Specific role of two NlpC/P60 endopeptidases in cell division and membrane vesicle formation in Deinococcus radiodurans

Highlights

• •Three NlpC/P60 endopeptidases identified in Deinococcus radiodurans.
• •CwlA is essential for cell viability and cell wall integrity.
• •CwlB is involved in cell septation whereas CwlC is dispensable.
• •Regulatory link between CwlA expression and the genotoxic stress response pathway.

Abstract

The bacterial cell wall is composed of peptidoglycan (PG), a sugar polymer cross-linked by short peptide stems. PG determines cell morphology and protects it from environmental stresses. Cell growth and division require a balance between synthesis and hydrolysis of the PG. One class of PG hydrolase is the NlpC/P60 superfamily which is broadly distributed in bacteria, archaea, eukaryotes and viruses. Deinococcus radiodurans, characterized by its extreme radioresistance, is a diderm bacterium with a thick layer of PG located between the inner and outer membrane. D. radiodurans exhibits three NlpC/P60 endopeptidases, their role in morphogenesis and cell cycle remains unexplored. In this work, we investigated the role of each endopeptidase in cell division to assess their specific role. Here, we showed that the CwlB protein is involved in cell division and that cwlA gene is essential for cell viability. The CwlC protein is not required for cell shape maintenance or cell division. We showed that CwlA protein is homogeneously localized around the cell except on septal region. CwlA-depleted cells lost viability, displayed morphological changes, and produced numerous membrane vesicles, similarly to cells exposed to sublethal mitomycin C (a DNA-damaging agent) or to DdrO depletion, the transcriptional repressor of the main genotoxic stress response in Deinococcus. We showed that cwlA expression is highly repressed under DdrO depletion. These data suggested a link between response pathway to genotoxic conditions and cell wall remodeling.

Graphical abstract

1. Introduction

The bacterial cell wall is dynamic and undergoes reorganization during vegetative growth, development, and cell division. During these processes, the wall is dismantled by a diverse set of enzymes that hydrolyze different bonds within the peptidoglycan (PG). These enzymes include glycosidases, such as lysozymes, which target the polysaccharide backbone, and peptidases which break down the cross-linking peptides. One class of PG hydrolase is the NlpC/P60 superfamily (Anantharaman and Aravind, 2003; Ishikawa et al., 1998). NlpC/P60-type endopeptidases are widely distributed among bacteria and participate in various processes such as cell growth, enabling the development of peptidoglycan through the insertion of new units, the maturation and recycling of peptidoglycan and its cleavage at the septum during cell division. Since over- or underactivity of these proteins lead to significant cellular damage, ranging from septation defects to cell lysis, the synthesis and activity of these proteins are tightly regulated throughout the cell cycle and cellular development (Vollmer et al., 2008).

The bacterium Deinococcus radiodurans is renowned for its extraordinary ability to withstand a broad spectrum of genotoxic treatments, including ionizing and ultraviolet radiation, mitomycin C, desiccation, and oxidative stress. When exposed to extreme doses of γ-radiation causing hundreds of DNA breaks, D. radiodurans reassemble its genome within 2 to 3 h. This remarkable ability is supported by highly efficient DNA repair mechanisms, as well as other survival strategies, including significant nucleoid compaction, mechanisms protecting against protein oxidation and the induction of specific proteins following irradiation (Confalonieri and Sommer, 2011; Slade and Radman, 2011; Daly, 2012; Blanchard et al., 2017; Lim et al., 2019). In Deinococcus, the main response pathway to genotoxic conditions is regulated by a constitutively expressed metalloprotease IrrE and a transcriptional repressor DdrO (Ludanyi et al., 2014; Devigne et al., 2015; Wang et al., 2015; Lu et al., 2024). Upon exposure to genotoxic stress conditions, IrrE is activated and cleaves DdrO, leading to expression of about 40 repressed genes constituting the Radiation Desiccation Response (RDR) regulon (Makarova and Daly, 2010; Eugénie et al., 2021). The ddrO gene is essential for cell viability of D. radiodurans and its prolonged depletion by a conditional deletion system induced an apoptotic-like response (DNA degradation, defects in chromosome segregation, membrane vesicle formation) (Devigne et al., 2015).

Although D. radiodurans is Gram-positive, the structural organisation of its membrane is closer to that of Gram-negative bacteria, with the presence of a thick peptidoglycan layer topped by a structurally complex layer rich in lipids, proteins and carotenoids, itself covered by a dense carbohydrate layer (Farci et al., 2022 ; Rothfuss et al., 2006; Sexton et al., 2021). The peptidoglycan has a unique composition due to the presence of l-ornithine, a very rare component only found in a few bacterial membranes (Work and Griffiths, 1968). However, D. radiodurans lacks a lipopolysaccharide (LPS) layer, which is characteristic of Gram-negative bacteria (Beaud Benyahia et al., 2025). This bacterium, and more generally the Deinococcus-Thermus phylum, represents an intermediate stage in the evolutionary transition between bacteria with a single membrane and those with two (Gupta, 2011).

Beyond its well-documented resistance to high doses of ionizing radiation and long-term desiccation, D. radiodurans has garnered significant attention in recent years in the field of astrobiology. This bacterium is one of the few microorganisms capable of surviving harsh conditions of outer space, including exposure to cosmic and solar radiation, extreme vacuum, temperature fluctuations, desiccation, and microgravity (Ott et al., 2020). Notably, D. radiodurans cells have been shown to survive for three years on the exterior of the International Space Station. The formation of membrane vesicles (MVs) is observed when the bacteria were subjected to such extreme stress (Ott et al., 2020). Bacterial extracellular vesicles are nanoscale, membrane-bound structures ranging in size from 10 to 500 nm released by both Gram-negative and Gram-positive bacteria into their surrounding environment. These MVs play a critical role in a wide range of biological processes. They are key mediators of intercellular communication by acting as molecular carriers, enabling the transfer of genetic material such as DNA and RNA. Additionally, they are involved in the transport of virulence factors, toxins, and other bioactive molecules (Brown et al., 2015; Schwechheimer and Kuehn, 2015; Toyofuku et al., 2019).

In this work, we aimed to dissect the role of three endopeptidases containing NlpC/P60 domains (CwlA, CwlB, and CwlC) by analyzing the phenotype of single or double mutants during both exponential and stationary phases. The cwlC mutant displayed no detectable phenotype. In contrast, CwlB was found to play a key role in septal degradation, highlighting its involvement in cell division. Finally, a reduction in the amount of CwlA endopeptidase was associated with the formation of extracellular MVs, indicating a potential link between CwlA activity and membrane dynamics.

2. Materials and methods

2.1. Bacterial strains, plasmids, oligonucleotides, media

Bacterial strains and plasmids are listed in Table 1, Table 2, respectively. The Escherichia coli strain DH5α was used as the general cloning host and strain SCS110 was used to propagate plasmids prior to introduction into D. radiodurans via transformation (Meima et al., 2001). All D. radiodurans strains were derivatives of the wild-type strain R1 ATCC 13939. The deletion mutants were constructed by the tripartite ligation method (Mennecier et al., 2004). An antibiotic cassette (hygromycin or chloramphenicol resistance gene) and two regions of around 500 bp genomic fragments from upstream and downstream of the coding region of target gene were amplified by PCR using primer pairs that introduced BamHI and XbaI restriction sites. The antibiotic cassette flanked by the two genomic fragments were ligated together in molecular ratio 1/1/1 (100 ng of 500 bp fragments). The constructs were then introduced into D. radiodurans by transformation selecting for antibiotic resistance. This led to the replacement of the wild-type allele by the mutant counterpart via homologous recombination. D. radiodurans contains from 4 to 10 genome equivalents. Homogenotes of the deletion allele were obtained after two cycles of purification on selective medium. The same strategy was used to construct strains expressing neon green-tagged CwlA or CwlB proteins. The genetic structure and purity of the mutants were verified by PCR (Figure S1, S2 and S9). The sequence of oligonucleotides used for strain construction and diagnostic PCR are listed in Table S1. Chromosomal DNA of D. radiodurans was extracted using the NucleoSpin DNA Microbial Mini kit (Macherey-Nagel). PCR amplification of DNA fragments, using plasmid or genomic DNA as a template, was performed using Phusion DNA polymerase (Thermo Scientific) or GoTaq Flexi G2 (Promega).

Table 1.

Bacterial strains used in this study. ^a: strains were constructed by the tripartite ligation method.

Strains	Description	source or reference
E. coli
DH5α	supE44ΔlacU(Φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1	laboratory stock
SCS110	endA dam dcm supE44 Δ(lac-proAB) (F’traD36 proAB lacIqZΔM15)	laboratory stock
D. radiodurans
R1 / GY9613	ATCC 13939	laboratory stock
GY13739	R1/p11554	laboratory stock
GY13747	R1/p13841	laboratory stock
GY14164	∆ddrO Ωcat / p11891 (prepUTs::ddrO+)	Devigne et al.(2015)
GY18246	∆cwlAΩhph non-homogenotized	This work a
GY18249	∆cwlAΩhph/p17263 (prepUTs::cwlA+) clone 1	This work a
GY18250	∆cwlAΩhph/p17263 (prepUTs::cwlA+) clone 2	This work
GY18875	cwlA::DrNeongreenΩhph	This work a
GY19037	∆cwlB Ωcat	This work a
GY19042	∆cwlBΩcat/ p18736 (cwlB+)	This work
GY19040	∆cwlAΩhph/ p18731 (prepU::cwlA+)	This work
GY19044	∆cwlC Ωhph	This work a
GY19047	∆cwlB Ωcat ∆cwlC Ωhph	This work
GY19075	∆cwlB Ωcat ∆cwlC Ωhph/ p18736 (cwlB+)	This work
GY19094	cwlB::DrNeongreenΩhph	This worka

Table 2.

Plasmids used in this study.

plasmids	description	reference
pPS6	Source of cat cassette	laboratory stock
p11554	Shuttle vector E. coli/ D. radiodurans, SpcR	laboratory stock
p11830	Vector thermosensitive for replication in D. radiodurans, SpcR, prepUTs	Nguyen et al. (2009)
p12625	Source of hph cassette	laboratory stock
p13841	p11830 PSpac-term 116	Nguyen et al. (2009)
p11891	p13841: prepUTs::ddrO+	Devigne et al.(2015)
p17235	p11554 BamHI/BglII + multiple cloning site	this work
p17263	p13841 NdeI /XhoI+ drO_1728	this work
p17291	pEX-128 + DrNeongreen	Eurofins
p17292	p17291 AgeI/XhoI + hph cassette, source of DrNeongreen-hph	this work
p18731	p17235 NdeI/XhoI + PcwlA-cwlA	this work
p18736	p17235 BamHI/XbaI + PcwlB-cwlB	this work

D. radiodurans strains were grown at 30 °C (or 28 °C, 37 °C) in TGY2X (1 % tryptone, 0.2 % dextrose, 0.6 % yeast extract), or plated on TGY1X containing 1,5 % agar, and E. coli strains were grown at 37 °C in LB (Lysogeny Broth). When necessary, media were supplemented with the appropriate antibiotics used at the following final concentrations: chloramphenicol 3 µg/mL; spectinomycin 75 µg/mL, hygromycin 50µg/mL for D. radiodurans and 40 µg/mL of spectinomycin for E. coli.

2.2. Assay of genes essentiality

The essentiality of genes was evaluated in a growth experiment, in which the strains grown at 28 °C in liquid medium with spectinomycin were serially diluted, plated on TGY agar and incubated at 28 °C or 37 °C in the presence or the absence of spectinomycin.

2.3. CwlA and Ddro depletion

ΔcwlA and ΔddrO strains complemented by cwlA or ddrO, respectively, expressed from a plasmid with thermosensitive replication (prepUTs) were grown at permissive temperature (30 °C) with selective antibiotics (spectinomycin and hygromycin or choramphenicol), until they reached an A_650nm 0.25∼0.3. Cells were harvested by centrifugation and resuspended at the previous A_650nm levels in fresh medium without antibiotics and grown at permissive (30 °C) or non-permissive (37 °C) temperature. At 24 h, aliquots were removed for further analyses.

2.4. Fluorescence microscopy

Cell membranes were stained with N-(3-triethylammoniumumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (FM 4–64) at 0.01 mg/mL, and the nucleoid with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) at 2 mg/mL. FM 4–64 stains the lipid membranes with red fluorescence (excitation/emission ∼515/640 nm) and DAPI stains the nucleoid with blue fluorescence (excitation/ emission ∼350/470 nm). The stained cells were observed using a Leica DM RXA microscope. Images were captured with a CDD camera 5 MHz Micromax 1300Y (Roper Instruments).

2.5. Transmission electron microscopy (TEM)

Cells were fixed in 0.1 M cacodylate pH 7.4 buffer containing 2 % paraformaldehyde and 2 % glutaraldehyde at room temperature for 1 hour. Samples were washed 5 times in 0.1 M cacodylate buffer for 5 min and post-fixed in buffer A (1 % osmium tetroxid, 1.5 % potassium ferrocyanide) at room temperature for 1 hour. Then, cells were embedded in 2 % low melting agarose and dehydrated in a graded series of ethanol-water solution (30, 50, 70, 90 %) for 10 min each. Subsequently, cells are dehydrated with HMPA 90 % (hexamethylphosphoramide) – ethanol 10 %, HMPA 95 %-ethanol 5 %, HMPA 95 % - ethanol 3 % solution for 10 min each. Thin sections of the samples were stained with uranyless followed by lead citrate. Samples were viewed using a JEOL 1400 transmission electron microscope operated at 120 kV.

2.6. RNA seq analysis

RNA-seq data published in Eugénie et al. (Eugénie et al., 2021) were processed in a similar way. Only reads corresponding to RNA strands (R2 sequences) were used for these studies. Sequence alignments on the genomic sequence were performed by Bowtie2 software and differential analysis of gene expression between ∆ddrO/prepU_TSddrO⁺strain compared to the ∆ddrO/prepUddrO⁺ strain for each time point was performed using the DESeq2 package using (FC ≥2, p-value ≤0.01) as cutoff/parameters to highlight upregulated or downregulated genes.

3. Results

3.1. Sequence analysis of endopeptidase NlpC/P60 in D. radiodurans

NlpC/P60 endopeptidases belong to a cysteine peptidases family that hydrolyze peptide bonds within the peptidoglycan layer. NlpC/P60 enzymes often exhibit a modular architecture, combining the catalytic domain with additional domains such as LysM, SH3 or choline binding domain (Anantharaman and Aravind, 2003), which facilitate substrate recognition and protein localization. D. radiodurans, encodes three putative NlpC/P60 endopeptidases (Anantharaman and Aravind, 2003): DRO_1728 (DR1749), DRO_1316 (DR1325), and DRO_1350 (DR1316). We named these proteins CwlA (cell wall hydrolase A), CwlB, and CwlC, respectively. Search for known domains in the InterPro Database showed that these endopeptidases may be classified into two groups based on their N-terminal domain organization (Fig. 1A). The first group, comprising CwlA and CwlB, possesses a putative signal peptide, indicative of extracellular localization or membrane targeting, and one or two LysM domains, respectively. The LysM (Lysin Motif) domain is a small globular domain of approximately 40 amino acids, originally identified in enzymes degrading bacterial cell walls. This widespread protein module binds peptidoglycan by interacting with N-acetylglucosamine residues. The third protein, CwlC, contains two bacterial SH3-like domains but lacks the signal peptide. The bacterial SH3-like domain also interacts with peptidoglycan, typically recognizing pentapeptide cross-bridges. This non-covalent binding facilitates cell wall targeting (Kurochkina and Guha, 2012). All these three proteins harbor a C-terminal NlpC/P60 catalytic domain that hydrolyzes specific peptide cross-bridges within the peptidoglycan layer using a cysteine residue as a nucleophile. A highly conserved catalytic triad composed of cysteine, histidine, and a polar residue as asparagine, glutamine or histidine carries their enzymatic activity (Anantharaman and Aravind, 2003). CwlA, CwlB, and CwlC, as well as YdhO from E. coli and CwlT from B. subtilis, all sequences contain this catalytic triad, along with three conserved residues that contribute to the formation of catalytic core (tyrosine, aspartic acid, and serine) (Fig. 1B). Due to the conservation of catalytic sites, their modular organization and the functional roles of their respective domains, CwlA, CwlB and CwlC are likely involved in peptidoglycan remodelling in D. radiodurans, thus playing a potential role in processes such as cell wall maintenance and cell division.

Fig. 1.

In silico analysis of NlpC/P60 endopeptidases of D. radiodurans.

A. Domain architecture of the three putative endopeptidases CwlA, CwlB, and CwlC from D. radiodurans. The signal peptide (S), suggesting potential secretion or membrane targeting. LysM and SH3 domains are known to be involved in peptidoglycan binding. The NlpC/P60 domain represents the catalytic core responsible for peptidoglycan hydrolase activity. Domain positions and lengths were identified using the InterPro database (Blum et al., 2025).

B. The NlpC/P60 catalytic domains of YdhO from E. coli, CwlT from B. subtilis, and CwlA, CwlB, and CwlC from D. radiodurans. These domains were aligned using MAFFT (Katoh et al., 2019). Symbols below the alignment indicate the degree of conservation at each position: an asterisk (*) denotes positions with fully conserved residues across all sequences; a colon (:) indicates conservation between residues with strongly similar biochemical properties; and a period (.) marks positions with weakly similar residues. The strictly conserved catalytic triad—cysteine, histidine, and asparagine (or histidine/glutamine) —is highlighted in dark pink. Additionally, three conserved residues contributing to the formation of the catalytic core are highlighted in cyan blue.

3.2. cwlA is an essential gene for cell viability

To investigate the role of Cwl proteins in D. radiodurans, deletion mutants were constructed for each gene. Since D. radiodurans contains between 4 and 10 genome copies, the transformants were purified on selective medium to obtain homogenotes with the deleted allele present in all genome copies. Homogenotes of cwlB and cwlC deletion mutants and the ΔcwlBΔcwlC double mutant were easily obtained after two rounds of purification on selective medium, and the purity of the strains was confirmed by PCR (Fig. S1). For cwlA, hygromycin-resistant colonies were obtained, but PCR analysis of two candidates revealed that, even after three rounds of purification on hygromycin plates, both still retained the wild-type allele in addition to the ΔcwlA::Ωhph allele (Fig. S2). Further purification attempts failed to eliminate the wild-type allele, strongly suggesting that the CwlA protein is essential for cell viability. To further confirm that the cwlA gene is essential in D. radiodurans, we used a conditional gene inactivation system (Nguyen et al., 2009). In this system, ΔcwlA cells expressed the CwlA protein at 30 °C from a temperature-sensitive (repUTs) replication vector. We used, as control, similar construct encoding the essential DdrO protein (Fig. 2, lane 6). Shifting the culture to 37 °C, a non-permissive temperature, resulted in an inability of the plasmid to replicate during successive cell divisions, leading to the depletion of CwlA or DdrO. The cells failed to grow at the non-permissive temperature (Fig. 2), confirming that cwlA is essential for cell viability.

Fig. 2.

CwlA is essential for D. radiodurans viability. Strains were grown in liquid medium with spectinomycin at 28 °C. Sequential dilutions of cells were spotted on TGY plates in the presence or absence of spectinomycin at 28 °C (A) or 37 °C (B). Lane 1: strain GY13739 containing non-thermosensitive plasmid p11554 (prepU); lane 2: strain GY13747 containing thermosensitive plasmid p13841 (prepUTs); lanes 3 and 4: GY18249 and GY18250 respectively, two independent clones [ΔcwlA (prepUTs-cwlA⁺)]; lane 5: strain GY19040 [ΔcwlA (prepU-cwlA⁺)]; lane 6: strain GY14164 [ΔddrO(prepUTs-ddrO⁺)].

3.3. CwlB is involved in the degradation of septa

To further investigate the function of CwlB and CwlC proteins, we examined by epifluorescence microscopy the cell morphology of wild type, ΔcwlB, ΔcwlC and ΔcwlBΔcwlC mutant bacteria. In the absence of CwlC, the cell morphology was comparable to that of the wild-type strain in the exponential or stationary phase of growth (Fig. S3) whereas CwlB depleted cells exhibited markedly abnormal shapes (Fig. 3). The cell morphology of this mutant was strongly affected in the exponential or stationary phase of growth. In stationary growing phase, the ΔcwlB mutant displayed a severe cytokinesis defect by the presence of enlarged multicellular structures, including double tetrads (eight cells) and quadruple tetrads (sixteen cells). In the wild-type strain during this phase, 23.4 % of the cells were present as dyads and 73.8 % as tetrads. In contrast, no dyads were observed in the ΔcwlB mutant. Only 1.6 % of the cells formed tetrads, while the majority were organized as double tetrads (48 %) or quadruple tetrads (50 %); the latter morphology being absent in the wild-type strain (Fig. 3). During the exponential phase, ΔcwlB mutant cells also displayed noticeable defects in cell division (Fig. S4); however, these abnormalities were difficult to be classified into distinct morphological forms. Normal cell segregation was restored in the cwlB mutant upon complementation with a plasmid expressing the cwlB gene under the control of its native promoter (Fig. 3). In the double ΔcwlBΔcwlC mutant, the proportion of quadruple tetrads (80 %) increased compared to that observed in the single cwlB mutant (40 %) (Fig. S5). As well as for the single mutant ΔcwlB, the wild-type phenotype was restored when the ΔcwlBΔcwlC double mutant was complemented with a plasmid carrying the cwlB gene (Fig. S6). When CwlB is present, its activity appears sufficient to degrade the peptidoglycan at the septum; in its absence, CwlC may partially compensate for this function, albeit to a limited extent.

Fig. 3.

CwlB is required for the degradation of septa. A. Microscopy analyses of stationary phase cells from wild type, ΔcwlB and ΔcwlB/cwlB⁺mutant strains. The different structures observed are indicated by coloured arrows: dyads in red, tetrads in green, double tetrades in blue and quadruple tetrades in yellow. B The percentage of cells found in dyads (2 cells, red), tetrads (4 cells, green), double tetrads (8 cells, blue) and quadruple tetrads (16 cells, yellow) are illustrated in the pie charts.

The higher-order assemblies of cells observed in the ΔcwlB mutant suggest that, while septation may initiate, it fails to proceed to completion, resulting in incomplete separation of daughter cells. We hypothesized that CwlB may be involved in degrading peptidoglycan at the septum during cell division. To determine the subcellular localization of CwlB, we generated strains expressing CwlB fused to fluorescent tag (NeonGreen). These strains displayed a morphology indistinguishable from the wild-type strain (Fig. S7); however, no clear fluorescent signal was detected.

3.4. Membrane vesicle formation under CwlA depletion conditions

To investigate potential effects of CwlA in cell division, we used the conditional gene inactivation system. Fig. 4 illustrates the impact of CwlA depletion on the cellular morphology, visualized through fluorescent microscopy after 24 h of growth at either 30 °C or 37 °C. At 30 °C, no cells exhibited significant structural abnormalities and extracellular membrane vesicles were not observed under these conditions. In contrast, cells incubated at 37 °C displayed marked morphological alterations. DIC images revealed a subset of cells with irregular shapes and evidence of membrane protrusions (highlighted by blue arrows). The FM4–64 staining clearly showed the presence of extracellular MVs surrounding many of the cells. To better visualise the membrane vesicles, electron microscopy experiments were carried out. Compared to wild-type morphology, CwlA-depleted cells exhibited pronounced production of outer membrane vesicles, seen as numerous spherical structures budding from or surrounding the cellular surface (Fig. 5 and Fig. S8).

Fig. 4.

Formation of membrane vesicles following depletion of CwlA. Cells (∆cwlA/prepUTS::cwlA⁺⁾ in exponential growth phase cultivated at 30 °C in medium supplemented with spectinomycin were harvested by centrifugation, diluted in antibiotic-free medium and incubated at 30 °C (upper part) or at 37 °C (lower part). In each part, the first line contains the pictures of the Nomarski interference contrast (DIC), the second line, the pictures of membrane DNA staining (DAPI) and the third line the pictures of membrane staining (FM4–64). All pictures are the same scale (bar = 5 μm). Membrane vesicles are indicated by blue arrows.

Fig. 5.

Transmission electron microscopy photograph of membrane vesicles in D. radiodurans. Impact of CwlA depletion on D. radiodurans cells. A and C: Cells in exponential growth phase cultivated at 30 °C in medium supplemented with spectinomycin were harvested by centrifugation, diluted in antibiotic-free medium and incubated at 37 °C for 24 h. A1 and A2 -GY 18249 (ΔcwlA/ prepUTS::cwlA⁺); C – GY14164 (ΔddrO/prepUTS::ddrO⁺). B: Membrane vesicle formation in wild type cells in the presence of mitomycin C.

The widespread production of MVs observed upon CwlA depletion reveals a strong association between loss of CwlA and cell envelope alterations. This phenotype is consistent with defects in envelope homeostasis, potentially linked to impaired peptidoglycan remodeling, although a more general envelope stress response cannot be excluded.

The absence of phenotypes such as cellular aggregation or mislocalization of septa during the depletion of CwlA suggests that this endopeptidase is not specifically involved in cell division but rather acts continuously throughout cell development. To support this hypothesis, we constructed a strain expressing a recombinant CwlA fused to a neongreen tag. Easy production of homozygous strains cwlA::neogreen (Fig. S9) and the absence of particular phenotype in terms of growth or cellular morphology (Fig. 6) showed that the addition of neongreen tag did not impact the functionality of the protein. The protein CwlA is homogeneously localized around the cell (Fig. 6). When compared to cells with membranes labeled with FM4–64, septal regions were devoid of fluorescent signal as long as cell separation was not fully completed, suggesting that CwlA is involved throughout cell development and not specifically during septation.

Fig. 6.

Cellular localization of the endopeptidase CwlA. Microscopy images of CwlA-neongreen cells under standard growth conditions. DIC: phase contrast; DAPI: DNA staining; FM4–64: membrane staining.

3.5. Membrane vesicle formation in D. radiodurans following DNA damage by mitomycin C

CwlA depletion leads to the formation of MVs. Previous work has also shown their formation when D. radiodurans cells were subjected to stress (Devigne et al., 2015; Li et al., 2017; Pospíšil et al., 2020; Ott et al., 2020). For instance, a treatment with a sub-lethal dose of mitomycin C (15 µg/ml for 40 min, resulting in a survival of 90 %), a DNA damage compound, led to morphological changes including membrane blebbing (Li et al., 2017). In order to observe the structures of the MVs, transmission electron microscopy (TEM) experiments were carried out. TEM images revealed the formation of numerous membrane vesicles at the cell surface of D. radiodurans following treatment with the DNA-damaging agent mitomycin C (Fig. 5B and Fig. S10). Notably, small and spherical vesicular structures were observed budding from the outer membrane, uniformly distributed around the cells. The emergence of these MVs in response to mitomycin C-induced genotoxic stress suggests a link between DNA damage and cell wall remodeling. In Deinococcus, the main response pathway to genotoxic conditions is regulated by a constitutively expressed metalloprotease IrrE and a transcriptional repressor DdrO. We previously showed that the depletion of DdrO expressed from a heat-sensitive replication plasmid leads to a loss of viability of the strain when cells grow at non permissive temperature and is accompanied by the development of numerous cellular phenotypes: condensation and disrupted nucleoids, DNA fragmentation and budding of MVs (Devigne et al., 2015). Under the same DdrO depletion conditions, the electron microscopy analyses performed here showed that the MVs formed are similar to those observed in the presence of mitomycin or under CwlA depletion conditions (Fig. 5C).

3.6. CwlA is repressed under Ddro depletion conditions

In our previous work (Eugénie et al., 2021), we mapped the DdrO regulon by ChIP-seq and RNA-seq analysis. The transcriptome of D. radiodurans was analysed at 37 °C, during DdrO depletion over a 24 h-kinetic (1 h, 4 h, 6 h, 8 h, 16 h, 24 h) using 1 h as the reference point. Here, differential analysis was designed to highlight genes whose expression level varies at the same time point between the ΔddrO/prepU_TS ddrO⁺ strain developing an apoptotic-like cell death phenotype after a long lasting DdrO depletion (16 h and 24 h) and not in the ΔddrO/ddrO⁺control strain.

Tables 3 and S2 list the most highly expressed genes at 16 h and 24 h, respectively, after DdrO depletion. The prevalence of RDR (radiation/desiccation response) regulon genes among the most overexpressed genes was found at the both time points, with some of them showing high expression levels, such as the ddrA gene. Among the most repressed genes (Tables 4 and S3), cwlA was approximately 18 times less expressed under DdrO depletion conditions at 16 h, being the most down-regulated gene. This repression is likely indirect, since no DdrO-binding site was identified in the cwlA regulatory region and previously performed DdrO ChIP-seq analyses did not detect any interaction peak upstream of cwlA (Eugénie et al., 2021). In contrast, the expression of cwlB and cwlC remained unchanged. Deregulation of cwlA under DdrO depletion may disrupt the structural organization of the membrane (disrupted peptidoglycan formation and recycling, imbalance of membrane components, activation of autolysins), ultimately leading to MV formation. Since DdrO depletion led to a strong reduction in cwlA expression, the MVs observed under these conditions are most likely a consequence of the decreased amount of CwlA.

Table 3.

list of the 50 most upregulated genes at 16 h of DdrO depletion.

ID gene Eugénie et al.	ID gene White	Function	Fold change	P-value
DRO_C0016	DRC0016	Hypothetical protein	483,02	8,43E-229
DRO_0421	DR0423	DdrA	454,33	0
DRO_0003		DdrC	311,39	0
DRO_0070	DR0070	DdrB	138,41	0
DRO_A0342	DRA0346	PprA	132,10	0
DRO_C0011	DRC0012	helix-turn-helix transcriptional regulator, GerE family	117,01	1,87E-162
DRO_C0010		Hypothetical protein	91,64	1,43E-156
DRO_0323	DR0326	DdrD	61,11	0
DRO_1140		Hypothetical protein	41,74	1,37E-83
DRO_2308	DR2338	competence/damage-inducible protein cinA	40,25	2,73E-273
DRO_0899	DR0906	GyrB	35,72	3,88E-223
DRO_2310	DR2340	RecA	27,00	9,52E-150
DRO_1139		Hypothetical protein	26,34	8,08E-79
DRO_0596	DR0596	RuvB	25,27	1,78E-150
DRO_C0021		Hypothetical protein	22,28	2,26E-114
DRO_2249	DR2275	UvrB	21,23	2,39E-194
DRO_A0073	DRA0075	Transposase, putative	16,71	4,04E-79
DRO_A0077	DRA0079	Hypothetical protein	16,03	2,90E-33
DRO_1880	DR1902	Exodeoxyribonuclease V, subunit RecD, putative	15,66	3,12E-93
DRO_2145	DR2174	Leucyl-tRNA ligase	14,83	2,39E-168
DRO_2144	DR2173	Hypothetical protein	13,61	1,82E-62
DRO_1891	DR1913	GyrA	13,50	1,20E-94
DRO_0420	DR0422	Trans-aconitate methyltransferase	13,32	6,50E-127
DRO_2415	DR2441	Acetyltransferase	12,89	2,93E-59
DRO_2251	DR2277	Nickel transporter, permease protein	12,78	3,57E-155
DRO_1899	DR1921	sbcD	12,75	1,57E-81
DRO_A0167	DRA0165	Hypothetical protein	12,68	1,29E-84
DRO_1459	DR1477	RecN	12,56	1,24E-132
DRO_C0022	DRC0024	Hypothetical protein	12,35	3,39E-104
DRO_A0084	DRA0085	Hypothetical protein	12,35	9,78E-27
DRO_C0009	DRB0005	Transposase	12,09	5,84E-83
DRO_0595	DR0595	Hypothetical protein	10,97	1,58E-73
DRO_1279		Hypothetical protein	10,91	9,93E-58
DRO_2346		Hypothetical protein	10,89	3,95E-32
DRO_1900	DR1922	SbcC	10,82	2,04E-52
DRO_1673	DR1696	MutL	10,78	1,58E-72
DRO_A0081	DRA0083	Hypothetical protein	10,65	2,15E-36
DRO_A0080	DRA0082	Hypothetical protein	10,62	1,29E-18
DRO_1879	DR1901	Hypothetical protein	10,55	6,26E-65
DRO_A0082	DRA0084	Hypothetical protein	10,46	7,55E-28
DRO_A0341	DRA0345	Erythromycin esterase	10,35	6,03E-123
DRO_0614	DR0614	Hypothetical protein	10,19	1,23E-91
DRO_1796	DR1817	Phospho-2-dehydro-3-deoxyheptonate aldolase	9,78	NA
DRO_A0085	DRA0086	Hypothetical protein	10,19	1,23E-91
DRO_0438	DR0440	RuvC-like	9,60	2,18E-79
DRO_2142	DR2171	Hypothetical protein	9,58	3,31E-58
DRO_0437	DR0439	Heme biosynthesis protein HemY	9,49	2,05E-69
DRO_2416	DR2442	Acetylglutamate kinase	9,29	2,42E-49
DRO_0820	DR0825	50S ribosomal protein L31	9,19	2,66E-39
DRO_C0008	DRC0010	Hypothetical protein	8,62	3,42E-72

Table 4.

list of the 50 most downregulated genes at 16 h of DdrO depletion.

ID gene Eugénie et al.	ID gene White	Function	Fold change	P-value
DRO_1728	DR1749	Endopeptidase-related protein	−18,02	1,15E-196
DRO_1645	DR1668	Potassium transporter KtrB	−7,78	6,72E-63
DRO_0564	DR0566	Hypothetical protein	−7,00	2,05E-51
DRO_2236	DR2262	Hypothetical protein	−6,50	1,33E-33
DRO_1964	DR1990	Hypothetical protein	−6,50	4,43E-04
DRO_1965	DR1991	Pseudouridine synthase	−5,54	2,01E-36
DRO_0015	DR0014	16S rRNA methyltransferase	−5,41	2,11E-27
DRO_0363	DR0364	Peptide ABC transporter permease	−5,37	4,43E-67
DRO_2032	DR2058	Hypothetical protein	−5,21	5,27E-46
DRO_B0027	DRB0025	Sigma-B regulator RsbS	−5,05	1,05E-45
DRO_1839	DR1862	Translation factor Sua5	−4,81	8,12E-06
DRO_1017	DR1022	Hypothetical protein	−4,76	8,58E-44
DRO_2518	DR2546	Hypothetical protein	−4,72	2,11E-21
DRO_2324	DR2353	L-asparaginase	−4,72	2,77E-31
DRO_0127	DR0128	Nucleotide exchange factor GrpE	−4,46	2,17E-30
DRO_2579	DR2607	Molybdenum cofactor biosynthesis protein Moa	−4,44	4,63E-20
DRO_1963	DR1989	Hypothetical protein	−4,43	4,41E-17
DRO_0465	DR0466	Diguanylate cyclase	−4,42	4,68E-50
DRO_1810	DR1831	Hypothetical protein	−4,36	7,03E-30
DRO_0137	DR0137	Restriction endonuclease	−4,34	4,54E-28
DRO_1439	DR1455	MerR family transcriptional regulator	−4,31	1,11E-21
DRO_0364	DR0365	Peptide ABC transporter permease	−4,31	7,85E-43
DRO_2318	DR2347	Hypothetical protein	−4,28	1,11E-25
DRO_2209	DR2235	Hypothetical protein	−4,25	2,80E-37
DRO_1683	DR1705	Hydrolase	−4,19	7,34E-37
DRO_1958	DR1984	Thymidine kinase	−4,12	1,49E-24
DRO_0391	DR0391	Hypothetical protein	−4,10	2,92E-05
DRO_B0026	DRB0024	Sigma-B regulator RsbR	−4,09	1,11E-30
DRO_B0028	DRB0026	Sigma-B regulator RsbT	−4,09	6,68E-18
DRO_B0055	DRB0053	Dihydroxyacetone kinase subunit DhaK	−4,07	7,21E-26
DRO_0949	DR0956	Phosphoesterase	−4,02	1,60E-16
DRO_1941	DR1967	Enoyl-ACP reductase	−3,96	4,88E-24
DRO_0665	DR0670	Hypothetical protein	−3,91	8,11E-14
DRO_0125	DR0127	Hypothetical protein	−3,86	5,37E-29
DRO_2045	DR2072	Hypothetical protein	−3,82	1,47E-23
DRO_B0054		Hypothetical protein	−3,82	1,02E-16
DRO_1809	DR1830	Hypothetical protein	−3,80	5,61E-28
DRO_1227	DR1231	Hypothetical protein	−3,78	3,53E-26
DRO_1836	DR1858	Permease	−3,73	1,60E-30
DRO_1837		Hypothetical protein	−3,72	1,93E-21
DRO_2238	DR2264	MBL fold metallo-hydrolase	−3,71	1,04E-27
DRO_0799	DR0804	RNA polymerase subunit sigma	−3,68	3,13E-27
DRO_0123	DR0125	Acetyltransferase	−3,66	1,58E-34
DRO_1850	DR1873	Hypothetical protein	−3,65	4,52E-22
DRO_1824	DR1845	Hypothetical protein	−3,62	3,64E-31
DRO_0483	DR0484	Hypothetical protein	−3,61	2,35E-25
DRO_0118	DR0120	DNA processing protein DprA	−3,59	1,63E-09
DRO_1813	DR1834	rRNA (guanine-N2)-methyltransferase	−3,59	4,44E-15
DRO_2117		ABC transporter	−3,57	3,99E-33
DRO_0870	DR0875	Zinc metalloprotease	−3,56	2,09E-29
DRO_1887	DR1909	Lactate utilization protein	−3,55	1,71E-27

4. Discussion

Although D. radiodurans is a Gram-positive bacterium, the organisation of its membrane structure is similar to that of Gram-negative bacteria. Cell wall hydrolases are ubiquitous enzymes that play critical roles in cell division and bacterial enveloppe remodeling. Here, we show that CwlB plays a key role in cell separation in D. radiodurans. In mycobacteria, the NlpC/P60 endopeptidase RipA family is required for septal degradation, and its deletion leads to cells with multiple septa (Chao et al., 2013). Similar phenotypes were observed in Corynebacterium, where deletion of the cg1735, another NlpC/P60 member caused elongated, multiseptated cells (Gaday et al., 2022). The predicted signal peptide at the N-terminal sequence of CwlB suggests an extracytoplasmic, likely periplasmic, localization. Although fluorescence was not detected for the CwlB–NeonGreen fusion protein, the wild-type phenotype of the strain indicates that this fusion protein is functional, suggesting technical limitations such as low protein abundance or inefficient folding or maturation of the fluorophore. The precise subcellular localization of CwlB remains to be determined through a protein isolation approach based on subcellular localization.

Peptidoglycan metabolism and hydrolase activity are tightly regulated throughout the cell cycle to prevent deleterious effects (Vermassen et al., 2019). In D. radiodurans, the DNA damage response gene ddrI encodes a transcriptional regulator belonging to the cAMP receptor protein (CRP) family. These proteins are known to act as positive or negative transcriptional regulators. In silico predictions identified a putative DdrI binding site upstream of the cwlB (dr1325) coding region (Meyer et al., 2018). DdrI is strongly upregulated in stationary-phase cells (Meyer et al., 2018), data confirmed by transcriptomic analyses showing a strong ddrI gene upregulation in stationary phase compared to exponential phase (Eugénie et al., 2021). Cells lacking DdrI exhibit a pleiotropic phenotype, including growth defects, increased sensitivity to DNA-damaging agents, oxidative stress, and heat shock (Yang et al., 2016; Meyer et al., 2018). In addition, DdrI deficiency leads to a significant increase in two-tetrad-forming subpopulations suggesting that DdrI is crucial for accurate completion of cell division, particularly during stationary phase (Meyer et al., 2018). The formation of double tetrads is even more pronounced in the absence of CwlB. Based on these observations, we propose that DdrI activates cwlB expression.

In this study, we demonstrated the essentiality of the cwlA gene. The loss of viability due to the absence of cwlA is quite surprising since the inability to obtain the deletion of a gene encoding an NlpC/P60 endopeptidase has rarely been reported in other bacteria. One of the few examples of essentiality for a hydrolase concerns PcsB in S. pneumoniae, were depletion of PcsB is bacteriostatic and leads to growth arrest, aberrant cell phenotypes and uncontrolled peptidoglycan synthesis (Ng et al., 2004). In Listeria monocytogenes, deletion of the cwhA gene results in abnormal septa and cell filamentation during the exponential phase (Pilgrim et al., 2003). Cell filamentation is also described after inactivation of the lytF gene in Bacillus subtilis, a phenotype exacerbated in the absence of the second peptidase NlpC/P60 LytE (Bisicchia et al., 2007). Only the absence of both LytE and the endopeptidase CwlO is lethal (Bisicchia et al., 2007). Finally, in E. coli, the three endopeptidases Spr, YdhO and YebA are functionally redundant (Singh et al., 2012). The inability to obtain a cwlA deletion mutant in D. radiodurans suggests that, unlike in other bacteria, CwlA activity cannot be replaced by other proteins, despite the presence of two other potential NlpC/P60 endopeptidases, CwlB and CwlC.

We also reported that depletion of CwlA results in extensive formation of membrane vesicles (MVs), a phenotype previously observed under stress conditions, such as exposure to the DNA-damaging agent mitomycin C (Li et al., 2017) or to low earth orbit conditions outside the International Space Station (Ott et al., 2020). Transmission electron microscopy analysis in our study confirmed the presence of numerous spherical vesicles budding from the outer membrane following mitomycin C treatment. These MVs were evenly distributed around the cell surface, indicating a regulated process rather than random membrane disintegration.

Depletion of CwlA and DdrO, two proteins essential for cell viability, induces MV formation. In contrast, MVs were not detected when other essential proteins in D. radiodurans, such as DNA gyrase or HU protein, were depleted (Nguyen et al., 2009). Changes in cell morphology are observed, such as the appearance of anucleate cells in the absence of DNA gyrase or fragmentation of the nucleoid leading to cell lysis upon depletion of HU. The formation of MVs observed during CwlA or DdrO depletion therefore appears to depend on a specific pathway. In Pseudomonas aeruginosa, the formation of MVs has been observed when cells are exposed to ciprofloxacin or mitomycin C and explosive cell lysis is regulated by the RecA-dependent SOS response (Turnbull et al., 2016). DNA damage activates RecA, triggering the SOS response and leading to the production of endolysins that degrade the cell wall. While D. radiodurans exhibits a more controlled vesiculation process rather than explosive lysis, both systems highlight the critical role of DNA damage signaling in modulating cell envelope integrity and membrane vesicle production. In Deinococcus, the main response pathway to genotoxic conditions is regulated by the metalloprotease IrrE and the repressor DdrO (Wang et al., 2015; Blanchard et al., 2017). We show that DdrO depletion results in strong repression of cwlA. Given that loss of CwlA alone is sufficient to induce vesiculation, we propose that its downregulation during DdrO depletion contributes to the observed membrane abnormalities and outer MV formation. However, no RDR binding motif is present upstream of cwlA, suggesting that this effect is indirect. In many Gram-positive bacteria, regulation of peptidoglycan metabolism and the numerous associated hydrolases involves the essential two-component system WalK/WalR (Dubrac et al., 2008). In E. coli, the two-component system CpxR/CpxA regulates several amidases (Weatherspoon-Griffin et al., 2011). The regulation of the cwlA expression and other cell wall hydrolases in D. radiodurans may also involve a two-component regulatory system similar to WalK/WalR. About twenty genes predicted to encode sensor proteins with histidine kinase like domains along with numerous proteins containing a receptor domain are present in D. radiodurans (Makarova and Daly, 2010) but only a few have been studied (Im et al., 2013).

CwlA is distributed around the cells, probably within the periplasm. The presence of an NlpC/P60-type catalytic domain together with a LysM peptidoglycan-binding domain is consistent with a potential role for CwlA in peptidoglycan metabolism. Based on homology with other NlpC/P60 endopeptidases, CwlA may contribute to the hydrolysis of peptide bonds within PG and thus participate in PG remodeling processes associated with cell wall growth and/or turnover (Anantharaman and Aravind, 2003; Vermassen et al., 2019). Although no direct evidence for such a function is provided here, altered peptidoglycan homeostasis resulting from CwlA depletion could weaken the peptidoglycan layer. Given the thickness of the D. radiodurans cell wall, fragile areas could lead to localized breakage points across the entire wall resulting in the formation of bubbles and MVs. Alternatively, membrane vesicule formation could result from the accumulation of PG fragments or misfolded proteins in the periplasm, generating turgor pressure on the outer membrane and promoting vesiculation, as previously proposed (McBroom and Kuehn, 2007; Tashiro et al., 2009). Future experiments will be required to discriminate between these possibilities and to determine whether MV formation is a direct consequence of altered peptidoglycan remodeling or a secondary effect of envelope stress.

Finally, the MazEF toxin-antitoxin system may contribute to stress-induced vesiculation. MazEF mediates programmed cell death (PCD) under severe stress conditions and induces membrane blebbing and MV formation (Li et al., 2017), similar to phenotypes observed during CwlA or DdrO depletion . This suggests a potential interplay between MazEF, DdrO, and CwlA in coordinating stress responses. The convergence of phenotypes associated with CwlA or DdrO depletion, and MazEF activation suggests a regulatory network linking DNA damage response, cell wall homeostasis and PCD enabling D. radiodurans to balance survival and death under extreme conditions.

Beyond structural consequences, MV production may have functional implications. Vesicles are known to carry diverse biomolecules, including proteins, lipids, and nucleic acids, which can participate in stress response, signaling, and horizontal gene transfer. The MVs produced upon CwlA depletion or DNA damage may contain enzymes or signaling molecules involved in envelope remodeling or DNA repair. The overrepresentation of DNA repair proteins has recently been demonstrated in extracellular vesicles produced by Methanobrevibacter smithii (Baquero et al., 2025) suggesting a link between genotoxic stress response and vesiculation. The role of MV production remains to be fully elucidated, but it may serve dual purposes: expelling damaged components to promote survival to the bacterial population or facilitating cell death by disrupting membrane integrity.

CRediT authorship contribution statement

Tim Kamara: Investigation, Visualization, Writing – review & editing. Geoffrey Martinez: Investigation, Visualization, Writing – review & editing. Nicolas Eugénie: Investigation, Data curation. Murielle Dutertre: Resources. Fabrice Confalonieri: Funding acquisition, Data curation, Writing – review & editing. Esma Bentchikou: Conceptualization, Writing – review & editing. Pascale Servant: Conceptualization, Supervision, Writing – original draft, Writing – review & editing.

Declaration of competing interest

All authors have read and approved the final version of the manuscript.

We confirm that this work is not presently submitted to a journal different from Current Research in Microbial Sciences.