Structural Determinants and Functional Significance of Dimerization for Osmosensing Transporter ProP in Escherichia coli.

Osmosensing transporter ProP forestalls cellular dehydration by detecting environments with high osmotic pressure and mediating the accumulation of organic osmolytes by bacterial cells. It is composed of 12 transmembrane helices with cytoplasmic N- and C-termini. In Escherichia coli, dimers form when the C-terminal domains of ProP molecules form homodimeric, antiparallel, α-helical coiled coils. No dominant negative effect was detected when inactive and active ProP molecules formed heterodimers in vivo. Purification of ProP in detergent dodecylmaltoside yielded monomers, which were functional after reconstitution in proteoliposomes. With other evidence, this suggests that ProP monomers function independently whether in the monomeric or dimeric state. Amino acid replacements that disrupted or reversed the coiled coil did not prevent in vivo dimerization of ProP detected with a bacterial two-hybrid system. Maleimide labeling detected no osmolality-dependent variation in the reactivities of cysteine residues introduced to transmembrane helix (TM) XII. In contrast, coarse-grained molecular dynamic simulations detected deformation of the lipid around TMs III and VI, on the lipid-exposed protein surface opposite to TM XII. This suggests that the dimer interface of ProP includes the surfaces of TMs III and VI, not of TM XII as previously suggested by crosslinking data. Homology modeling suggested that coiled-coil formation and dimerization via such an interface are not mutually exclusive. In previous work, alterations to the C-terminal coiled coil blocked co-localization of ProP with phospholipid cardiolipin at E. coli cell poles. Thus, dimerization may contribute to ProP targeting, adjust its lipid environment, and hence indirectly modify its osmotic stress response.

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.

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

Osmosensing by transporter ProP is modulated by its cardiolipin (CL)-dependent concentration at the poles of Escherichia coli cells. Other contributors to this phenomenon were sought with the BACterial Two-Hybrid System (BACTH). The BACTH-tagged variants T18-ProP and T25-ProP retained ProP function and localization. Their interaction confirmed the ProP homo-dimerization previously established by protein crosslinking. YdhP, YjbJ and ClsA were prominent among the putative ProP interactors identified by the BACTH system. The functions of YdhP and YjbJ are unknown, although YjbJ is an abundant, osmotically induced, soluble protein. ClsA (CL Synthase A) had been shown to determine ProP localization by mediating CL synthesis. Unlike a deletion of clsA, deletion of ydhP or yjbJ had no effect on ProP localization or function. All three proteins were concentrated at the cell poles, but only ClsA localization was CL-dependent. ClsA was shown to be N-terminally processed and membrane-anchored, with dual, cytoplasmic, catalytic domains. Active site amino acid replacements (H224A plus H404A) inactivated ClsA and compromised ProP localization. YdhP and YjbJ may be ClsA effectors, and interactions of YdhP, YjbJ and ClsA with ProP may reflect their colocalization at the cell poles. Targeted CL synthesis may contribute to the polar localization of CL, ClsA and ProP.

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

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.

Contributions of Coulombic and Hofmeister Effects to the Osmotic Activation of Escherichia coli Transporter ProP.

Osmosensing transporters mediate osmolyte accumulation to forestall cellular dehydration as the extracellular osmolality increases. ProP is a bacterial osmolyte-H(+) symporter, a major facilitator superfamily member, and a paradigm for osmosensing. ProP activity is a sigmoid function of the osmolality. It is determined by the osmolality, not the magnitude or direction of the osmotic shift, in cells and salt-loaded proteoliposomes. The activation threshold varies directly with the proportion of anionic phospholipid in cells and proteoliposomes. The osmosensory mechanism was probed by varying the salt composition and concentration outside and inside proteoliposomes. Data analysis was based on the hypothesis that the fraction of maximal transporter activity at a particular luminal salt concentration reflects the proportion of ProP molecules in an active conformation. ProP attained the same activity at the same osmolality when diverse, membrane-impermeant salts were added to the external medium. Contributions of Coulombic and/or Hofmeister salt effects to ProP activation were examined by varying the luminal salt cation (K(+) and Na(+)) and anion (chloride, phosphate, and sulfate) composition and then systematically increasing the luminal salt concentration by increasing the external osmolality. ProP activity increased with the sixth power of the univalent cation concentration, independent of the type of anion. This indicates that salt activation of ProP is a Coulombic, cation effect resulting from salt cation accumulation and not site-specific cation binding. Possible origins of this Coulombic effect include folding or assembly of anionic cytoplasmic ProP domains, an increase in local membrane surface charge density, and/or the juxtaposition of anionic protein and membrane surfaces during activation.

YehZYXW of Escherichia coli Is a Low-Affinity, Non-Osmoregulatory Betaine-Specific ABC Transporter.

Transporter-mediated osmolyte accumulation stimulates the growth of Escherichia coli in high-osmolality environments. YehZYXW was predicted to be an osmoregulatory transporter because (1) osmotic and stationary phase induction of yehZYXW is mediated by RpoS, (2) the Yeh proteins are homologous to the components of known osmoregulatory ABC transporters (e.g., ProU of E. coli), and (3) YehZ models based on the structures of periplasmic betaine-binding proteins suggested that YehZ retains key betaine-binding residues. The betaines choline-O-sulfate, glycine betaine, and dimethylsulfoniopropionate bound YehZ and ProX with millimolar and micromolar affinities, respectively, as determined by equilibrium dialysis and isothermal titration calorimetry. The crystal structure of the YehZ apoprotein, determined at 1.5 Å resolution (PDB ID: 4WEP ), confirmed its similarity to other betaine-binding proteins. Small and nonpolar residues in the hinge region of YehZ (e.g., Gly223) pack more closely than the corresponding residues in ProX, stabilizing the apoprotein. Betaines bound YehZ-Gly223Ser an order of magnitude more tightly than YehZ, suggesting that weak substrate binding in YehZ is at least partially due to apo state stabilization. Neither ProX nor YehZ bound proline. Assays based on osmoprotection or proline auxotrophy failed to detect YehZYXW-mediated uptake of proline, betaines, or other osmolytes. However, transport assays revealed low-affinity glycine betaine uptake, mediated by YehZYXW, that was inhibited at high salinity. Thus, YehZYXW is a betaine transporter that shares substrate specificity, but not an osmoregulatory function, with homologues like E. coli ProU. Other work suggests that yehZYXW may be an antivirulence locus whose expression promotes persistent, asymptomatic bacterial infection.

Analysis of strains lacking known osmolyte accumulation mechanisms reveals contributions of osmolytes and transporters to protection against abiotic stress.

Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K(+) glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high- and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.

Salinity-dependent impacts of ProQ, Prc, and Spr deficiencies on Escherichia coli cell structure.

ProQ is a cytoplasmic protein with RNA chaperone activities that reside in FinO- and Hfq-like domains. Lesions at proQ decrease the level of the osmoregulatory glycine betaine transporter ProP. Lesions at proQ eliminated ProQ and Prc, the periplasmic protease encoded by the downstream gene prc. They dramatically slowed the growth of Escherichia coli populations and altered the morphologies of E. coli cells in high-salinity medium. ProQ and Prc deficiencies were associated with different phenotypes. ProQ-deficient bacteria were elongated unless glycine betaine was provided. High-salinity cultures of Prc-deficient bacteria included spherical cells with an enlarged periplasm and an eccentric nucleoid. The nucleoid-containing compartment was bounded by the cytoplasmic membrane and peptidoglycan. This phenotype was not evident in bacteria cultivated at low or moderate salinity, nor was it associated with murein lipoprotein (Lpp) deficiency, and it differed from those elicited by the MreB inhibitor A-22 or the FtsI inhibitor aztreonam at low or high salinity. It was suppressed by deletion of spr, which encodes one of three murein hydrolases that are redundantly essential for enlargement of the murein sacculus. Prc deficiency may alter bacterial morphology by impairing control of Spr activity at high salinity. ProQ and Prc deficiencies lowered the ProP activity of bacteria cultivated at moderate salinity by approximately 70% and 30%, respectively, but did not affect other osmoregulatory functions. The effects of ProQ and Prc deficiencies on ProP activity are indirect, reflecting their roles in the maintenance of cell structure.

Impacts of the osmolality and the lumenal ionic strength on osmosensory transporter ProP in proteoliposomes.

H(+) symporter ProP serves as a paradigm for the study of osmosensing. ProP attains the same activity at the same osmolality when the medium outside cells or proteoliposomes is supplemented with diverse, membrane-impermeant solutes. The osmosensory mechanism of ProP has been probed by varying the solvent within membrane vesicles and proteoliposomes. ProP activation was not ion specific, did not require K(+), and could be elicited by large, uncharged solutes polyethylene glycols (PEGS). We hypothesized that ProP is an ionic strength sensor and lumenal macromolecules activate ProP by altering ion activities. The attainable range of lumenal ionic strength was expanded by lowering the phosphate concentration within proteoliposomes. ProP activity at high osmolality, but not the osmolality, yielding half-maximal activity (Π(1/2)/RT), decreased with the lumenal phosphate concentration. This was attributed to acidification of the proteoliposome lumen due to H(+)-proline symport. The ionic strength yielding half-maximal ProP activity was more anion-dependent than Π(1/2)/RT for proteoliposomes loaded with citrate, sulfate, phosphate, chloride, or iodide. The anion effects followed the Hofmeister series. Lumenal bovine serum albumin (BSA) lowered the lumenal ionic strength at which ProP became active. Osmolality measurements documented the non-idealities of solutions including potassium phosphate and other solutes. The impacts of PEGS and BSA on ion activities did not account for their impacts on ProP activity. The effects of the tested solutes on ProP appear to be non-coulombic in nature. They may arise from effects of preferential interactions and macromolecular crowding on the membrane or on ProP.

ProQ is an RNA chaperone that controls ProP levels in Escherichia coli.

Transporter ProP mediates osmolyte accumulation in Escherichia coli cells exposed to high osmolality media. The cytoplasmic ProQ protein amplifies ProP activity by an unknown mechanism. The N- and C-terminal domains of ProQ are predicted to be structurally similar to known RNA chaperone proteins FinO and Hfq from E. coli. Here we demonstrate that ProQ is an RNA chaperone, binding RNA and facilitating both RNA strand exchange and RNA duplexing. Experiments performed with the isolated ProQ domains showed that the FinO-like domain serves as a high-affinity RNA-binding domain, whereas the Hfq-like domain is largely responsible for RNA strand exchange and duplexing. These data suggest that ProQ may regulate ProP production. Transcription of proP proceeds from RpoD- and RpoS-dependent promoters. Lesions at proQ affected ProP levels in an osmolality- and growth phase-dependent manner, decreasing ProP levels when proP was expressed from its own chromosomal promoters or from a heterologous plasmid-based promoter. Small RNA molecules are known to regulate cellular levels of sigma factor RpoS. ProQ did not act by changing RpoS levels since proQ lesions did not influence RpoS-dependent stationary phase thermotolerance and they affected ProP production and activity similarly in bacteria without and with an rpoS defect. Taken together, these results suggest that ProQ does not regulate proP transcription. It may act as an RNA-binding protein to regulate proP translation.

Transmembrane helix I and periplasmic loop 1 of Escherichia coli ProP are involved in osmosensing and osmoprotectant transport.

Osmoregulatory transporters stimulate bacterial growth by mediating osmoprotectant uptake in response to increasing osmotic pressure. The ProP protein of Escherichia coli transports proline and other osmoprotectants. Like LacY, ProP is a member of the major facilitator superfamily and a H(+)-solute symporter. ProP is regulated by osmotic pressure via a membrane potential-dependent mechanism. A homology model predicts that ionizable and polar residues, highly conserved among ProP homologues, cluster deep within the N-terminal helix bundle of ProP. Chemical labeling of introduced cysteine (Cys) residues supported the homology model by confirming the predicted positions of transmembrane helix I (TMI) and periplasmic loop 1. Replacements of residues in the putative polar cluster impaired or altered ProP function, suggesting that they are important for osmosensing and may interact with the transport substrates. Asn34, Glu37, Phe41, Tyr44, and Ala48 line the most polar face of TMI; Tyr44 is on the periplasmic side of the putative polar cluster, and Ala59 is in periplasmic loop 1. The N-ethylmaleimide reactivities of Cys introduced at positions 41, 44, 48, and 59 increased with osmotic pressure, whereas the reactivities of those at cytoplasm-proximal positions 34 and 37 did not. Replacements of polar cluster residues that blocked transport also affected the NEM reactivity of Cys44 and its osmolality dependence. This report and previous work suggest that conformational changes associated with osmosensing may shift the equilibria between outward- and inward-facing transport pathway intermediates.

Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality.

ProP is an osmosensory transporter. The activities of ProP and ProP*, a cysteine-less, His(6)-tagged ProP variant, increase with osmotic pressure in cells and proteoliposomes. In proteoliposomes, ProP activity is osmolality-dependent only if the magnitude of the membrane potential (DeltaPsi) exceeds 100 mV. Some amino acid replacements rendered ProP activity osmolality-insensitive [e.g., Y44M in transmembrane segment 1 (TMI); S62C in periplasmic loop 1 (loop P1)], whereas others elevated the osmolality at which ProP activates (e.g., A59C). This suggested that the environments and/or conformations of TMI and loop P1 might be osmolality-dependent. This report correlates structural dynamics of ProP with osmoregulation of its transport activity. Residues in periplasmic loops were replaced with Cys, and changes in their environments were detected by monitoring their reactivities with N-ethylmaleimide (NEM). Increasing osmolality markedly increased the NEM reactivity of some Cys residues (e.g., C59, loop P1; C415-C418, loop P6) but not others (e.g., C293, loop P4; C348, loop P5). The NEM reactivity of C62 was insensitive to osmolality, as expected. Substitution Y44M rendered the transport activities of ProP*-A59C and ProP*-Q415C, and the NEM reactivities of the introduced Cys, osmolality-insensitive. Furthermore, osmolality did not affect the reactivity of C59 in cells lacking a protonmotive force, consistent with evidence that DeltaPsi is required for osmosensing by ProP. These results indicate that the osmotically induced increases in NEM reactivity of C59 and C415 in energized bacteria are due to a conformational change of ProP in response to osmolality. They therefore constitute the first direct evidence of an osmotically induced conformational change associated with osmosensing by a transporter.

Roles of K+, H+, H2O, and DeltaPsi in solute transport mediated by major facilitator superfamily members ProP and LacY.

H (+)-solute symporters ProP and LacY are members of the major facilitator superfamily. ProP mediates osmoprotectant (e.g., proline) accumulation, whereas LacY transports the nutrient lactose. The roles of K (+), H (+), H 2O, and DeltaPsi in H (+)-proline and H (+)-lactose symport were compared using right-side-out cytoplasmic membrane vesicles (MVs) from bacteria expressing both transporters and proteoliposomes (PRLs) reconstituted with pure ProP-His 6. ProP activity increased as LacY activity decreased when osmotic stress (increasing osmolality) was imposed on MVs. The activities of both transporters decreased to similar extents when Na (+) replaced K (+) in MV preparations. Thus, K (+) did not specifically control ProP activity. As with LacY, an increasing extravesicular pH stimulated ProP-mediated proline efflux much more than ProP-mediated proline exchange from de-energized MVs. In contrast to that of LacY, ProP-mediated exchange was only 2-fold faster than ProP-mediated efflux and was inhibited by respiration. In the absence of the protonmotive force (Deltamu H (+) ), efflux of lactose from MVs was much more sensitive to increasing osmolality than lactose exchange. Thus, H 2O may be directly involved in proton transport via LacY. In the absence of Deltamu H (+) , proline efflux and exchange from MVs were osmolality-independent. In PRLs with a DeltapH of 1 (lumen alkaline), ProP-His 6 was inactive when the membrane potential (DeltaPsi) was zero, was active but insensitive to osmolality when DeltaPsi was -100 mV, and became osmolality-sensitive as DeltaPsi increased further to -137 mV. ProP-His 6 had the same membrane orientation in PRLs as in cells and MVs. ProP switches among "off", "on", and "osmolality-sensitive" states as the membrane potential increases. Kinetic parameters determined in the absence of Deltamu H (+) represent a ProP population that is predominantly off.

Cardiolipin controls the osmotic stress response and the subcellular location of transporter ProP in Escherichia coli.

The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiled-coil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.

Cardiolipin promotes polar localization of osmosensory transporter ProP in Escherichia coli.

The osmolality required to activate osmosensory transporter ProP and the proportion of cardiolipin (CL) among the phospholipids of Escherichia coli rise with growth medium osmolality. Most CL synthesis has been attributed to the cls gene product. Transcription of cls increased with osmolality. The proportion of CL was low and osmolality-independent in cls(-) bacteria. It increased more dramatically on the transition to stationary phase in cls(-) than cls(+) bacteria. Thus, Cls is responsible for osmoregulated CL synthesis and other enzymes may contribute to CL accumulation during stationary phase. The proportion of phosphatidylglycerol (PG) was elevated and it increased with medium osmolality in cls(-) bacteria. A cls defect impaired growth of E. coli on solid and in liquid media at low and, more strongly, at high osmolality. Bacteria cultured at high osmolality without osmoprotectant were shorter and rounder than those cultured at low osmolality or with glycine betaine. Fluorescence microscopy showed that CL and ProP colocalize at the poles and near the septa of dividing E. coli cells. The polar localization of ProP was independent of its expression level but correlated with the proportion and polar localization of CL. Association with CL (and not PG) may be required for polar ProP localization.

Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli.

Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted structure of ProP, reveal the dimerization interface, and show that the structure of TM XII influences the osmolality at which ProP activates.