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

In diderm bacteria, the outer membrane (OM) must invaginate in step with septal peptidoglycan (PG) remodeling during cytokinesis. One OM lipoprotein, DolP, has been shown to localize at the cell division site and to facilitate the daughter cell separation. Yet how DolP is recruited to the division site remains unclear at the molecular level. Here, we show that DolP arrives 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 a previously underappreciated immobile state enriched at the septal and polar regions. Importantly, DolP does not comigrate with the processive FtsW-FtsI-FtsN complex. Mutations of the cardiolipin-binding surface caused loss of mid-cell enrichment and reduced the immobile fraction. Cardiolipin’s preference for high negative curvature enriches it at the division site, creating a trapping environment for DolP. Additionally, the active septal constriction but not the late divisome proteins recruit DolP. Together, these findings indicate that cardiolipin-mediated immobilization underlies DolP’s septal enrichment.