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An early origin of iron–sulfur cluster biosynthesis machineries before Earth oxygenation

Abstract

Iron–sulfur (Fe–S) clusters are ubiquitous cofactors essential for life. It is largely thought that the emergence of oxygenic photosynthesis and progressive oxygenation of the atmosphere led to the origin of multiprotein machineries (ISC, NIF and SUF) assisting Fe–S cluster synthesis in the presence of oxidative stress and shortage of bioavailable iron. However, previous analyses have left unclear the origin and evolution of these systems. Here, we combine exhaustive homology searches with genomic context analysis and phylogeny to precisely identify Fe–S cluster biogenesis systems in over 10,000 archaeal and bacterial genomes. We highlight the existence of two additional and clearly distinct ‘minimal’ Fe–S cluster assembly machineries, MIS (minimal iron–sulfur) and SMS (SUF-like minimal system), which we infer in the last universal common ancestor (LUCA) and we experimentally validate SMS as a bona fide Fe–S cluster biogenesis system. These ancestral systems were kept in archaea whereas they went through stepwise complexification in bacteria to incorporate additional functions for higher Fe–S cluster synthesis efficiency leading to SUF, ISC and NIF. Horizontal gene transfers and losses then shaped the current distribution of these systems, driving ecological adaptations such as the emergence of aerobic lifestyles in archaea. Our results show that dedicated machineries were in place early in evolution to assist Fe–S cluster biogenesis and that their origin is not directly linked to Earth oxygenation.

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Fig. 1: Processes and components of Fe-S cluster biogenesis pathways.
Fig. 2: SMS is a bona fide Fe–S cluster biogenesis system.
Fig. 3: Taxonomic distribution of the five different Fe–S cluster biogenesis systems.
Fig. 4: Phylogenetic tree of SUF/SMS and MIS/NIF systems.
Fig. 5: Inferred scenario for the origin and evolution of Fe–S synthesis machineries.

Data availability

Supplementary data are available at https://figshare.com/articles/dataset/Garcia_et_al_2022_Supplementary_data/20209877. Source data are provided with this paper.

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Acknowledgements

We thank members of the SAMe and of the EBMC units for discussion. This project was supported by Institut Pasteur and CNRS and by grants from the ANR-10-LABX-62-IBEID, the LabEx ARCANE (ANR-11-LABX-0003–01) and the CBH- EUR-GS (ANR-17-EURE-0003).

Author information

Authors and Affiliations

Authors

Contributions

P.S.G., F.D., E.B., S.O.d.C., S.G. and F.B. designed the experiment. P.S.G., F.D., S.O.d.C. and M.D. performed the experiments. P.S.G., F.D., S.O.d.C., E.B., S.G. and F.B. analysed the data. P.S.G., S.G. and F.B. wrote the manuscript. P.S.G., S.G. and F.B. revised the manuscript.

Corresponding authors

Correspondence to Simonetta Gribaldo or Frédéric Barras.

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Nature Ecology & Evolution thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Biochemical analysis of SMS system.

A. Production of SMS hybrid fusion proteins used for Bacterial 2-hybrid. E. coli strains transformed with the indicated plasmids were grown in LB at 37 °C and expression was induced with 0.5 mM IPTG for two hours. Total cell extracts were separated on a 10% SDS–PAGE and proteins were detected by Western blot with an anti-T18 monoclonal antibody (sc-13582, Santa Cruz) or an anti-T25 polyclonal antibody (gift from D. Ladant). B. Quantification of the two-hybrid assay. BTH101 strain transformed by the indicated two-hybrid plasmids together with a placZ-GFP compatible plasmid were incubated overnight at 30 °C in LB supplemented with 0.5 mM IPTG. The activities correspond to the ratio between GFP fluorescence and the OD600 of 4 replicates and are given in arbitrary units (A.U.). The mean is represented and the error bars stand for standard deviations. C. UV-visible spectroscopy analysis of reconstituted Methanococcus jannaschii SmsCB (2.3 mg/ml) in 100 mM Tris-HCL, 150 mM NaCl pH 8. Iron and sulfur content: 3.7 ± 0.1 Fe and 3.5 ± 0.15 S/ SufC2B2.

Extended Data Fig. 2 Taxonomic distribution of all components of the five different systems on the phylogeny (cladogram) of Bacteria.

Bootstrap values > 80% are indicated by dots on branches. The size of dots corresponds to the ratio of presence of each system in each clade/phylum. The colour of dots corresponds to the biochemical role of each component as in Fig. 1.

Extended Data Fig. 3 Taxonomic distribution of all components of the five different systems on the phylogeny (cladogram) of Archaea.

Bootstrap values > 80% are indicated by dots on branches. The size of dots corresponds to the ratio of presence of each system in each clade/phylum. The colour of dots corresponds to the biochemical role of each component as in Fig. 1.

Extended Data Fig. 4 Taxonomic distribution of components of ISC and SUF on the phylogeny (cladogram) of Gammaproteobacteria.

Bootstrap values > 80% are indicated by dots on branches. Black squares correspond to the presence of at least one sequence.

Extended Data Fig. 5 Full phylogenetic tree of SUF/SMS (phylogram), corresponding to Fig. 4a.

Colours of branches correspond to the colour scheme used in Fig. 3 (Bacteria: Terrabacteria: marine blue, Gracilicutes: light blue, CPR: dark blue, Fusobacteria: grey blue; Archaea: Cluster I: orange and II: red). Bootstrap values > 80% are indicated by dots on branches. The scale bar indicates the average of number substitutions per site. The genomic context of genes coding for SUF and SMS components is indicated in front of the tree, centred around SufB and SmsB (brown and light brown, respectively). SufA: pink, SufC: yellow, SufD: dark green, SufS: light blue, SufE: turquoise SufT: dark blue, SufU: violet, SmsC: light green).

Extended Data Fig. 6 Phylogeny of SufS.

Bootstrap values > 80% are indicated by dots on branches. The scale bar indicates the average number of substitutions per site. Colour of leaves corresponds to major groups in Bacteria (Terrabacteria: marine blue, Gracilicutes: light blue, CPR: dark blue, Fusobacteria: grey blue) and in Archaea (Cluster I: orange and II: red). The group of gammaproteobacterial CsdA is indicated by a grey rectangle.

Extended Data Fig. 7 Phylogeny of SufU.

Bootstrap values > 80% are indicated by dots on branches. The scale bar indicates the average number of substitutions per site. Colour of leaves corresponds to major groups in Bacteria (Terrabacteria: marine blue, Gracilicutes: light blue, CPR: dark blue, Fusobacteria: grey blue) and in Archaea (Cluster I: orange and II: red).

Extended Data Fig. 8 Phylogeny of SufE.

Bootstrap values > 80% are indicated by dots on branches. The scale bar indicates the average number of substitutions per site. Colour of leaves corresponds to major groups in Bacteria (Terrabacteria: marine blue, Gracilicutes: light blue, CPR: dark blue, Fusobacteria: grey blue) and in Archaea (Cluster I: orange and II: red). The group of gammaproteobacterial CsdE is indicated by a grey rectangle.

Extended Data Fig. 9 Phylogenetic trees of MIS/NIF.

Left panel: phylogeny of MisS/NifS. The genomic context of genes of MIS/NIF is indicated in front of the tree, using MisS/NifS as markers (MisS: light red, MisU: blue, NifS: dark green, NifU: dark blue). Right panel: phylogeny of a concatenation of NifU/NifS and the closest group of MisU/MisS. The genomic context of genes of MIS/NIF is indicated in front of the tree, using MisS/NifS as markers (MisS: dark brown, MisU: light brown, NifS: light blue, NifU: dark blue, IscAnif: violet, other Nif genes (ClpX, CysE1, NifB, NifD, NIfE, NifF, NifH, NifK, NifM, NifN, NifQ, NifV, NifW, NifX, NifZ): grey). Bootstrap values > 80% are indicated by dots on branches. The scale bar indicates the average of number of substitutions per site. Colour of leaves corresponds to phyla or classes for Proteobacteria.

Extended Data Fig. 10 Full phylogenetic tree of the ISC system based on the concatenation of IscS, IscU, IscX, HscA, HscB, CyaY, and Fdx.

Bootstrap values > 80% are indicated by dots on branches. The scale bar indicates the average number of substitutions per site. Colour of leaves corresponds to phyla or classes for Proteobacteria. The genomic context of genes of ISC is indicated in front of the tree, using IscU as a marker (IscS: cyan, IscU: blue, IscA: dark green, HscB: light green, HscA: yellow, Fdx: brown, CyaY: violet).

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and discussion.

Reporting Summary

Peer Review File

Supplementary Tables

Supplementary Table 1: Taxonomic distribution of the different components of Fe–S synthesis machineries identified in the 10,865 prokaryote genomes. A total of 4,027 proteomes were not annotated by the NCBI pipeline and have been annotated using Prodigal. The identifiers in format X.n_m correspond to identifiers generated by Prodigal. Supplementary Table 2: List of prokaryote genomes subsampled for phylogenetic analyses. Supplementary Table 3: List of the analysed archaeal and bacterial genomes. Supplementary Table 4: Absence/presence of SUF, MIS and SMS components and six conserved Fe–S proteins in lactobacillales genomes with a ‘Chromosome’ or ‘Complete genome’ status. The number of copies for each protein is indicated (grey cases correspond to at least one copy). Genomes with no Fe–S biogenesis system and no conserved Fe–S protein are framed. Supplementary Table 5: Models predicted by IQ-TREE for the different datasets. Supplementary Table 6: E. coli strains, plasmids and oligonucleotides used in this study. Supplementary Table 7: List of SmsB and SmsC used for functional analysis.

Source data

Source Data

Unprocessed gels for Fig. 2b.

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Garcia, P.S., D’Angelo, F., Ollagnier de Choudens, S. et al. An early origin of iron–sulfur cluster biosynthesis machineries before Earth oxygenation. Nat Ecol Evol 6, 1564–1572 (2022). https://doi.org/10.1038/s41559-022-01857-1

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