ProP‐ProP and ProP‐phospholipid interactions determine the subcellular distribution of osmosensing transporter ProP in Escherichia coli

Abstract

Osmosensing transporter ProP protects bacteria from osmotically induced dehydration by mediating the uptake of zwitterionic osmolytes. ProP activity is a sigmoidal function of the osmolality. ProP orthologues share an extended, cytoplasmic C-terminal domain. Orthologues with and without a C-terminal, α-helical coiled-coil domain respond similarly to the osmolality. ProP concentrates at the poles and septa of Escherichia coli cells in a cardiolipin (CL)-dependent manner. The roles of phospholipids and the C-terminal domain in subcellular localization of ProP were explored. Liposome association of peptides representing the C-terminal domains of ProP orthologues and variants in vitro was compared with subcellular localization of the corresponding orthologues and variants in vivo. In the absence of coiled-coil formation, the C-terminal domain bound liposomes and ProP concentrated at the cell poles in a CL-independent manner. The presence of the coiled-coil replaced those phenomena with CL-dependent binding and localization. The effects of amino acid replacements on lipid association of the C-terminal peptide fully recapitulated their effects on the subcellular localization of ProP. These data suggest that polar localization of ProP results from association of its C-terminal domain with the anionic lipid-enriched membrane at the cell poles. The coiled-coil domain present on only some orthologues renders that phenomenon CL-dependent.

Cardiolipin synthase A colocalizes with cardiolipin and osmosensing transporter ProP at the poles of Escherichia coli cells

“…That is, intense green FlAsH fluorescence is concentrated at the poles in approximately one‐half of labelled clsA + cells and less than one‐fifth of labelled clsA – cells expressing MVCCPGCC‐ProP. This clsA ‐dependent ProP localization has been demonstrated by visual inspection of micrographs and by computer‐based analysis of the distribution of fluorescence within cells (Table ; Figs and ) (Romantsov et al ., ; Romantsov et al ., ). More cells are classified as having ProP concentrated at the cell poles by computer‐based image analysis than by visual inspection, but the same dependence on CL is observed with both approaches.…”

“…YjbJ is a small, highly abundant protein of known structure (Pineda‐Lucena et al ., ) and unknown function. ClsA‐mediated CL synthesis had been implicated in ProP localization and function (Romantsov et al ., ; Romantsov et al ., ; Romantsov et al ., ; Romantsov et al ., ).…”

“…FlAsH‐EDT 2 ‐labelled, N‐terminally MVCCPGCC‐tagged ProP concentrates at E. coli cell poles in a CL‐dependent manner (Romantsov et al ., ; Romantsov et al ., ; Romantsov et al ., ; Romantsov et al ., ). That is, intense green FlAsH fluorescence is concentrated at the poles in approximately one‐half of labelled clsA + cells and less than one‐fifth of labelled clsA – cells expressing MVCCPGCC‐ProP.…”

“…Recent data suggest that ProP concentrates at the cell poles because CL headgroups bind to the homodimeric, antiparallel, α‐helical coiled‐coil formed by adjacent ProP monomers (Romantsov et al ., ). Further, direct interaction between ProP and ClsA is possible (Fig.…”

“…Bacteria were cultivated without l ‐arabinose, labelled with fluorescent probe NAO (10‐ N ‐nonyl acridine orange) or FlAsH‐EDT 2 (Fluorescein Arsenical Helix binder, bis‐EDT adduct), and imaged as described previously (Romantsov et al ., ; Romantsov et al ., ). FlAsH fluorescence was detected for approximately 50% of the cells in each population and cells with septa (6–7% of fluorescent cells) were excluded from further analysis.…”