Salt-Dependent Interactions between the C-Terminal Domain of Osmoregulatory Transporter ProP of Escherichia coli and the Lipid Membrane.

Osmosensing transporter ProP detects the increase in cytoplasmic cation concentration associated with osmotically induced cell dehydration and mediates osmolyte uptake into bacteria. ProP is a 12-transmembrane helix protein with an α-helical, cytoplasmic C-terminal domain (CTD) linked to transmembrane helix XII (TM XII). It has been proposed that the CTD helix associates with the anionic membrane surface to lock ProP in an inactive conformation and that the release of the CTD may activate ProP. To investigate this possible activation mechanism, we have built and simulated a structural model in which the CTD was anchored to the membrane by TM XII and the CTD helix was associated with the membrane surface. Molecular dynamics simulations showed specific intrapeptide salt bridges forming when the CTD associated with the membrane. Experiments supported the presence of the salt bridge Lys447-Asp455 and suggested a role for these residues in osmosensing. Simulations performed at different salt concentrations showed weakened CTD-lipid interactions at 0.25 M KCl and gradual stiffening of the membrane with increasing salinity. These results suggest that salt cations may affect CTD release and activate ProP by increasing the order of membrane phospholipids.

Cultivation at high osmotic pressure confers ubiquinone 8-independent protection of respiration on Escherichia coli.

Ubiquinone 8 (coenzyme Q8 or Q8) mediates electron transfer within the aerobic respiratory chain, mitigates oxidative stress, and contributes to gene expression in Escherichia coli In addition, Q8 was proposed to confer bacterial osmotolerance by accumulating during growth at high osmotic pressure and altering membrane stability. The osmolyte trehalose and membrane lipid cardiolipin accumulate in E. coli cells cultivated at high osmotic pressure. Here, Q8 deficiency impaired E. coli growth at low osmotic pressure and rendered growth osmotically sensitive. The Q8 deficiency impeded cellular O2 uptake and also inhibited the activities of two proton symporters, the osmosensing transporter ProP and the lactose transporter LacY. Q8 supplementation decreased membrane fluidity in liposomes, but did not affect ProP activity in proteoliposomes, which is respiration-independent. Liposomes and proteoliposomes prepared with E. coli lipids were used for these experiments. Similar oxygen uptake rates were observed for bacteria cultivated at low and high osmotic pressures. In contrast, respiration was dramatically inhibited when bacteria grown at the same low osmotic pressure were shifted to high osmotic pressure. Thus, respiration was restored during prolonged growth of E. coli at high osmotic pressure. Of note, bacteria cultivated at low and high osmotic pressures had similar Q8 concentrations. The protection of respiration was neither diminished by cardiolipin deficiency nor conferred by trehalose overproduction during growth at low osmotic pressure, but rather might be achieved by Q8-independent respiratory chain remodeling. We conclude that osmotolerance is conferred through Q8-independent protection of respiration, not by altering physical properties of the membrane.

Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP.

ProP is a member of the major facilitator superfamily, a proton-osmolyte symporter, and an osmosensing transporter. ProP proteins share extended cytoplasmic carboxyl terminal domains (CTDs) implicated in osmosensing. The CTDs of the best characterized, group A ProP orthologs, terminate in sequences that form intermolecular, antiparallel α-helical coiled coils (e.g., ProPEc, from Escherichia coli). Group B orthologs lack that feature (e.g., ProPXc, from Xanthomonas campestris). ProPXc was expressed and characterized in E. coli to further elucidate the role of the coiled coil in osmosensing. The activity of ProPXc was a sigmoid function of the osmolality in cells and proteoliposomes. ProPEc and ProPXc attained similar activities at the same expression level in E. coli. ProPEc transports proline and glycine betaine with comparable high affinities at low osmolality. In contrast, proline weakly inhibited high-affinity glycine-betaine uptake via ProPXc. The KM for proline uptake via ProPEc increases dramatically with the osmolality. The KM for glycine-betaine uptake via ProPXc did not. Thus, ProPXc is an osmosensing transporter, and the C-terminal coiled coil is not essential for osmosensing. The role of CTD-membrane interaction in osmosensing was examined further. As for ProPEc, the ProPXc CTD co-sedimented with liposomes comprising E. coli phospholipid. Molecular dynamics simulations illustrated association of the monomeric ProPEc CTD with the membrane surface. Comparison with the available NMR structure for the homodimeric coiled coil formed by the ProPEc-CTD suggested that membrane association and homodimeric coiled-coil formation by that peptide are mutually exclusive. The membrane fluidity in liposomes comprising E. coli phospholipid decreased with increasing osmolality in the range relevant for ProP activation. These data support the proposal that ProP activates as cellular dehydration increases cytoplasmic cation concentration, releasing the CTD from the membrane surface. For group A orthologs, this also favors α-helical coiled-coil formation that stabilizes the transporter in an active form.