The function of CozE proteins is linked to lipoteichoic acid biosynthesis in Staphylococcus aureus.

Coordinated membrane and cell wall synthesis is vital for maintaining cell integrity and facilitating cell division in bacteria. However, the molecular mechanisms that underpin such coordination are poorly understood. Here we uncover the pivotal roles of the staphylococcal proteins CozEa and CozEb, members of a conserved family of membrane proteins previously implicated in bacterial cell division, in the biosynthesis of lipoteichoic acids (LTA) and maintenance of membrane homeostasis in Staphylococcus aureus. We establish that there is a synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc <sub>2</sub> DAG. By contrast, in cells lacking LtaA, the flippase of Glc <sub>2</sub> DAG, the essentiality of CozE proteins was alleviated, suggesting that the function of CozE proteins is linked to the synthesis and flipping of the glycolipid anchor. CozE proteins were indeed found to modulate the flipping activity of LtaA in vitro. Furthermore, CozEb was shown to control LTA polymer length and stability. Together, these findings establish CozE proteins as novel players in membrane homeostasis and LTA biosynthesis in S. aureus.IMPORTANCELipoteichoic acids are major constituents of the cell wall of Gram-positive bacteria. These anionic polymers are important virulence factors and modulators of antibiotic susceptibility in the important pathogen Staphylococcus aureus. They are also critical for maintaining cell integrity and facilitating proper cell division. In this work, we discover that a family of membrane proteins named CozE is involved in the biosynthesis of lipoteichoic acids (LTAs) in S. aureus. CozE proteins have previously been shown to affect bacterial cell division, but we here show that these proteins affect LTA length and stability, as well as the flipping of glycolipids between membrane leaflets. This new mechanism of LTA control may thus have implications for the virulence and antibiotic susceptibility of S. aureus

CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus.

Staphylococcus aureus needs to control the position and timing of cell division and cell wall synthesis to maintain its spherical shape. We identified two membrane proteins, named CozEa and CozEb, which together are important for proper cell division in S. aureus. CozEa and CozEb are homologs of the cell elongation regulator CozE <sup>Spn</sup> of Streptococcus pneumoniae. While cozEa and cozEb were not essential individually, the ΔcozEaΔcozEb double mutant was lethal. To study the functions of cozEa and cozEb, we constructed a CRISPR interference (CRISPRi) system for S. aureus, allowing transcriptional knockdown of essential genes. CRISPRi knockdown of cozEa in the ΔcozEb strain (and vice versa) causes cell morphological defects and aberrant nucleoid staining, showing that cozEa and cozEb have overlapping functions and are important for normal cell division. We found that CozEa and CozEb interact with and possibly influence localization of the cell division protein EzrA. Furthermore, the CozE-EzrA interaction is conserved in S. pneumoniae, and cell division is mislocalized in cozE <sup>Spn</sup> -depleted S. pneumoniae cells. Together, our results show that CozE proteins mediate control of cell division in S. aureus and S. pneumoniae, likely via interactions with key cell division proteins such as EzrA