SepF is the FtsZ anchor in archaea, with features of an ancestral cell division system

Abstract

Most archaea divide by binary fission using an FtsZ-based system similar to that of bacteria, but they lack many of the divisome components described in model bacterial organisms. Notably, among the multiple factors that tether FtsZ to the membrane during bacterial cell constriction, archaea only possess SepF-like homologs. Here, we combine structural, cellular, and evolutionary analyses to demonstrate that SepF is the FtsZ anchor in the human-associated archaeon Methanobrevibacter smithii. 3D super-resolution microscopy and quantitative analysis of immunolabeled cells show that SepF transiently co-localizes with FtsZ at the septum and possibly primes the future division plane. M. smithii SepF binds to membranes and to FtsZ, inducing filament bundling. High-resolution crystal structures of archaeal SepF alone and in complex with the FtsZ C-terminal domain (FtsZ_CTD) reveal that SepF forms a dimer with a homodimerization interface driving a binding mode that is different from that previously reported in bacteria. Phylogenetic analyses of SepF and FtsZ from bacteria and archaea indicate that the two proteins may date back to the Last Universal Common Ancestor (LUCA), and we speculate that the archaeal mode of SepF/FtsZ interaction might reflect an ancestral feature. Our results provide insights into the mechanisms of archaeal cell division and pave the way for a better understanding of the processes underlying the divide between the two prokaryotic domains.

Fig. 1. SepF co-localizes with FtsZ during the M. smithii cell cycle.

M. smithii cells were permeabilized with PeiW and immunostained with anti-MsSepF and anti-MsFtsZ antibodies. a 3D Structured Illumination Microscopy (SIM) maximum projections of a non-constricting M. smithii cell (left panel) and a constricting cell (right panel) stained with anti-MsSepF (cyan) and anti-MsFtsZ antibodies (magenta). Front views of single channels and the overlay are depicted (above) as well as the side view shifted by 90° (below). White dotted lines represent the cell outlines and scale bars are 0.5 µm. b Phase contrast (Phase) and corresponding epifluorescence images of representative co-labeled M. smithii cells (SepF in cyan, FtsZ in magenta and an overlay of both). Cells are arranged from non-constricting to constricting from 1 to 6. White dotted lines represent the cell outlines deduced from the corresponding phase contrast images and the scale bars are 1 µm. c Mean fluorescence intensity plots of cells grouped into three classes according to the detected FtsZ fluorescent maxima (0–1, 2 or 3 maxima detected) with the corresponding SepF (cyan) and FtsZ (magenta) mean fluorescence intensity [a. u.] of each group plotted against the normalized cell length [0-1]. d Schematic view of SepF (cyan) and FtsZ (magenta) localization pattern during the life cycle of M. smithii. (i) In non-constricting cells, both proteins co-localize at the septation plane. (ii) and (iii) SepF progressively moves to the future division plane prior FtsZ. (iv) As constriction is almost completed, FtsZ and SepF co-localize at the prospective septation plane of the daughter cells. The data shown here are representative for experiments performed in triplicate.

Fig. 2. MsSepF binds to membranes and to FtsZ_CTD inducing filament bundling.

a Domain organization of SepF from M. smithii with an N-terminal amphipathic helix (M), flexible linker (L) and putative C-terminal FtsZ-binding core (C). b Negative stain electron microscope images of SUVs (100 µmol L⁻¹) and MsSepF (50 µmol L⁻¹) with (i) or without (ii) FtsZ_CTD (100 µmol L⁻¹). c Negative stain electron microscope images of FtsZ (30 µmol L⁻¹) and GTP (3 mmol L⁻¹) with (upper panel) or without (lower panel) MsSepF_core (20 µmol L⁻¹). b and c Panels show original image with an enlarged inlet that is marked by black dotted square next to it. Scale bars are 100 nm (original images) and 50 nm (inlets). These experiments were performed two times. d Boxplots showing filament diameter measurements [nm] for FtsZ + GTP (n = 130) and FtsZ + GTP + SepF_core (n = 130). Box is the inter quartile range, where the lower edge is 25^th percentile and the upper edge the 75^th percentile. Whiskers show the range between the lowest value and the highest value. Line inside each box indicates the median and x indicates the mean. Black circles are outliers. A 2-sample t-test was conducted, the result was found to be −23.8032 and the test resulted in a critical t-value of t(alpha) of 1.9692 for an alpha of 0.025. A significant difference of the diameter was found at a 5% level of significance, because the H₀ was rejected, as the modulus of the critical value was > α/2. (*) indicates that means of diameter are significantly different.

Fig. 3. Structural characterization of MsSepF_core.

a Crystal structure of the MsSepF dimer composed of two identical monomers, color-coded according to secondary structure (helices, green; strands, yellow). Each protomer consists of a 5-stranded β-sheet flanked by 2 α-helices and a helical turn (η1). In each protomer, the C-terminal strand β5 (in purple) forms part of the opposing β-sheet. b Comparison of functional SepF dimer interfaces in Bacteria and Archaea, as defined by the complexes with FtsZ. The α interface has only been found in the crystal structures of bacterial SepF dimers such as those of C. glutamicum (PDB 6SCP, shown in the figure) and B. subtilis (PDB 3ZIH), whereas the β interface has been found in all Archaeal structures (e.g. Archaeoglobus fulgidus (PDB: 3ZIE), Pyrococcus furiosus (PDB: 3ZIG), see also Supplementary Fig. 8). The C-terminal secondary structure element of the crystal structures (β5 in M. smithii and α3 in C. glutamicum) are depicted in purple.

Fig. 4. The SepF-FtsZ_CTD complex.

a Surface representation of the crystal structure of FtsZ_CTD (green) bound to MsSepF_core (blue). b Detailed protein-protein interactions within the binding pocket. The side-chains of contact residues (d < 5 Å) are shown in stick representation and hydrogen bonds are represented by gray dotted lines. c Cartoon representations of the different functional dimers of archaeal MsSepF (left panel) and bacterial CgSepF (right panel) bound to FtsZ_CTD (for clarity, only one of the two bound peptides is shown in stick representation). The N-terminal half of FtsZ_CTD (in red) binds to a similar groove of the SepF monomer formed between secondary structure elements α2, β3 and η1 (only in MsSepF), shown in turquoise. Highly conserved positions in archaeal and bacterial FtsZ_CTD are labeled. d Sequence conservation logo of archaeal (left) and bacterial (right) FtsZ_CTD sequences. The arrows indicate the highly conserved residues across Archaea or Bacteria that are labeled in panel c.

Fig. 5. SepF is widely present in Archaea and co-occurs with FtsZ.

a Distribution of FtsZ1, FtsZ2, SepF, FtsA and ESCRT-III (CdvB) homologs on a schematic reference phylogeny of the Archaea. FtsZ1 and FtsZ2 homologs are present in most archaeal lineages (magenta), and frequently in two or more copies each (dark magenta). Semicircles indicate that the corresponding protein could not be identified in all the taxa of the corresponding clade and may be either due to true absences or partial genomes. The presence of SepF (turquoise) correlates to that of FtsZ in the majority of taxa. A FtsA homolog was only identified in Methanopyri (orange). Homologs of ESCRT-III (CdvB and homologs) (purple) are only present in the Asgard superphylum and in most representatives of the TACK superphylum, except for Korarchaeota and Thermoproteales. For full data see Supplementary Data 1. b Distribution of FtsZ, SepF and FtsA homologs on a schematic reference phylogeny of Bacteria. FtsZ (magenta) is present in most bacterial phyla, with the exception of some phyla within the PVC superphylum (Planctomycetes, Omnitrophica and Chlamydiae), which are known to have specific FtsZ-less cytokinesis. Both SepF and FtsA are also absent in Planctomycetes and Chlamydiae. Two copies of FtsZ can be identified in Atribacteria, Synergistetes, Biopolaricaulota, Armatimonadetes and Aquificae (dark magenta). SepF is present in some phyla (Synergistetes, Caldiserica, Coprothermobacterota, Margulisbacteria, Melainabacteria, Cyanobacteria, Actinobacteria, Eremiobacteraeota, Firmicutes, Armatimonadetes, Abditibacteriota and Fusobacteria), most belonging to the Terrabacteria. In contrast, all Gracilicutes except Fusobacteria, and the remaining Terrabacteria phyla have only FtsA, while Cyanobacteria, Melainabacteria and Saganbacteria have only SepF. Finally, some members of Synergistetes, Caldiserica, Coprothermobacterota, Margulisbacteria, Actinobacteria, Eremiobacteraeota, Firmicutes, Armatimonadetes, Abditibacteriota and Fusobacteria have both FtsA and SepF. Semicircles indicate that the corresponding protein could not be identified in all the analyzed taxa in the linage displayed. * indicates uncultured Candidate phyla for which many genomes are incomplete. These phyla were not included in phylogenetic reconstructions. For full data see Supplementary Data 2.