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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.

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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.

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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).

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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.

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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.

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Correspondence to Simonetta Gribaldo.

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Taib, N., Megrian, D., Witwinowski, J. et al. Genome-wide analysis of the Firmicutes illuminates the diderm/monoderm transition. Nat Ecol Evol 4, 1661–1672 (2020). https://doi.org/10.1038/s41559-020-01299-7

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