# Combining live fluorescence imaging with in situ cryoelectron tomography sheds light on the septation process in Deinococcus radiodurans

Lorenzo Gaifas, Jean-Philippe Kleman, Françoise Lacroix, Erin Schexnaydre, Jennyfer Trouve, Cécile Morlot, Linda Sandblad, Irina Gutsche, Joanna Timmins

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## To cite this version:

Lorenzo Gaifas, Jean-Philippe Kleman, Françoise Lacroix, Erin Schexnaydre, Jennyfer Trouve, et al. Combining live fluorescence imaging with in situ cryoelectron tomography sheds light on the septation process in Deinococcus radiodurans. Proceedings of the National Academy of Sciences of the United States of America, 2025, 122 (19), pp.e2425047122. 10.1073/pnas.2425047122. hal-05193537

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# Combining live fluorescence imaging with in situ cryoelectron tomography sheds light on the septation process in *Deinococcus radiodurans*

Lorenzo Gaifas<sup>a,1</sup> <sup>ID</sup>, Jean-Philippe Kleman<sup>a,1</sup> <sup>ID</sup>, Françoise Lacroix<sup>a,1</sup>, Erin Schexnaydre<sup>b,c</sup>, Jennyfer Trouve<sup>a</sup>, Cecile Morlot<sup>a</sup> <sup>ID</sup>, Linda Sandblad<sup>b,c</sup> <sup>ID</sup>, Irina Gutsche<sup>a,b,2</sup> <sup>ID</sup>, and Joanna Timmins<sup>a,2</sup> <sup>ID</sup>

Affiliations are included on p. 10.

Edited by Joe Lutkenhaus, University of Kansas Medical Center, Kansas City, KS; received November 29, 2024; accepted March 24, 2025

Cell division is a fundamental biological process that allows a single mother cell to produce two daughter cells. In walled bacteria, different modes of cell division have been reported that are notably associated with distinctive cell shapes. In all cases, division involves a step of septation, corresponding to the growth of a new dividing cell wall, followed by splitting of the two daughter cells. The radiation-resistant *Deinococcus radiodurans* is a spherical bacterium protected by a thick and unusual cell envelope. It has been reported to divide using a distinctive mode of septation in which two septa originating from opposite sides of the cell progress with a flat leading edge until meeting and fusing at mid-cell. In the present study, we have combined conventional and superresolution fluorescence microscopy of live bacteria with in situ cryogenic electron tomography of bacterial lamellae to investigate the septation process in *D. radiodurans*. This work provides important insight into i) the complex architecture and multilayered composition of the cell envelope of this bacterium, ii) the unusual “sliding doors” septation process and iii) the sequence of events and molecular mechanisms underlying septal closure, including the synthesis of a FtsZ-dependent peptidoglycan layer that rigidifies and straightens the growing septa.

bacterial cell envelope | cell division | cryo-FIB milling | cryo-ET | fluorescence microscopy

Bacterial cell division occurs through binary fission (1) and involves two major steps: i) septation, corresponding to the inward growth of a new dividing cell wall across the cell, known as septum or cross-wall, and ii) separation or splitting of the two daughter cells notably through the action of cell wall hydrolases. These two steps can be either concomitant or dissociated in time (2–5). When splitting is sufficiently delayed compared to septation, the cross-wall advances centripetally like a closing iris until full closure, resulting in a clearly visible septum at the division site. In contrast, when septal synthesis and cleavage proceed at a similar rate, the division site undergoes an apparent “constriction”, with a reduction of its diameter without visible septum formation.

Bacterial cell division is tightly coupled to the synthesis and remodeling of the cell envelope, whose composition defines the classification between gram-positive (or monoderm) and gram-negative (or diderm) bacteria. In all bacteria, the cell envelope includes a common and essential constituent called the peptidoglycan (PG) that forms a cross-linked mesh-like structure, which confers cell shape and protects the cell from turgor pressure (6). Gram-positive bacteria that are missing a second outer membrane bilayer usually possess a thick and multilayered PG, while gram-negative bacteria have a thin predominantly single-layered PG (6). Until recently, most gram-negative bacteria were reported to divide through constriction. However, cryoelectron tomography (cryo-ET) studies have revealed that gonococci and *Escherichia coli* actually use a combination of constriction and septation to divide, a mode of division that is likely not restricted to these two species (5, 7). Similarly, in the gram-positive bacterium *Streptococcus pneumoniae*, both modes of cell division have been reported to be at play (8). In contrast, other gram-positive bacteria, including *Staphylococcus aureus* or *Bacillus subtilis*, divide through complete septation, with a septum composed of two adjacent cross-walls separated by a low-density region (9–14).

These distinct modes of cell division rely on both common and species-specific molecular mechanisms and division factors. In most bacteria, cell division begins with the assembly at the division site of the highly conserved FtsZ protein into an annular structure known as the Z-ring on the cytoplasmic side of the inner membrane. This Z-ring then acts as a scaffold for the recruitment of several membrane-associated division and cell wall

## Significance

In bacteria, cell division is intimately linked to the architecture of the outer cell envelope. In *Deinococcus radiodurans*, a bacterium well-known for its remarkable radiation-resistance, the cell envelope is complex and composed of multiple layers. Using a combination of advanced fluorescence microscopy and cryogenic electron tomography, this study reveals the composition and structural organization of the various layers composing *D. radiodurans'* cell envelope and uncovers the mechanisms that drive its unique mode of division, involving the inward growth of two septa from opposite sides of the cell. These findings enhance our understanding of a fundamental process required for bacterial proliferation and raise questions about the evolutionary advantages of this unusual division mode and its role in the organism's remarkable resilience.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Copyright © 2025 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

¹L.G., J.-P.K., and F.L. contributed equally to this work.

²To whom correspondence may be addressed. Email: irina.gutsche@ibs.fr or Joanna.timmins@ibs.fr.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2425047122/-/DCSupplemental.

Published May 6, 2025.
synthesis factors (including the penicillin-binding proteins or PBP5s) that together form the divisome (15–17). After the divisome has assembled, the Z-ring constricts and PG is synthesized at the leading edge of the invaginating membrane, dividing the mother cell into two daughter cells. In addition to the divisome complex, a second distinct PG synthesis machinery, known as the elongasome, is responsible for peripheral (or lateral) cell wall synthesis respectively (3, 18).

The cell envelope composition of the spherical bacterium, *Deinococcus radiodurans*, known for its outstanding resistance to DNA-damaging agents (19–21), remains a matter of debate (22–30). It is known to possess an unusual cell wall that stains gram-positive (22, 23, 29, 30), but yet is composed of both an inner and an outer plasma membrane interspersed by a thick multilayered region (12, 23, 31). The septation process in *D. radiodurans* is also unlike that of other cocci. Based on electron micrographs of freeze-cleaved *D. radiodurans* cells, Murray *et al*. reported that division is not initiated from the whole circumference of the cell, but instead from two discrete regions on opposite sides of the cell. Two septa with a flat leading edge then progress across the cell until they fuse at mid-cell to close the slit (13). This observation was more recently supported by 3D confocal microscopy imaging of labeled *D. radiodurans* cell membranes (32).

Exposure of *D. radiodurans* to high doses of radiation causes significant damage to the genome and immediate cell cycle arrest (33), suggesting that cell growth and division in this organism are tightly regulated and coordinated with genome maintenance. *D. radiodurans* cells divide in two alternative perpendicular planes (13, 34) and their cell cycle can be classified into 6 phases (Fig. 1A). Starting from an elliptical and largely symmetric pair of cells (or diad) in Phase 1, the cells grow and septal closure progresses until tetrads are formed in Phase 6, which, in exponential phase, are very short-lived and rapidly split into two diads to initiate a new cell cycle (32). As a result, in this organism, the two major steps of cell division, i.e., i) septation and ii) splitting of the daughter cells, actually occur in separate cell cycles, with septal closure taking place in cycle n and splitting of the cells in cycle n + 1 (Fig. 1A). This temporal separation makes *D. radiodurans* particularly well suited as a model to study septation.

In the present study, we have combined conventional and single-molecule localization microscopy (SMLM) of live cells with in situ cryo-ET of bacterial lamellae obtained by cryofocused ion beam (FIB) milling, to image dividing *D. radiodurans* cells. This work unveils the different layers of the cell envelope at various stages of the division process and unambiguously demonstrates that septation proceeds by a “sliding doors” mechanism. We further show that PG assembly in *D. radiodurans* occurs in both septal regions and throughout the whole periphery of the diad, but involves two distinct machineries. To our surprise, remarkable membranous protrusions were frequently observed in our tomograms at the tips of the septa, which may constitute a preformed dual membrane layer that is subsequently filled, thickened, and rigidified by PG synthesis. Finally, this rigidification step appears to be associated with the assembly of FtsZ filaments at the tips of the growing septa.

## Results

**Composition, structure, and maturation of *D. radiodurans* cell envelope.** To decipher the structure and composition of the unusual cell envelope of *D. radiodurans*, we have combined SMLM on live cells with cryo-ET on cryo-FIB-milled cell lamellae (Fig. 1B and C). Three distinct cell envelope regions can be observed in our cryo-ET data (Fig. 1B). The peripheral, fully matured,

cell envelope is the thickest. The new growing septum is on the contrary the thinnest, and the old septum (synthesized during the previous cell cycle) located between the two cells composing a *D. radiodurans* diad, corresponds to an intermediate stage of cell wall maturation (Fig. 1B and C).

The peripheral cell envelope bears two lipid bilayers that can be seen as dark lines in the cryo-ET data (Fig. 1B) and could be distinguished on a few occasions in the superresolved PAINT images of Nile Red stained *D. radiodurans* (Fig. 1C, Right panel). The distance between the inner (IM) and outer (OM) cell membranes was in good agreement between the two techniques and was found to be ~100 nm (Fig. 1B and C). A more in-depth analysis of the cell envelope composition was facilitated by the calculation of straightened cell wall projections of the cryo-ET images using a recently developed tool, blik (35). Density profiles of the peripheral envelope (SI Appendix, Fig. S1) revealed that this region is composed of 5 distinct layers: i) the IM, ii) a low-density periplasmic space, iii) a high-density PG layer, iv) an intermediate layer, known as the SlpA layer (31), and v) the OM (Figs. 1B and 2A). In addition, in several tomograms, an additional S-layer was seen as the outermost layer of this complex cell envelope (Figs. 1B, 2A and SI Appendix, Figs. S2 and S3). A distinctive white line was observed separating the PG layer from the SlpA layer, and V-shaped invaginations of the OM were seen regularly along the peripheral cell envelope and throughout the bacteria's cell surface.

Measurements made on numerous tomograms (SI Appendix, Figs. S2 and S3) allowed us to precisely define the architecture of this cell envelope region. When present, the S-layer was typically located at 17.9 ± 0.8 nm (n = 5) above the OM. The SlpA layer was found to be 34.9 ± 1.5 nm (n = 16) in thickness in good agreement with the estimated dimensions of its major constituent, SlpA, a trimeric porin-like protein that is embedded in the OM and connects to the PG layer via a long coiled-coil domain (31). The PG layer was found to be 43.9 ± 4.8 nm (n = 16) in thickness and located approximately 14.8 ± 3.2 nm (n = 16) above the IM, with a low-density region nestled in between these two essential layers. Interestingly, these measurements revealed that the IM bilayer was significantly thicker than the OM (5.8 ± 0.5 vs 5.0 ± 0.5 nm; n = 16), suggesting distinct lipid compositions (Fig. 2B).

A similar procedure was used to analyze the composition of the growing and old septa (Fig. 1B and 2A). The outermost layers of the peripheral cell envelope (S-layer, OM, and SlpA layer) were missing in these regions, and instead septa were composed of a single continuous PG layer sandwiched between two low-density periplasmic layers and bordered on either side by the IM (Fig. 2A). These three layers were continuous with those of the peripheral cell envelope. While the IM bilayer and low-density periplasmic space showed similar thicknesses in these two locations, the PG layer displayed significant variations, ranging from 26 to 51 nm in the old septa and as low as 12 nm in the growing septa. In both septal regions, the PG layer was typically thicker at the cell periphery than at the leading edge of the septum (Fig. 2C) or the center of the diad. Moreover, in the old septa, the mean thickness of the PG layer was also found to vary substantially as a function of the phase of the cell cycle (Fig. 2D), suggesting a progressive thickening of this layer until the splitting of the daughter cells.

At the junction between the peripheral cell envelope and the root of the growing septum, we observed that the PG and low-density periplasmic layers followed the IM, while the outermost layers (SlpA layer and OM) were restricted to the peripheral cell envelope and were often seen to form V-shaped invaginations (Fig. 2E and SI Appendix, Fig. S4A for more examples). 3D visualization of the tomograms revealed that these invaginated outer layers extended inward toward the center of the diad during the splitting of the two
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<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Structures of exponentially growing *D. radiodurans* cell walls during cell division. (A) Schematic diagram of exponentially growing *D. radiodurans*. Transition from a diad (two-cell unit) to a tetrad (four-cell unit) involves 6 distinct phases (P1-P6). The septation process occurs during phases P3 to P6 of cycle *n* (Left, red), while the splitting of this newly synthesized cell wall only occurs in the following cell cycle, cycle *n* + 1 (Right, red). Early-, mid-, and late-phases of septation are annotated in blue. (B) Central slice of a typical cryoelectron tomogram of an actively dividing wild-type (WT) *D. radiodurans* diad. Three different types of cell envelope regions can be seen: i) the multilayered peripheral region composed notably of the inner (IM, black line) and outer (OM, red line) membranes and a discontinuous S-layer (purple line), ii) the “old” septum dividing the two cells, and iii) the “new” growing septa originating from opposite sides of the cell. The mean distance between the two membranes in the peripheral region was determined to be 95 nm. The light gray density in the center of the cell corresponds to the nucleoid, the darker spherical form is a stress granule and the small dark densities distributed throughout the cytoplasm (and excluded from the nucleoid) are ribosomes. (C) Two examples of super-resolved PAINT images of Nile Red stained WT *D. radiodurans* diads. As in (A), three distinct regions of the cell envelope can be distinguished. On a few occasions, the two lipid bilayers of the peripheral cell envelope could be resolved (as in the Right image). The mean distance between these two layers was determined to be 100 nm.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** In-depth analysis of the architecture and composition of the cell envelope of *D. radiodurans*. (A) Straightened views of the peripheral region (Top), old (Middle) and growing (Bottom) septa used to measure the thickness of the various layers, shown schematically to the right of the images (SI Appendix, Fig. S1 for more details regarding the measurements). Left: Top panels show a straightened region and Lower panels show the same region with segmentations of the various layers. The IM is highlighted in black, the OM in red, the S-layer in purple, the SlpA layer in yellow, the peptidoglycan (PG) in green and the low-density periplasmic space (LD) in light gray. The “white” line connecting the SlpA and PG layers is indicated with a black arrow. (B) Mean and SD of the thickness of the IM and OM within the peripheral cell envelope. Individual points correspond to the mean thickness calculated from measures made on individual tomograms (N = 16). Horizontal and vertical bars correspond respectively to the mean and SD of the mean thickness of each bilayer. Statistical test: Welch's *t* test. ***P = 0.0002. (C) Plot illustrating the variation in PG thickness as a function of the distance from the root of the growing septa. Individual points correspond to single measurements made on numerous cells and at different positions along the septa. (D) Plot illustrating the phase-dependent thickness of the PG layer in the old septum (d2 in SI Appendix, Fig. S1). Individual points correspond to the mean thickness calculated from measures made on individual tomograms (N < 6 for each phase). Horizontal and vertical bars correspond respectively to the mean and SD of the mean PG thickness for each phase. (E) Two illustrations of junctions between the peripheral cell envelope and the growing septa. Left panels show 2D slices of a tomogram and Right panels show the same regions with segmentations of the various layers (colors as in A). (F) 2D slices extracted from different Z values of a given tomogram, illustrating the progressive splitting of daughter cells that starts at the cell periphery of the diad (Right) and then progresses inward toward the center of the diad (Left). The OM is delineated in red.</figcaption></figure>
daughter cells, forming bubble-like structures (Fig. 2 E and F and SI Appendix, Fig. S4B for more examples).

**Structure of Septal Tips.** A close inspection of the tomograms revealed that the tips of the growing septa exhibited particular structures. A majority of septa (40 of the 64 septa visible in our tomograms) were slightly tapered at their tips and the low-density periplasmic space was significantly thinner in these regions, bringing the PG layer very close to (in some cases even contacting) the IM (Fig. 3A). Strikingly, in nearly 40% of the growing septa (n = 77), membrane protrusions were observed at their tips (Fig. 3 B and C and SI Appendix, Fig. S5 for more examples). These structures, mostly observed in cells at early stages of septation (27 out of 30 instances), appear to be composed solely of the low-density layer delimited on either side by the IM, and adopt either outstretched tube-like structures or more circular loop-like arrangements. Occasionally, these circular structures even appeared to be fully separated from the tip of the septum (Fig. 3B). In all cases, they appear to be very flexible, probably as a result of the absence of PG to rigidify the protrusion. This intrinsic flexibility may explain why the tips of the growing septa appeared as open ends in the superresolved images of Nile Red-labeled *D. radiodurans* cells (Fig. 3 D, Left panel). This suggests that either the Nile Red dye poorly labeled these highly curved membrane bilayers [likely exhibiting altered membrane fluidity that is known to affect Nile Red staining (36)] or that the tips were very mobile and not captured in live imaging experiments; both of these phenomena may also be at play. On a few occasions, we did capture poorly defined Nile Red labeling at the tips of growing

septa that may correspond to such flexible membrane protrusions (Fig. 3 D, Right panel and Fig. 1 C, Right panel).

**Septation Through a Sliding Doors Mechanism.** Using timelapse 3D video confocal microscopy of Nile Red stained *D. radiodurans* bacteria immobilized in various orientations on agarose pads (SI Appendix, Fig. S6), we followed the division process in live cells (Movie S1). Septation was found to proceed in several steps. First, two septa originating from opposite sides of the cell grow inward with a flat leading edge (Fig. 4). As the septa grow, their leading edge progressively becomes more curved. Finally, when the two septa come close to each other, fusion starts first at the periphery of the cell, forming a cat's eye structure, and then rapidly proceeds through a zipping mechanism toward the cell center. 3D PAINT images of Nile Red labeled *D. radiodurans* cells confirmed these observations, allowing to capture snapshots of dividing cells exhibiting septa with flat or slightly curved leading edges embracing a central slit that stretches across the cell (Fig. 4A). Kymographs of individual septation events were derived from these acquisitions to probe the kinetics of septal closure (Fig. 4B and SI Appendix, Fig. S7). Septation was found to be a linear process in which the external and internal septa (Fig. 1B) grow respectively at rates of 7.3 ± 0.5 nm.min⁻¹ and 4.1 ± 0.7 nm.min⁻¹ until full closure. Interestingly, the external septum not only grows at a faster rate, as can be seen from the asymmetric V-shaped structure of the kymographs, it also starts growing ahead of the internal septum (Δt in Fig. 4B). This asymmetry in the growth of the internal and external septa was also visible in the

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Close-up view of septal tips. (A and B) Examples of typical septa tip morphologies: (A) tapered leading edge with a well-defined and rigid PG layer (green) stretching almost to the IM (black) and (B) tubular (Top), curved or circular (Bottom) membrane protrusions extending the growing septa. Left panels show 2D slices of a tomogram and right panels show the same regions with segmentations of the various layers (colors as in Fig. 2A). (C) 2D slices extracted at different Z values from a given tomogram illustrating the change in size and shape of these membrane protrusions as a function of the position in the tomogram. Top panels show 2D slices and lower panels show the same regions with segmentations of the PG and IM layers (colors as in Fig. 2A). (D) Close-up views of the leading edges of growing septa captured by PAINT microscopy of Nile Red stained WT *D. radiodurans*. Left image: septal tips appear to be open-ended. Right image: blurry septal tips are visible pointing in opposite directions. These structures may correspond to the membrane protrusions observed in the tomograms (B and C).</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Septation through a sliding doors mechanism. (A) 3D superresolved PAINT image of a Nile Red stained WT *D. radiodurans* diad. Top panel: the 3D volume was obtained by combining 2 stacks of images collected at different focus heights. The flat leading edge is very clear in the left cell while the right cell, which is more advanced in its division process, illustrates the formation of a cat's eye structure stretching across the cell. This structure is also visible in the side view (Lower panel). (B) Kymograph analysis of the septation process in Nile Red stained *D. radiodurans*. Top panel: example of a typical kymograph obtained for a dividing diad. The coordinates of the borders of the fluorescent signal were used to determine the length of the external (bright green) and internal (orange) septa as a function of time. The lag time (Δt) between the start of growth (dashed red line) for the external septa versus the internal septa was also measured. Lower panel: Plot illustrating the linear growth of the external (bright green) and internal (orange) septa as a function of time. Data points correspond to the mean lengths of measured septa (4 < N < 12) at each time point. Error bars correspond to the SD. *** (P-value: 0.0004). (C) Examples of segmented septa in two tomograms, illustrating the flat edge of the growing septa (Left panel, green) that progressively becomes more curved (Right panel, blue) as the division process advances. (D) 2D slices extracted from various tomograms illustrating different stages of the septation process (early, mid- or late). Left panels show 2D slices of a tomogram and Right panels show the same regions with segmentations of the various layers (colors as in Fig. 2A). The light gray area in the Right panels corresponds to the nucleoid. All slices are arranged with the internal septa on the Left. (E) 3D superresolved dSTORM image of PG-labeled (through incorporation of azido-D-Alanine) WT *D. radiodurans* diads. Top panel: PG synthesis occurs both in the septa and in the peripheral cell wall, with a strong PG synthesis activity detected at the leading edge (intense ring and arch). Lower panel: side view of a PG-labeled *D. radiodurans* diad, illustrating the incorporation throughout the septal region and the formation of the cat's eye structure. (F) Schematic model of the sliding doors mechanism of septation in *D. radiodurans*. The Top panels illustrate the growth of the membrane (red), while the lower panels show the closely related growth of the PG layer (green) and the strong PG synthesis activity at the leading edge of the septa.</figcaption></figure>
tomograms (Fig. 4 C and D) and is likely explained by the diad configuration of *D. radiodurans* cells and their expansion that is concomitant with the septation process (Fig. 1A). Indeed, the external septum must grow further than the internal one to reach the site of fusion located on the plane that intersects the point of maximal curvature of *D. radiodurans* cells. The flat or slightly curved leading edges of the growing septa were also observed in the tomograms (Fig. 4C).

In addition to staining the cell membrane with Nile Red, we also labeled the PG by growing *D. radiodurans* cells in the presence of azido-D-alanine (aDA) during a short (10 min) “pulse labeling” period. The incorporated aDA moiety was subsequently conjugated to fluorescent probes suitable for either confocal or dSTORM imaging (8, 37) by copper-free click chemistry (*SI Appendix*, Fig. S8). The aDA pulse-labeling pattern was very similar to that of Nile Red, with efficient incorporation in both the growing and old septal regions, as well as in the peripheral cell envelope (Fig. 4E and *SI Appendix*, Fig. S9), as observed previously using fluorescently labeled D-alanine (32). In both the 3D dSTORM and confocal images, the observation of tilted cells offering a front or side view (as opposed to the more common top view) of the septation plane further revealed that PG labeling is enriched at the leading edge of the growing septa, suggesting it corresponds to the major site of septal PG synthesis in these cells (Fig. 4E and F and *SI Appendix*, Fig. S9). This was particularly visible at late stages of the cell cycle [Fig. 1A, Phases 5 and 6]), where an intense band of labeled PG could be seen at the site of fusion (*SI Appendix*, Fig. S9C). This finding was confirmed by performing a pulse-chase experiment in which aDA pulse-labeled cells were further grown for 45 min in the absence of the probe (*SI Appendix*, Fig. S10A). In the absence of the chase, Nile Red and PG-labeling were largely superimposed. In contrast, after the chase, most Nile Red-stained septa showed incomplete PG labeling, indicating that septa having incorporated aDA at their leading edge during the pulse period were extended by unlabeled PG during the chase period. Altogether, these observations indicate that new PG is added to the existing layer in an inward direction until the opposing septa are close enough to fuse (Fig. 4F and *SI Appendix*, Fig. S10B). On the contrary, peripheral PG labeling was largely unaffected by the chase, suggesting that aDA incorporation (and thus PG synthesis or remodeling) occurs throughout the periphery of the diads, leading only to a progressive dilution of the labeling intensity (*SI Appendix*, Fig. S10B).

Close examination of the tomograms also revealed that the relative position and shape of the growing septa changed as a function of their length (Fig. 4D). At early stages of the septation process, short septa were not always precisely facing each other and their tips were often bent and bearing membrane protrusions (Figs. 3 B and C and 4D), while at later stages, septa were remarkably straight and fully aligned to ensure their fusion (Fig. 4D). The presence of a thick PG layer in the growing septa appears to be a prerequisite for the formation of these straight and well-aligned septa, indicating that PG synthesis may provide the necessary rigidity for septal closure. This final fusion step was captured in two of the tomograms and was found to proceed first through fusion of the IM and subsequently through synthesis of PG to fill the gap and “glue” the two septa together (Fig. 4D and *SI Appendix*, Fig. S3). Taken together, these data allow us to propose a model of septation through a sliding doors mechanism, which is illustrated schematically in Fig. 4F.

**PG Synthesis in the Peripheral and Septal Regions Are Performed by Distinct Machineries.** To better understand the molecular mechanisms underlying this unusual mode of septation, we treated *D. radiodurans* cultures with a β-lactam antibiotic, ampicillin, and

observed the growth of untreated and treated Nile Red labeled cells by 3D confocal timelapse microscopy for a 3 h period (Fig. 5A and Movies S2 and S3). Ampicillin is known to bind to the active sites of certain PBPs, thereby inhibiting their enzymatic PG synthesis function (38). To our surprise, septation, but not cell growth was arrested by ampicillin treatment (Fig. 5 A–D). Indeed, the growth of the cell perimeter was unaffected by this treatment during this 3 h period, while growth of both the internal and external septa was rapidly arrested (Fig. 5 C and D). After 1 h, the septa even started to shorten, suggesting they may be undergoing disassembly and retraction (Fig. 5D). As a result of this change in the balance between cell growth and septation, cells progressively became distorted (more elongated with a slight bulge at sites of division), as evidenced by the substantially increased diameter (d) to perimeter (p) ratio after 3 h of treatment with ampicillin (Fig. 5E). The specific inhibition of septal growth by ampicillin suggests that the PBPs responsible for septal PG synthesis and maturation are distinct from those involved in peripheral PG synthesis (Fig. 5F).

Next, we repeated the aDA pulse-labeling experiment on cells pregrown for either 1 or 2 h in the presence of ampicillin before aDA incorporation (*SI Appendix*, Fig. S11). In agreement with our timelapse experiments, aDA was still incorporated in the peripheral region, albeit to a reduced level compared to untreated cells. Septal labeling, on the other hand, was restricted to bright dots (in 2D) or distinctive rings (in 3D) positioned at the division site (*SI Appendix*, Fig. S11). In untreated cells, such PG-labeled rings were typically seen in ~10% of cells, corresponding to cells at the onset of division. In ampicillin-treated cells, and more particularly in samples pregrown for 2 h in the presence of the β-lactam antibiotic, over 30% of cells exhibited such PG-labeled rings (N > 300; *SI Appendix*, Fig. S11B). Initial septal PG synthesis thus appears to be unaffected by ampicillin, while subsequent extension of the septa is arrested by this treatment. Distinct sets of PBPs may therefore be involved in the initiation and extension stages of septation (Fig. 5F).

**FtsZ Is Present at the Tips of Septa.** We investigated the location of FtsZ, one of the key players in bacterial cell division, in dividing *D. radiodurans* cells using both fluorescence microscopy and cryo-ET. Two strategies were used to fluorescently label FtsZ: i) immunolabeling of an endogenously HA-tagged FtsZ or ii) endogenous tagging of FtsZ with a photoconvertible fluorescent protein, mEos4B. The latter was compatible with live cell imaging, but this genetically modified strain of *D. radiodurans* showed altered cell morphology (forming many large cells with likely impaired septation) and only a small fraction of cells exhibited fluorescence signal and visible Z-rings (Fig. 6A). In contrast, the strain expressing HA-tagged FtsZ showed no major morphological defects (*SI Appendix*, Fig. S12), and FtsZ could be detected by either confocal microscopy or dSTORM, following anti-HA antibody immunolabeling of fixed and permeabilized cells (Fig. 6A and B). Both strategies indicated that *D. radiodurans* FtsZ forms ring- or oval-shaped structures, some of which appear to be incomplete and/or display weaker curvature in regions likely corresponding to septal leading edges. In support of these observations, dual labeling of FtsZ and the cell membrane showed that FtsZ locates to the leading edge of growing septa at all stages of septation, including very early stages when septa are not yet visible by fluorescence microscopy (Fig. 6B).

In several tomograms, a double-arched structure was seen in the cytoplasm at ~15 nm from the IM border of the septal leading edge, which likely corresponds to FtsZ (outer arch) and its cellular partner, the membrane-anchored FtsA (12, 39–42) (inner arch; Fig. 6C and *SI Appendix*, Fig. S13). These two arches follow the curvature of the
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Distinct machineries are involved in septal and peripheral PG synthesis. (A) Snapshots of untreated (Left) and ampicillin-treated (Right) Nile Red stained *D. radiodurans* extracted from the time-lapse experiment (Movies S2 and S3). (B) Schematic diagram of a *D. radiodurans* diad, illustrating the measurements performed on different features to probe the effect of ampicillin treatment. (C) Effects of ampicillin treatment on the growth rate of the external (green) and internal (orange) septa, and of the peripheral cell wall (black) derived from measurements of the cell perimeter (N > 25). Statistical test: One-way ANOVA. ns: nonsignificant, ****P-value < 0.0001. (D) Plot illustrating the inhibition of both the external (green) and internal (orange) septal growth by treatment with 1 µg/mL ampicillin. (E) Comparison of the diameter:perimeter (d/p) ratio of untreated (black) and ampicillin treated (t = 3 h) cells (red). Data points correspond to individual measurements made on N > 45 cells. Horizontal bars correspond to the mean values. Statistical test: unpaired t test. ****P-value < 0.0001. (F) Schematic model of the two PG synthesis machineries at play in *D. radiodurans*. Peripheral PG synthesis and remodeling (orange) that is insensitive to ampicillin is likely to occur throughout the peripheral envelope of the diads. In contrast, septal PG synthesis (purple) is very sensitive to ampicillin and is located mainly at the leading edge of the growing septa.</figcaption></figure>

septal tip, with the outer FtsZ arch typically between 20 and 50 nm in length and an interfilament distance of ~5 nm. As in our fluorescence microscopy data, these structures were observed at all stages of the septation process from budding septa all the way to almost fusing septa. In the side projections of the tomograms, both FtsA and FtsZ were observed to form long, straight filaments along the flat leading edge of the closing septa, with a typical FtsA-FtsZ distance of ~7 to 8 nm, in good agreement with previous reports of in situ FtsZ filaments (39, 40) (Fig. 6C). Interestingly, when membrane protrusions were present at septal tips, FtsZ was not observed on these protrusions, but was occasionally seen in Z-slices either above or below these structures (SI Appendix, Fig. S13 and Movie S4), which may explain some of the discontinuous ring structures observed by fluorescence microscopy (Fig. 6A and B).

Discussion

In this study, we have combined fluorescence microscopy with in situ
cryo-ET imaging of *D. radiodurans* to follow the process of septation
in this bacterium. This work provides important insight into i) the
complex architecture of its cell envelope along the cell cycle, ii) its
distinct mode of septation, and iii) the sequence of events underlying
membrane and PG synthesis during septal growth. Furthermore, it
starts to unveil the key role of FtsZ in this division process.

*D. radiodurans* possesses a unique cell envelope including an outer
S-layer, which has been the object of numerous studies over the past
decades (22–31). Like other members of the Deinococcus-Thermus

phylum, *D. radiodurans* exhibits features of both gram-positive and
gram-negative bacteria, and may be considered as a primitive form
of diderm bacterium (4). *D. radiodurans* is indeed lacking lipopol-
ysaccharides commonly found in gram-negative bacteria, possesses
a thick PG layer (35 to 55 nm) typical of gram-positive bacteria and
yet its cell envelope is composed of two membrane bilayers charac-
teristic of gram-negative bacteria (43). The exact composition and
structure of this unusual cell envelope has been the object of much
controversy in recent years, notably regarding the outer layers linking
the PG to the outermost S-layer hexagonal lattice structure. Our
in-depth cryo-ET analysis of the complex architecture of *D. radio-
durans* cell envelope fully supports the model recently proposed by
Bharat and colleagues, in which the whole peripheral cell envelope
is ~100 nm in thickness and composed of two membranes in
between which can be found a thin periplasmic space and the thicker
PG and SlpA layers (31). The S-layer forms an additional coat
located ~18 nm above the OM (24). The predicted length of the
SlpA protein assembly (~28 to 29 nm) is in good agreement with
our measured thickness of the SlpA layer (35 nm). Interestingly, in
our tomograms, we observe a distinctive white line between the PG
and SlpA layers that may correspond to the sites at which the flexible
N-terminal SLH domain of SlpA attaches to the PG layer.

In *D. radiodurans*, daughter cell separation driven by the activity of autolysins (44) [four of which have been identified in *D. radiodurans* (45)] is uncoupled from septation, with these two phenomena occurring in successive cell cycles. Furthermore, we show that three cell envelope regions can be distinguished in dividing *D. radiodurans*,
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Localization of the key cell division factor, FtsZ, in *D. radiodurans*. (A) 3D fluorescence imaging of FtsZ in *D. radiodurans*. Left: 3D super-resolved PALM imaging of *D. radiodurans* cells expressing FtsZ-mEos4B, which forms well-defined ring-like structures. Middle and Right: 3D confocal images (Middle) and superresolved dSTORM images (Right) of immunolabeled *D. radiodurans* cells expressing FtsZ-HA. (B) Two-color labeling of *D. radiodurans* cells expressing FtsZ-HA (green) and stained with Nile Red (red). From Left to Right: illustrations of different stages of the division process (early, mid- and late). At all stages, FtsZ localizes to the leading edge of the septa. Top panels: Top views, Lower panels: side views. (C) Left: 2D slice of a typical tomogram of *D. radiodurans* illustrating the double-arched structure of FtsA (purple) and FtsZ (blue) in the vicinity of the IM (black) on the cytoplasmic side of the growing septum. The top panel shows a 2D slice through a tomogram and the lower panel the same region with segmentations of the various layers (colors as in Fig. 2A) and the double-arched structure. Right: side view of the FtsA and FtsZ filaments highlighted in the Right panel in purple and blue respectively.</figcaption></figure>

displaying different compositions and characteristics that reflect their stage of maturation. Contrary to *S. aureus*, in which two distinct PG layers (one for each daughter cell) are readily observed in growing septa (5, 10), all *D. radiodurans* septa are composed of a single PG layer, which thickens during septal growth and even after closure of the septal slit. The SlpA and OM layers of the outer envelope are only added at a late division stage, when the splitting of the cells is initiated. Different from *E. coli*, in which the full cell envelope is synthesized within one division cycle (5), the assembly of the cell envelope in *D. radiodurans* therefore encompasses two complete cell cycles. The temporal dissociation between the synthesis of the inner layers (IM and PG) and that of the outer layers (SlpA and OM), which is coordinated with septum splitting, may be required to allow incorporation of the abundant SlpA protein onto the PG layer and its attachment to the OM. These two additional layers efficiently protect *D. radiodurans* from its external environment and must therefore be completed before the splitting of the daughter cells. Our 3D cryo-ET data suggest that the splitting process is initiated from the periphery of the cell at sites of OM invagination and then moves inward toward the center of the diad until full separation of the two daughter cells.

PG synthesis and its subsequent remodeling are key processes in bacterial septation. In *D. radiodurans*, PG synthesis occurs in

both septal and peripheral cell envelope regions (32). Here, by observing aDA-labeled cells with high-resolution fluorescence microscopy techniques, we show that the leading edge of the septa constitutes the main site of septal PG synthesis in dividing *D. radiodurans* and that it proceeds in an inward direction until the opposing septa meet and fuse. These growing septa are very often tapered with a thinner PG layer at the leading edge than at the root of the septum. This has also been reported in *S. aureus* (10, 46, 47), suggesting that PG synthesis may not be restricted to the leading edge of the septa. As has been reported for the ovococci, *Lactococcus lactis* (48), and *S. pneumoniae* (49, 50), we found that ampicillin treatment specifically impedes septation and not peripheral cell wall expansion needed for cell growth. In ovococci, this effect has been attributed to the specific inhibition of the class B PBP, PBP2x, that is part of the divisome and is involved in septation (48, 49, 51). The genome of *D. radiodurans* encodes for two class A PBPs and one class B PBP, which is annotated as PBP2 (DR_1868). Although little is known so far about the respective roles of these PBPs in *D. radiodurans*, our observations suggest that *D. radiodurans* PBP2 may be the target of ampicillin and thus largely responsible for septal PG synthesis at the leading edge of the growing septa.

Whereas in most cocci and ovococci studied so far septation advances centripetally like a closing iris, with the present study, we provide evidence that septation in *D. radiodurans* is quite distinct. It proceeds via a sliding doors mechanism, also described in an earlier study as a "septal curtain" (13), in which the two septa originating from opposite sides of the cell grow inward with a flat leading edge. As septation progresses, the leading edge becomes more curved and eventually, when the two septa come close to each other, fusion occurs and the septal disk is filled. Why *D. radiodurans* uses such a distinct mode of septation remains to date a mystery. Physical constraints associated with the relatively large size (2 to 3 µm in diameter) of *D. radiodurans*, its cell morphology (diads and tetrads) or its mode of division in two alternating perpendicular planes may have contributed to the development of this unusual division mechanism.

With such a septation process, ensuring the opposing septa are correctly aligned and meet for septal fusion constitute major challenges for the bacteria. The latter is, in part at least, achieved by differential growth rates of the external and internal septa. Indeed, we observed that the septum originating from the external side of the diad grows at a faster rate and initiates its growth shortly before the internal septum originating from the old septum to compensate for the longer distance needed for it to reach the site of fusion. This study also identifies two important factors that may constitute a physical link between the opposing septa: i) the flexible membrane protrusions at the septal tips, and ii) the FtsZ rings that encircle the division plane. These two mutually exclusive features are present at the leading edge of *D. radiodurans* septa. Membrane protrusions were detected in many instances at early stages of the septation process, adopting a variety of conformations. Interestingly, similar structures were reported in early electron microscopy studies of *D. radiodurans* (34, 52) and other bacteria (53, 54), but were later considered as artifacts of chemical fixation procedures used for sample preparation (55–57). In our case, these structures were observed in situ in near-native conditions (vitrified cells), strongly suggesting that they are not artifacts. Instead, we propose that these membrane extensions may provide the necessary flexibility to facilitate the alignment of opposing septa during the initial steps of septation. In contrast, we found that the presence of FtsZ filaments at the septal tips was associated with more rigid and well-aligned septa, suggesting that the assembly of the FtsZ ring may constitute a critical step in the proper alignment of opposing septa, possibly through a direct bridging
<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Schematic model of the septation process in *D. radiodurans*. Top and side views are illustrated at various stages of the division process. For simplicity, only the IM (red) and the PG layer (green) are highlighted. FtsZ is shown in blue with incomplete Z-rings as dashed lines and complete Z-rings as a full line. Flexible membrane protrusions are visible at early stages of the septation process, and are progressively filled with PG, providing the necessary rigidity to achieve a solid and well-aligned cross-wall. FtsZ plays a key role in targeting the ampicillin-sensitive PG synthesis machinery to the leading edge of the growing septa and may also facilitate the proper alignment of opposing septa by bridging them via Z-ring formation.</figcaption></figure>

of the two opposing sliding doors. Importantly, FtsZ was only observed at the leading edge of septa containing a well-defined central PG layer that extended almost to the lipid bilayer. This suggests that the Z-ring may promote septal extension by recruiting the PG synthesis machinery to the septal tips, as has been reported for many other bacteria [reviewed in (58–60)]. However, FtsZ may not be needed for septal initiation since aDA-labeled rings corresponding to early stages of septum synthesis were still observed in the presence of ampicillin, which is known to inhibit FtsZ-associated PBPs (61).

Although FtsZ was never seen on the membrane protrusions themselves, in several tomograms FtsZ was observed on the leading edges of such septa in Z-slices located either above or below the protrusions. The leading edge of a given sliding door is thus likely to be both dynamic and heterogeneous, composed of flexible regions bearing membrane protrusions and more rigid areas in which FtsZ can assemble to drive PG synthesis. We thus propose a model for *D. radiodurans* septation (Fig. 7) in which septal PG synthesis is initiated by ampicillin-insensitive PG synthases, and then further extended by ampicillin-sensitive PBPs that are likely part of the divisome in a FtsZ-dependent manner. In this model, FtsZ-independent membrane synthesis would precede FtsZ-driven PG assembly to create membrane protrusions that would facilitate alignment of the opposing septal sites at early stages of the division process. The subsequent assembly of FtsZ filaments at the leading edge of the septa, followed by the formation of the Z-ring, would then act as a cue for the recruitment and activation of PG synthesis in order to progressively fill and rigidify the growing septa. Further studies will certainly be needed to decipher the precise details of

this process and the specific role of FtsZ and other key players in this complex and intriguing septation of *D. radiodurans*.

## Materials and Methods

Detailed methods are provided in *SI Appendix*. Bacterial strains used in this study are listed in *SI Appendix*, Table S1. All *D. radiodurans* strains were derivatives of the WT strain R1 ATCC 13939 and the genetically engineered strain of *D. radiodurans* expressing FtsZ fused to mEos4B (DR-FtsZ-mEos4B) was obtained by the tripartite ligation method as described recently (62). *D. radiodurans* cells were grown to exponential phase at 30 °C in TGY2X medium supplemented with the appropriate antibiotics. For confocal and SMLM, *D. radiodurans* cells were deposited on agarose pads and were imaged respectively on an Olympus IX81 inverted microscope equipped with a Yokogawa CSU-X1 confocal head or on an Olympus IX83 inverted motorized microscope equipped with a SAFE 360 (Abbelight) SMLM set up. Detailed labeling protocols and imaging parameters are provided in *SI Appendix*. For cellular cryo-ET imaging, cryo-FIB milled lamellae of flash frozen *D. radiodurans* cells were prepared using a Scios cryo-FIB/scanning electron microscope dual-beam microscope and were transferred to a Titan Krios microscope operated at 300 keV and equipped with a BioQuantum postcolumn energy filter (Gatan, Pleasanton, CA) and a K2 Summit direct detector for acquisition of cryo-ET data. Details of the cryo-ET sample preparation and analysis are provided in *SI Appendix*.

**Data, Materials, and Software Availability.** All study data are included in the article and/or supporting information.

**ACKNOWLEDGMENTS.** The Institut de Biologie Structurale (IBS) acknowledges integration into the Interdisciplinary Research Institute of Grenoble. The optical imaging was carried out on the M4D imaging platform of the Grenoble InstructERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology, supported by the French Infrastructure for Integrated Structural Biology (ANR-10-INBS-0005-02) and the Grenoble Alliance for Integrated Structural & Cell Biology Labex, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) that also funded L.G.'s PhD position. We thank the M4D platform at IBS for their help for data acquisition and processing, and the Abbelight team for help with data acquisition. Electron microscopy sample preparation, cryo-focused ion beam milling, and cryo-electron tomography data collection were performed at the Umeå Centre for Electron Microscopy (UCEM), the Umeå Cryo-EM node of SciLifeLab national research infrastructure. I.G. thanks the UCEM staff for training and support. The fluorescence microscopy work benefitted from funding from the Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Radiobiology program and the Agence Nationale de la Recherche (Grants N°ANR-22-CE11-0029-01 to J.T. and ANR-23-CE11-0029 to C.M.). The electron microscopy work was supported by visiting professorship fundings from the Molecular Infection Medicine Sweden, the Wenner Gren foundation and the Swedish Research Council (VR) Tage Erlander (Grant N° DNR 2022-00308) to I.G.

Author affiliations: <sup>a</sup>University of Grenoble Alpes, Commissariat à l'énergie atomique et aux énergies alternatives, CNRS, Institut de Biologie Structurale, Grenoble F-38000, France; <sup>b</sup>Department of Chemistry, Umeå University, Umeå SE-901 87, Sweden; and <sup>c</sup>SciLifeLab research infrastructure at Umeå University, Umeå Centre for Electron Microscopy, Umeå SE-901 87, Sweden

Author contributions: L.G., J.-P.K., I.G., and J. Timmins designed research; L.G., J.-P.K., F.L., E.S., I.G., and J. Timmins performed research; J. Trouve, C.M., and L.S. contributed new reagents/analytic tools; L.G., J.-P.K., I.G., and J. Timmins analyzed data; and L.G., J.-P.K., C.M., I.G., and J. Timmins wrote the paper.

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