P. furiosus proteome response to oxidative stress.

Volcano plots depicting the Rbr (PF1283) expression level change (based on Label-Free Quantification, LFQ) under aerobic versus anaerobic conditions. In panels (a-d), red dots denote statistically significantly up- and down-regulated proteins at 11h, 15h, 18h, and 33h of culture growth time. The horizontal dotted line indicates significance threshold of 0.05 for FDR-corrected p-values determined by Student’s t-test and a permutation test. Vertical lines represent fold change (FC) threshold set to ≥1. (e, f) Change in the expression profiles of rubrerythrin and encapsulin under anaerobic and aerobic conditions. Volcano plots data is listed in Supplemental Table 2.

Oxidative stress-induced tubules (OSITs) formation upon oxidative stress.

(a) Cryo-tomogram slice of P. furiosus cell grown under anaerobic conditions after 33 h cultivation then exposed to oxygen containing environment. Scale bar: 100 nm. (b) 3D isosurface rendering of segmented cell shown in a. cyan: OSIT, pink: VLPs, yellow: cell membrane, green: S-layer.

RubL domain contributes to OSIT formation.

(a) Left: cryo-EM density map of 2×2 Rbr tetramer patch in OSIT, colored by fitted subunit. Right: atomic model of four Rbr tetramers built into cryo-EM map. A RubL domain is marked by a circle. (b) Interface of neighboring tetramers formed by RubL domains of Rbr monomers from adjacent tetramers. Dotted lines indicate hydrogen bonds.(c) Comparison of our atomic model in OSIT with crystal structure of oxidized Rbr (3MPS). Arrow indicates repositioning of Tyr156 side chain.

Proposed model for electron transfer upon oxidative stress.

SOR firstly responds by reducing superoxide into hydrogen peroxide. The resulting hydrogen peroxide oxidizes Rbr, which triggers OSIT formation. OSITs trap VLPs and form VLP-OSIT super-complexes. VLPs can sequester Fe(OH)3 in their lumens to prevent catalysis of the harmful Fenton reaction. In the core of VLPs formed by FL-Enc, the FL domains, which assemble to dodecamers, can oxidize Fe2+ to Fe3+, which gets contained. The resulting electrons may flow across the VLP shell to Rbr’s di-iron FL domain, which has a higher redox potential. These electrons in turn can be accepted by further H2O2 molecules.

Response to oxidative stress in P. furiosus of Ferritin like domain containing proteins.

(a) Growth curves of WT and Δrbr P. furiosus cells in anaerobic environment over 24 h. (b) 8 rubredoxin domain containing proteins detected and quantified by proteomics at 4 time points under anaerobic and aerobic conditions. Of all these proteins uniquely PF1283 is highly more abundant under aerobic conditions. The proteins are annotated according to their encoding genes.

Cryo-tomogram slices of P. furiosus cells in response to oxidative stress.

(a) Cryo-tomogram slice of P. furiosus cell under anaerobic conditions after 33 h cultivation. Along the z-direction the slices covers 1.73 nm. Red arrow: VLP. Scale bar: 100 nm. (b) Cryo-tomogram slice of P. furiosus cell under anaerobic conditions after 33 h cultivation then exposed to oxygen-containing environment. Scale bar: 100 nm. (c) 3D isosurface rendering of segmented cell shown in b. cyan: OSIT, pink: VLPs, purple: ribosomes, yellow: cell membrane, green: S-layer. Scale bar: 100 nm. (d) Analysis of OSIT width and length distribution in all tomograms with OSITs (N=421 OSIT, 120 tomograms).

Identification of OSIT composition by MS and subtomogram averaging.

(a) Intensity based quantification by label free proteomics shows the 25 most abundant proteins (Uniprot IDs) in the enriched OSIT sample, with Rbr being the most prominent (Supplementary Table 2). (b) OSIT subtomogram averaging indicates heterogeneity of the OSITs. The tomograms are aligned using Aretomo, particles were manually picked in IMOD, then converted the coordinates for Warp with a rise of every 80 angstrom. Subtomograms were reconstructed in Warp then applied 3D classification in Relion 3.1.4. The 3D classification shows 10 classes with various nanometers in diameter.

Helical reconstruction workflow for OSIT processing.

(a) Workflow of the helical reconstruction detailed in Materials and Methods. Scale bar: 50nm. (b) Fourier shell correlation (FSC) curves of the 3D reconstruction of 3×3 patch density map.

3D density map of Helical reconstruction of OSITs.

(a) CryoEM density map of OSIT in top view. Left: CryoEM density map of OSIT in top view. Right: cross-section view of OSIT with subunits indicated as found in best-resolved class. (b) CryoEM density map of OSIT in side view. (c) cryoEM density of OSIT with enlarged view of circled area in (b).

Cryo-EM single-particle analysis and validation.

(a) Single-particle analysis workflow to reconstruct 2×2 patch Rbr density. (b) Fourier Shell Correlation (FSC), Guinier plot with fitted B-factor and particle orientation distribution plots for the final Rbr reconstruction. (c) Domain annotation and model/map fit of Rbr.

OSIT dissociation upon 5mM DTT treatment imaged by negative stain EM.

(a) untreated enriched OSIT fraction and (b) enriched OSIT fraction treated with 5mM DTT at low magnification (scale bar: 500 nm). (c) enriched, untreated OSIT. The arrow indicates OSITs. (d) enriched OSIT treated with 5mM DTT at higher magnification (scale bar: 100nm).

Sequence alignment and peptide mapping of Ferritin-like encapsulin shell fusion protein.

Alignment of separate Enc (PF1192), the FL domain (PF1191) and FL-Enc fusion protein shows specific sequence interconnecting FL and Enc domains. Tryptic peptides identified by bottom-up MS (blue bars) yielded 64.9% sequence coverage, covering unique peptide sequences (green bar) responsible for fusion of FL and Enc domains.

Proteome changes in Δrbr cells.

Violins indicate changes of all proteins at different timepoints. Rbr can only be detected in the WT cells, consistent with the knock-out. Enc, which serves as a control, is detected in both cell types and does not change significantly.

Influence of rbr knockout on P. furiosus cells.

(a) Cryo-EM image of the oxidative stressed Δrbr cell shows intense inorganic aggregates inside the cell. Scale bar: 100nm. (b) High Angle Annular Dark Field (HAADF) cryo-EM image of the oxidatively stressed Δrbr cells used for EDX scanning. Two positions with strong signal likely arising from inorganic aggregates were selected for EDX, as well as a control area without strong HAADF signal. Scale bar: 100nm. (c) HAADF cryo-EM images of (b) after EDX scanning, indicating removal of vitrified water and cell organic components by the electron beam. Scale bar: 500nm. (d-f) EDX spectra in the areas marked “background”, “1” and “2” in b.