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

Abstract

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 a-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 K M for proline uptake via ProPEc increases dramatically with the osmolality. The K M 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 a-helical coiled-coil formation that stabilizes the transporter in an active form.

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration onEscherichia coli

“…The osmoadaptive mechanism that protects respiration may be related to the mechanism by which osmotic upshifts inhibit respiration. By measuring DPH fluorescence anisotropy, Cul-ham et al (5) showed that osmotic upshifts decreased the membrane fluidity of liposomes comprised of E. coli phospholipid. Such a decrease may render the intramembrane diffusion of electron carriers rate-limiting for respiration, and osmotic adaptation of the respiratory system may be designed to address that limitation.…”

“…Following extrusion, liposomes (in rehydration buffer) were supplemented with 0.06% DPH (w/w, relative to lipid; ϳ0.2 mol%) (Molecular Probes, Inc., Eugene, OR) and incubated for 18 -20 h in the dark at room temperature. Fluorescence measurements were performed at the specified temperature using a PTI QuantaMaster QM-8 steady state fluorimeter as described (5). Duplicate measurements from two independent experiments were combined and analyzed as described (5).…”

“…Fluorescence measurements were performed at the specified temperature using a PTI QuantaMaster QM-8 steady state fluorimeter as described (5). Duplicate measurements from two independent experiments were combined and analyzed as described (5). The data were analyzed by nonlinear regression with the polynomial quadratic function using SigmaPlot 12.5.…”

“…For example, osmosensing transporters activate to mediate osmolyte uptake as other transporters inactivate at high osmotic pressure. Evidence suggests that osmotically induced changes to physical properties of the cytoplasmic membrane contribute to the activation of osmosensing transporters (5)(6)(7). However, the biochemical basis for the inhibition and subsequent restoration of other energy linked functions is not understood.…”

Exploiting Substrate Promiscuity of Ectoine Hydroxylase for Regio- and Stereoselective Modification of Homoectoine

“…Escherichia coli possesses two osmotically regulated uptake systems for a variety of compatible solutes: the proton-solute symporter ProP, a member of the major facilitator superfamily (MFS) and the ABC-type (ATP-binding cassette) transporter ProU (Lucht and Bremer, 1994; Wood et al, 2001). Both transporters can also serve as uptake systems for ectoine and 5-hydroxyectoine (Jebbar et al, 1992; Czech et al, 2016; Culham et al, 2018). For our studies on the osmostress protective properties of homoectoine (Nagata, 2001), we used the well-known E. coli K-12 laboratory strain MG1655 (Blattner et al, 1997) and an isogenic set of mutant strains derived from MG1655 with defects in either ProP or ProU, or in both transport systems (Supplementary Table S1).…”