Recruitment of the outer-membrane lipoprotein DolP to the division site via anionic phospholipid-mediated diffusion-state switching

In diderm bacteria, the outer membrane (OM) must invaginate in concert with septal peptidoglycan (PG) remodeling during cytokinesis. One OM lipoprotein, DolP, has been shown to localize at the cell division site and facilitate the daughter cell separation. Yet how DolP is recruited remains unclear at the molecular level. Here, we show that DolP arrives at mid-cell concomitantly with the late divisome protein FtsN. Utilizing single-particle tracking Photoactivated Localization Microscopy (spt-PALM), we investigated the dynamics of individual DolP molecules in living Escherichia coli cells. Single-molecule analysis revealed two diffusion states: a diffusive state across the cell envelope and an immobile state enriched at the septal and polar regions. Because anionic phospholipids are known enriched at regions of high negative curvature, we tested mutations in the DolP anionic phospholipid-binding surface and found they abolished mid-cell enrichment and reduced the immobile fraction. Importantly, DolP's localization is independent of division proteins like EnvC and NlpD, and DolP does not comigrate with the core septal synthesis complex FtsW-FtsI-FtsN complex. Instead, DolP enrichment requires an actively constricting septum. Together, these findings support a model in which anionic phospholipid-mediated diffusion-state switching drives DolP enrichment at the division site.