# CozEs Control Membrane Homeostasis During Sporulation in Bacillus subtilis

Paprapach Wongdontree, Dimitri Juillot, Charlène Cornilleau, Arnaud

Chastanet, Rut Carballido-López

► To cite this version:

Paprapach Wongdontree, Dimitri Juillot, Charlène Cornilleau, Arnaud Chastanet, Rut Carballido-López. CozEs Control Membrane Homeostasis During Sporulation in Bacillus subtilis. 2025 Great Wall Symposium, Sep 2025, Catania, Italy. hal-05304697

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Submitted on 8 Oct 2025

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Distributed under a Creative Commons Attribution 4.0 International License
# CozEs Control Membrane Homeostasis During
Sporulation in *Bacillus subtilis*

<figure><img src="image_2.png" /></figure>

Paprapach Wongdontree, Dimitri Juillot, Charlène Cornilleau, Arnaud Chastanet, Rut Carballido-López

Micalis Institute, INRAe, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France.

@paprapach.bsky.social & @carballido-lab.bsky.social

## INTRODUCTION

Sporulation is a critical survival strategy that enables bacteria to withstand stress conditions such as heat, nutrient deprivation, and antibiotics. While spore formation has been extensively studied, the regulation of membrane organization during this process remains poorly understood. CozE proteins are essential for cell size and cell division in the non-spore-forming bacteria *Streptococcus pneumoniae* and *Staphylococcus aureus*, but their role in spore-forming bacteria has not been explored. In *Bacillus subtilis*, five CozE homologs were identified in our laboratory. Deletion of all homologs appears to be lethal. Under standard laboratory growth conditions, inactivation of four homologs (*Δ4cozE*) does not impair growth or cell size of vegetatively growing cells, but causes cell shape defect and severe lysis during sporulation. Our results suggest that inactivation of CozEs impairs the ability to maintain membrane integrity during sporulation. Remarkably, in *Δ4cozE* cells, anionic phospholipid mislocalizes and is not properly transferred from the mother cell to the forespore, ultimately blocking successful spore formation. Our findings identify CozE proteins as key factors in membrane dynamics and cell fate determination during sporulation.

**Keywords**: CozEs, cardiolipin, phosphatidylglycerol, lipid, membrane, sporulation, *Bacillus subtilis*

## RESULTS

### Sporulation defects of Δ4cozE mutants

<figure><img src="image_3.png" /><figcaption>*Δ4cozE* cells display cell shape defects and lysis during sporulation.</figcaption></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

* Deletion of *cozE* proteins impairs cell length regulation during sporulation. At T2, the average length of *Δ4cozE* cells is the same than during vegetative growth (T0), while the length of WT sporulating cells reduced from T2. Impaired cell division likely contributes to the increased cell length [1]. During sporulation, some of *Δ4cozE* cells show spherical shapes (black arrow).

* During sporulation induction by the resuspension method [2,3], *Δ4cozE* undergoes extensive cell lysis (white arrows), indicating that CozE is important for successful sporulation.

<figure><img src="image_6.png" /><figcaption>*Δ4cozE* cells have delayed entry into sporulation.</figcaption></figure>

* Spo0A is a master sporulation regulator which is activated by phosphorylation via a phosphorelay [4].

* WT cells initiate Spo0A induction at T2, whereas *Δ4cozE* shows only weak Spo0A signaling. At T3, Spo0A induction is detectable in a subset of *Δ4cozE* cells but many of them remains lower than WT, indicating delayed sporulation.

## SUMMARY & CONCLUSION

* Our results show that **CozE** homologs play a critical role in **B. subtilis** during sporulation.

* We propose that **CozE** proteins act as novel regulators of membrane remodeling, ensuring proper allocation of lipids from the mother cell to the forespore.

### Membrane defects of Δ4cozE mutants

<figure><img src="image_7.png" /><figcaption>CozE inactivation disrupts membrane homeostasis during sporulation.</figcaption></figure>

Membrane was labelled by FM4-64 dye

* Membrane staining revealed that sporulating **Δ4cozE** cells exhibit **membrane accumulation** at the cell poles and septum (white arrows).

* TEM images confirm lipid accumulation in *Δ4cozE* during sporulation, consistent with the fluorescence microscopy results.

* Notably, **Δ4cozE** cells exhibit **mispositioned and incomplete septa** (black arrows) and **membrane invaginations** (green arrow) in the septal region, suggesting that aberrant lipid production and/or organization may impair proper cell division.

<figure><img src="image_8.png" /></figure>

* Total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) [5] showed that the **mother cell** of *Δ4cozE* cells lose the **ability to maintain membrane fluidity** during sporulation (T2).

* These observations suggest that **CozE** proteins may play an **important role in maintaining membrane homeostasis** during sporulation.

<figure><img src="image_9.png" /><figcaption>CozE inactivation leads to anionic phospholipid mislocalization during sporulation.</figcaption></figure>

* The main lipids in spores are phosphatidylglycerol (PG) and cardiolipin (CL), which are anionic phospholipids [6]. NAO is not a CL-specific dye but rather binds to anionic phospholipids [7].

* **Δ4cozE** cells show **mislocalization of anionic phospholipids** during sporulation, as observed using NAO.

* These results suggest that **CozE** proteins are involved in the proper localization and **mobilization** of at least anionic **phospholipids** from the mother cell to the forespore.

## REFFERENCES

[1] Gulsoy, I.C., Saaki, T.N.V., Wenzel, M., Syvertsson, S., Morimoto, T., Siersma, T.K., and Hamoen, L.W. (2025) Minimization of the *Bacillus subtilis* divisome suggests FtsZ and SepF can form an active Z-ring, and reveals the amino acid transporter BraB as a new cell division influencing factor. *PLoS Genet* **21**: e1011567

[2] Sterlini, J.M., and Mandelstam, J. (1969) Commitment to sporulation in *Bacillus subtilis* and its relationship to development of actinomycin resistance. *Biochemical Journal* **113**: 29-37.

[3] Lablaine, A., Juillot, D., Condon, C., and Carballido-López, R. (2025) Real-Time Nanoscale Investigation of Spore Coat Assembly in *Bacillus subtilis*. *Communications biology* **8**

[4] Chastanet, A., Vitkup, D., Yuan, G.-C., Norman, T.M., Liu, J.S., and Losick, R.M. (2010) Broadly heterogeneous activation of the master regulator for sporulation in *Bacillus subtilis*. *Proc Natl Acad Sci USA* **107**: 8486-8491.

[5] Barbotin, A., Billaudieu, C., Sezgin, E., and Carballido-López, R. (2024) Quantification of membrane fluidity in bacteria using TIR-FCS. *Biophysical Journal* **123**: 2484-2495.

[6] Kawai, F., Hara, H., Takamatsu, H., Watabe, K., and Matsumoto, K. (2006) Cardiolipin Enrichment in Spore Membranes and Its Involvement in Germination of *Bacillus subtilis* Marburg. *Genes Genet Syst* **81**: 69-76.

[7] Pogmore, A.-R., Seistrup, K.H., and Strahl, H. (2018) The Gram-positive model organism *Bacillus subtilis* does not form microscopically detectable cardiolipin-specific lipid domains. *Microbiology* **164**: 475-482.

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