biol. Evol. 26, 335–343 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Lake, J. A. Evidence for an early prokaryotic endosymbiosis. Nature 460, 967–971 (2009).

    CAS  PubMed  Google Scholar 

  31. 31.

    Vollmer, W. Bacterial outer membrane evolution via sporulation? Nat. Chem. Biol. 8, 14–18 (2012).

    CAS  Google Scholar 

  32. 32.

    Tocheva, E. I. et al. Peptidoglycan remodeling and conversion of an inner membrane into an outer membrane during sporulation. Cell 146, 799–812 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Sutcliffe, I. C. A phylum level perspective on bacterial cell envelope architecture. Trends Microbiol. 18, 464–470 (2010).

    CAS  PubMed  Google Scholar 

  34. 34.

    Raymann, K., Brochier-Armanet, C. & Gribaldo, S. The two-domain tree of life is linked to a new root for the Archaea. Proc. Natl Acad. Sci. USA 112, 6670–6675 (2015).

    CAS  PubMed  Google Scholar 

  35. 35.

    Cavalier-Smith, T. & Chao, E. E. Y. Multidomain ribosomal protein trees and the planctobacterial origin of Neomura (eukaryotes, archaebacteria). Protoplasma 621–753 https://doi.org/10.1007/s00709-019-01442-7 (2020).

  36. 36.

    Hug, L. A. et al. A new view of the tree of life. Nat. Microbiol. 1, 16048 (2016).

    CAS  PubMed  Google Scholar 

  37. 37.

    Vincent, A. T. et al. The mycobacterial cell envelope: a relict from the past or the result of recent evolution? Front. Microbiol. 9, 2341 (2018).

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Gaisin, V. A., Kooger, R., Grouzdev, D. S., Gorlenko, V. M. & Pilhofer, M. Cryo-Electron tomography reveals the complex ultrastructural organization of multicellular filamentous chloroflexota (Chloroflexi) bacteria. Front. Microbiol. 11, 1373 (2020).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Sutcliffe, I. C. Cell envelope architecture in the chloroflexi_ a shifting frontline in a phylogenetic turf war. Environ. Microbiol. 13, 279–282 (2011).

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Blobel, G. Intracellular protein topogenesis. Proc. Natl Acad. Sci. USA 77, 1496–1500 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform. 11, 119 (2010).

    Google Scholar 

  42. 42.

    Finn, R. D. et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44, D279–D285 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Johnson, L. S., Eddy, S. R. & Portugaly, E. Hidden Markov model speed heuristic and iterative HMM search procedure. BMC Bioinform. 11, 431 (2010).

    Google Scholar 

  44. 44.

    Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    PubMed  PubMed Central  Google Scholar 

  46. 46.

    Criscuolo, A. & Gribaldo, S. BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments. BMC Evol. Biol. 10, 210 (2010).

    PubMed  PubMed Central  Google Scholar 

  47. 47.

    Nguyen, L. T., Schmidt, H. A., Von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Minh, B. Q., Nguyen, M. A. T. & Von Haeseler, A. Ultrafast approximation for phylogenetic bootstrap. Mol. Biol. Evol. 30, 1188–1195 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Lartillot, N. & Philippe, H. A Bayesian mixture model for across-site heterogeneities in the amino-acid replacement process. Mol. Biol. Evol. 21, 1095–1109 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Letunic, I. & Bork, P. Interactive Tree of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 47, W256–W259 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Huerta-Cepas, J. et al. Fast genome-wide functional annotation through orthology assignment by eggNOG-mapper. Mol. Biol. Evol. 34, 2115–2122 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Huerta-Cepas, J. et al. EGGNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res. 44, D286–D293 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. 53.

    Miele, V., Penel, S. & Duret, L. Ultra-fast sequence clustering from similarity networks with SiLiX. BMC Bioinform. 12, 116 (2011).

    Article  Google Scholar 

  54. 54.

    Miele, V. et al. High-quality sequence clustering guided by network topology and multiple alignment likelihood. Bioinformatics 28, 1078–1085 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  55. 55.

    R Core Team R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2016).

  56. 56.

    Garcia, P. S., Jauffrit, F., Grangeasse, C. & Brochier-Armanet, C. GeneSpy, a user-friendly and flexible genomic context visualizer. Bioinformatics 35, 329–331 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  57. 57.

    Abby, S. S., Néron, B., Ménager, H., Touchon, M. & Rocha, E. P. C. MacSyFinder: a program to mine genomes for molecular systems with an application to CRISPR–Cas systems. PLoS ONE 9, e110726 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  58. 58.

    Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. 59.

    Coleman, G. A. et al. A rooted phylogeny resolves early bacterial evolution. Preprint at BioRxiv https://doi.org/10.1101/2020.07.15.205187 (2020).

Download references

Acknowledgements

S.G., C.B. and J.W. acknowledge funding from the French National Research Agency (ANR), project Fir-OM (grant no. ANR-16-CE12-0010) and from the Institut Pasteur Programmes Transversaux de Recherche (grant no. PTR 39–16). D.M. and D.P. were supported by the Pasteur-Paris University (PPU) International PhD Program. This work used the computational and storage services (TARS cluster) provided by the IT department at Institut Pasteur, Paris.

Author information

Affiliations

Authors

Contributions

S.G. conceived the study. N.T. and D.M. carried out all comparative genomics and phylogenomic analyses. J.W. helped with the annotation of the OM markers. D.P. and G.B. helped with the genome reconstruction of two uncultured Limnochordia genomes in an earlier version of the study. P.A. assembled the DB Bacteria and calculated the reference tree shown in Fig. 6. C.B. helped with functional annotation and overall supervision. N.T., C.B. and S.G. wrote the paper, with contributions from D.M. and J.W. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Simonetta Gribaldo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4.

Reporting Summary

Supplementary Tables

All nine supplementary tables merged in one file. Each sheet corresponds to one supplementary table.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Taib, N., Megrian, D., Witwinowski, J. et al. Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition. Nat Ecol Evol (2020). https://doi.org/10.1038/s41559-020-01299-7

Download citation

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing