Figure 5. Phospholipid binding is required for DolP recruitment to division sites.

(A) Fluorescence microscopy of ΔdolP cells expressing either DolP^WT::mCherry or DolP^W127E::mCherry from the pET17b plasmid after growth to mid-exponential phase (OD₆₀₀ ~0.4–0.8). Scale bars represent 2 μM and both phase contrast and the mCherry channel are shown in greyscale and red respectively. White arrows highlight division site localisation of DolP^WT-mCherry. Demographic representations of the DolP^WT-mCherry or DolP^W127E-mCherry fluorescence intensities measure along the medial axis of the cells. Images of >500 cells were analysed using the MicrobeJ software and sorted according to length where the y-axis represents relative cellular position with 0 being mid-cell and 3 or −3 being the cell poles (Ducret et al., 2016). (B) Thin layer chromatography of phospholipids extracted from either E. coli BW25113 (WT), ΔrcsFΔlpp, ΔrcsFΔlppΔpgsA (referred to as ΔpgsA) or ΔclsAΔclsBΔclsC (referred to as ΔclsABC) strains. The rcsF and lpp genes must be removed in order to prevent toxic build-up of Lpp on the IM in the pgsA mutant. Phospholipids were separated using chloroform:methanol:acetic acid (65:25:10) as the mobile phase before staining with phophomolybdic acid and charring.( C) Fluorescence microscopy of ΔpgsA or ΔclsABC cells expressing DolP^WTmCherry from the pET17b plasmid after growth to mid-exponential phase (OD₆₀₀ ~0.4–0.8). White arrows highlight DolP-mCherry mislocalisation.