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 and the osmotic stress responses of bacteria

Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a non-electrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls⁻ bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H⁺-lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.     

Characterization of Staphylococcus aureus cardiolipin synthases 1 and 2 and their contribution to accumulation of cardiolipin in stationary phase and within phagocytes

In many bacteria, including Staphylococcus aureus, progression from the logarithmic to the stationary phase is accompanied by conversion of most of bacterial membrane phosphatidylglycerol (PG) to cardiolipin (CL). Phagocytosis of S. aureus by human neutrophils also induces the conversion of most bacterial PG to CL. The genome of all sequenced strains of S. aureus contains two open reading frames (ORFs) predicting proteins encoded with ～30% identity to the principal CL synthase (cls) of Escherichia coli. To test whether these ORFs (cls1 and cls2) encode cardiolipin synthases and contribute to CL accumulation in S. aureus, we expressed these proteins in a cls strain of E. coli and created isogenic single and double mutants in S. aureus. The expression of either Cls1 or Cls2 in CL-deficient E. coli resulted in CL accumulation in the stationary phase. S. aureus with deletion of both cls1 and cls2 showed no detectable CL accumulation in the stationary phase or after phagocytosis by neutrophils. CL accumulation in the stationary phase was due almost solely to Cls2, whereas both Cls1 and Cls2 contributed to CL accumulation following phagocytosis by neutrophils. Differences in the relative contributions of Cls1 and Cls2 to CL accumulation under different triggering conditions suggest differences in the role and regulation of these two enzymes.     

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.     

The osmotic activation of transporter ProP is tuned by both its C-terminal coiled-coil and osmotically induced changes in phospholipid composition

Transporter ProP of Escherichia coli (ProPEc) senses extracellular osmolality and mediates osmoprotectant uptake when it is rising or high. A replica of the ProPEc C terminus (Asp468-Arg497) forms an intermolecular alpha-helical coiled-coil. This structure is implicated in the osmoregulation of intact ProPEc, in vivo. Like that from Corynebacterium glutamicum (ProPCg), the ProP orthologue from Agrobacterium tumefaciens (ProPAt) sensed and responded to extracellular osmolality after expression in E. coli. The osmotic activation profiles of all three orthologues depended on the osmolality of the bacterial growth medium, the osmolality required for activation rising as the growth osmolality approached 0.7 mol/kg. Thus, each could undergo osmotic adaptation. The proportion of cardiolipin in a polar lipid extract from E. coli increased with extracellular osmolality so that the osmolality activating ProPEc was a direct function of membrane cardiolipin content. Group A ProP orthologues (ProPEc, ProPAt) share the C-terminal coiled-coil domain and were activated at low osmolalities. Like variant ProPEc-R488I, in which the C-terminal coiled-coil is disrupted, ProPEc derivatives that lack the coiled-coil and Group B orthologue ProPCg required a higher osmolality to activate. The amplitude of ProPEc activation was reduced 10-fold in its deletion derivatives. The coiled-coil structure is not essential for osmotic activation of ProP per se. However, it tunes Group A orthologues to osmoregulate over a low osmolality range. Coiled-coil lesions may impair both coiled-coil formation and interaction of ProPEc with amplifier protein ProQ. Cardiolipin may contribute to ProP adaptation by altering bulk membrane properties or by acting as a ProP ligand.     

Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP,LacY, ProW,AqpZ, MscS,and MscL

Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).Modern developments in fluorescence microscopy have led to a new understanding of the organization of bacterial cells, particularly protein and lipid localization (21, 56). Analysis of the subcellular localization of diverse proteins and lipids has shown that they are not uniformly distributed. The phospholipid cardiolipin (CL) localizes at the poles and septal regions (36), and there is evidence for segregation of phosphatidylethanolamine (PE) from phosphatidylglycerol (PG) in the membranes of living Escherichia coli cells (69). Localization of many proteins that are integral or peripheral to the cytoplasmic membrane has been studied by fusing them to green fluorescent protein (GFP) (or its derivatives), and it is possible to classify the fusion proteins according to their subcellular localization. The first group, comprised of proteins that are concentrated at the cell poles, includes chemoreceptors (31, 62), the lactose permease LacY (43), and the metabolic sensor kinases DcuS and CitA (55). Members of the second group form helices that extend from pole to pole and include MreB (25), MinD (57), the Sec protein export system (58), and RNase E, which is the main component of the RNA degradosome in E. coli (67). Other proteins may appear to be similarly distributed due to their association with the Sec system (58). Members of the third group are uniformly distributed and include the mechanosensitive channel MscL (45) and the sensor kinase KdpD (32).The polar localization of proteins appears to be a critical feature of the complicated internal localization of bacteria. For example, it is important for temporally and spatially accurate placement of the septum during cell division (15). However, the mechanism of protein organization at bacterial cell poles is still unclear, and in many cases its functional role has not been determined. Do the poles merely serve as a receptacle for proteins, superstructures, or membrane domains with no functional effects, or is this location functionally important for membrane proteins and lipids?Recent evidence indicates that the subcellular localization of the transporter ProP in E. coli is related to membrane phospholipid composition, cardiolipin localization, and ProP function (51, 52). E. coli cells from cultures grown to exponential phase contain mostly the zwitterionic phospholipid PE (approximately 75 mol%) and the anionic phospholipids PG (approximately 20 mol%) and CL (approximately 5 mol%) (8). (Note that cardiolipin is diphosphatidylglycerol.) However, the phospholipid composition depends on the bacterial growth conditions. We found that the proportion of CL among E. coli lipids varies directly with growth medium osmolality (68), and increased CL synthesis was at least partially attributed to regulation of the cls locus encoding cardiolipin synthase (52). There is residual CL in cls bacteria, indicating that there is an alternative pathway for CL synthesis (51). The CL-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to show that CL clusters at the poles and septa in growing E. coli cells (36, 52). This result was corroborated by analyzing the phospholipid composition of E. coli minicells (DNA-free cells resulting from asymmetric cell division) (24, 51).ProP is an osmosensory transporter that senses increasing osmolality and responds by mediating the cytoplasmic accumulation of organic osmolytes (e.g., proline, glycine betaine, and ectoine). Biochemical regulation of the ProP protein ensures that ProP activity increases with increasing assay medium osmolality (49). We showed that ProP and CL colocalize at the poles and near the septa of dividing E. coli cells and that the polar concentration of ProP correlates with the polar concentration of CL (52). Moreover, we showed that the osmolality required to activate ProP increased in parallel to the CL content when E. coli was cultivated in media with increasing osmolality (51, 52, 68). The osmolality required to activate ProP was also a direct function of CL content in proteoliposomes reconstituted with purified ProP (51). We concluded that concentration at the cell poles controlled the osmoregulatory function of ProP by placing the transporter in a cardiolipin-rich environment.To determine whether CL-dependent membrane protein localization is a general phenomenon in E. coli, we compared the subcellular localization of ProP with that of its paralogue LacY, a well-characterized lactose transporter (16). LacY and ProP are both members of the major facilitator superfamily and H⁺ symporters. LacY transports the nutrient lactose, and LacY activity decreases while ProP activity increases with increasing osmolality (9). Nagamori et al. reported polar localization of a LacY-GFP fusion protein in E. coli (43). We confirmed this observation and demonstrated that, in contrast to the behavior of ProP, the polar concentration of LacY did not correlate with the polar concentration of CL (51).In this work we further explored the relationship between CL and protein localization in E. coli. We compared ProP with other proteins related to cellular osmoregulation. Bacteria use arrays of osmoregulatory mechanisms to survive and function when the osmotic pressure of their environment changes. In E. coli, the aquaporin AqpZ mediates transmembrane water flux, the transporters ProP, ProU, BetT, and BetU mediate organic osmolyte accumulation at high osmotic pressure, and the mechanosensitive (MS) channels MscL and MscS mediate solute efflux in response to osmotic downshock (71). Localization of these proteins might be expected since AqpZ might influence cell morphology changes by accelerating water flux at particular positions on the cell surface and the pressure sensitivities of MscL and MscS are known to depend on membrane curvature in vitro (18).For ProP and LacY, we labeled the inserted peptide tag CCPGCC with the biarsenical fluorescein reagent FlAsH-EDT₂ (fluorescein arsenical helix binder, bis-EDT adduct) (1, 2) to examine the subcellular localization of AqpZ, the integral membrane component ProW of the osmoregulatory ATP-binding cassette (ABC) transporter ProU, and the MS channel proteins MscS and MscL in cls⁺ and cls bacteria. Fluorescence microscopy was used to determine the proportion of cells with labeled protein concentrated at the poles as a function of bacterial CL content and protein expression level. For ProP, the frequency with which MscS was concentrated at cell poles was proportional to the level and polar concentration of CL. LacY concentrated at the cell poles at a high and CL-independent frequency. The frequencies with which AqpZ, MscL, and ProW concentrated at the cell poles and septa were low (up to 12%) and CL independent.     

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.     

Osmosensor ProP of Escherichia coli responds to the concentration,chemistry, and molecular size of osmolytes in the proteoliposome lumen

Transporter ProP of Escherichia coli mediates the cellular accumulation of organic zwitterions in response to increased extracellular osmolality. We compared and characterized the osmoregulation of ProP activity in cells and proteoliposomes to define the osmotic shift-induced cellular change(s) to which ProP responds. ProP-(His)(6) activity in cells and proteoliposomes was correlated with medium osmolality, not osmotic shift, turgor pressure, or membrane strain. Both K(M) and V(max) for proline uptake via ProP-(His)(6) increased with increasing medium osmolality, as would be expected if osmolality controls the proportions of transporter with inactive and active conformations. The osmolality yielding half-maximal ProP-(His)(6) activity was higher in proteoliposomes than in cells. The osmolality response of ProP is also attenuated in bacteria lacking soluble protein ProQ. Indeed, the catalytic constant (k(cat)) for ProP-(His)(6) in proteoliposomes approximated that of ProP in intact bacteria lacking ProQ. Thus, the proteoliposome system may replicate a primary osmosensory response that can be further amplified by ProQ. ProP-(His)(6) is designated as an osmosensor because its activity is dependent on the osmolality, but not the composition, of the assay medium to which the cell surface is exposed. In contrast, ProP-(His)(6) activity was dependent on both the osmolality and the composition of the lumen in osmolyte-loaded proteoliposomes. For proteoliposomes containing inorganic salts, glucose, or poly(ethylene glycol) 503, transporter activity correlated with total lumenal cation concentration. In contrast, for proteoliposomes loaded with larger poly(ethylene glycol)s, the osmolality, the lumenal cation concentration, and the lumenal ionic strength at half-maximal transporter activity decreased systematically with poly(ethylene glycol) radius of gyration (range 0.8-1.8 nm). These data suggest that ProP-(His)(6) responds to osmotically induced changes in both cytoplasmic K(+) levels and the concentration of cytoplasmic macromolecules.     

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.     

Localization of Anionic Phospholipids in Escherichia coli Cells

Cardiolipin (CL) is an anionic phospholipid with a characteristically large curvature and is of growing interest for two primary reasons: (i) it binds to and regulates many peripheral membrane proteins in bacteria and mitochondria, and (ii) it is distributed asymmetrically in rod-shaped cells and is concentrated at the poles and division septum. Despite the growing number of studies of CL, its function in bacteria remains unknown. 10-N-Nonyl acridine orange (NAO) is widely used to image CL in bacteria and mitochondria, as its interaction with CL is reported to produce a characteristic red-shifted fluorescence emission. Using a suite of biophysical techniques, we quantitatively studied the interaction of NAO with anionic phospholipids under physiologically relevant conditions. We found that NAO is promiscuous in its binding and has photophysical properties that are largely insensitive to the structure of diverse anionic phospholipids to which it binds. Being unable to rely solely on NAO to characterize the localization of CL in Escherichia coli cells, we instead used quantitative fluorescence microscopy, mass spectrometry, and mutants deficient in specific classes of anionic phospholipids. We found CL and phosphatidylglycerol (PG) concentrated in the polar regions of E. coli cell membranes; depletion of CL by genetic approaches increased the concentration of PG at the poles. Previous studies suggested that some CL-binding proteins also have a high affinity for PG and display a pattern of cellular localization that is not influenced by depletion of CL. Framed within the context of these previous experiments, our results suggest that PG may play an essential role in bacterial physiology by maintaining the anionic character of polar membranes.     

Osmosensing and osmoregulatory compatible solute accumulation by bacteria

Bacteria inhabit natural and artificial environments with diverse and fluctuating osmolalities, salinities and temperatures. Many maintain cytoplasmic hydration, growth and survival most effectively by accumulating kosmotropic organic solutes (compatible solutes) when medium osmolality is high or temperature is low (above freezing). They release these solutes into their environment when the medium osmolality drops. Solutes accumulate either by synthesis or by transport from the extracellular medium. Responses to growth in high osmolality medium, including biosynthetic accumulation of trehalose, also protect Salmonella typhimurium from heat shock. Osmotically regulated transporters and mechanosensitive channels modulate cytoplasmic solute levels in Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Lactobacillus plantarum, Lactococcus lactis, Listeria monocytogenes and Salmonella typhimurium. Each organism harbours multiple osmoregulatory transporters with overlapping substrate specificities. Membrane proteins that can act as both osmosensors and osmoregulatory transporters have been identified (secondary transporters ProP of E. coli and BetP of C. glutamicum as well as ABC transporter OpuA of L. lactis). The molecular bases for the modulation of gene expression and transport activity by temperature and medium osmolality are under intensive investigation with emphasis on the role of the membrane as an antenna for osmo- and/or thermosensors.     

Solution structure of the C-terminal antiparallel coiled-coil domain from Escherichia coli osmosensor ProP

Bacteria respond to increasing medium osmolality by accumulating organic solutes that are compatible with cellular functions. Transporter ProP of Escherichia coli, a proton symporter and a member of the major facilitator superfamily, senses osmotic shifts and responds by importing osmolytes such as glycine betaine. ProP contains a cytoplasmic, C-terminal extension that is essential for its activity. A peptide corresponding to the C-terminal extension of ProP forms a homodimeric alpha-helical coiled-coil even though some of its heptad a positions are not occupied by hydrophobic amino acid residues. Unexpectedly, amino acid replacement R488I, occurring at a heptad a position, destabilized the coiled-coil formed by the ProP peptide and attenuated the response of the intact transporter to osmotic upshifts in vivo. Thus, ProP was proposed to dimerize via an antiparallel coiled-coil. We used nuclear magnetic resonance (NMR) spectroscopy to determine the structure of the synthetic peptide corresponding to residues 468-497 of ProP. This region did form an antiparallel coil-coil in which critical residue R488 specifies the antiparallel coiled-coil orientation by forming stabilizing salt-bridges. Charged residues (both acidic and basic) are clustered on the c/g surface of the coiled-coil whereas polar residues are distributed on the b/e surface. This causes the structure to be bent, in contrast to other known antiparallel coiled-coils (those from the hepatitis delta antigen (PDB ID code 1A92) and the bovine F(1) ATPase inhibitor protein (PDB ID code 1HF9)). The coiled-coil and its possible importance for osmosensing are discussed.     

Formation of an antiparallel, intermolecular coiled coil is associated with in vivo dimerization of osmosensor and osmoprotectant transporter ProP in Escherichia coli

Membrane transporter ProP from Escherichia coli senses extracellular osmolality and responds by mediating the uptake of osmoprotectants such as glycine betaine when osmolality is high. Earlier EPR and NMR studies showed that a peptide replica of the cytoplasmic ProP carboxyl terminus (residues D468-R497) forms a homodimeric, antiparallel, alpha-helical coiled coil in vitro stabilized by electrostatic interactions involving R488. Amino acid replacement R488I disrupted coiled-coil formation by the ProP peptide, elevated the osmolality at which ProP became active, and rendered the osmolality response of ProP transient. In the present study, either E480 or K473 was replaced with cysteine (Cys) in ProP, a Cys-less, fully functional, histidine-tagged ProP variant, to use Cys-specific cross-linking approaches to determine if antiparallel coiled-coil formation and dimerization of the intact protein occur in vivo. The Cys at positions 480 would be closer in an antiparallel dimer than those at positions 473. These replacements did not disrupt coiled-coil formation by the ProP peptide. Partial homodimerization of variant ProP-E480C could be demonstrated in vivo and in membrane preparations via Cys-specific cross-linking with dithiobis(maleimidoethane) or by Cys oxidation to cystine by copper phenanthroline. In contrast, these reagents did not cross-link ProP with Cys at position 133 or 241. Cross-linking of ProP with Cys at position 473 was limited and occurred only if ProP was overexpressed, consistent with an antiparallel orientation of the coiled coil in the intact protein in vivo. Although replacement E480C did not alter transporter activity, replacement K473C reduced the extent and elevated the threshold for osmotic activation. K473 may play a role in ProP structure and function that is not reflected in altered coiled-coil formation by the corresponding peptide. Substitution R488I affected the activities of ProP-(His)(6), ProP-E480C, and ProP-K473C as it affected the activity of ProP. Surprisingly, it did not eliminate cross-linking of Cys at position 480, and it elevated cross-linking at position 473, even when ProP was expressed at physiological levels. This suggested that the R488I substitution may have changed the relative orientation of the C-termini within the dimeric protein from antiparallel to parallel, resulting in only transient osmotic activation. These results suggest that ProP is in monomer-dimer equilibrium in vivo. Dimerization may be mediated by C-terminal coiled-coil formation and/or by interactions between other structural domains, which in turn facilitate C-terminal coiled-coil formation. Antiparallel coiled-coil formation is required for activation of ProP at low osmolality.     

Isolation,purification and characterization of Cardiolipin synthase from Mycobacterium phlei {PRIVATE}

It has been observed that mycobacterial species has high content of cardiolipin (CL) in their cell membranes more so pathogenic mycobacteria and in bacteria CL activates polymerases, gyrases by removing the bound ADP. Therefore, in the present study cardiolipin synthase (cls) which catalyses the formation of CL was isolated purified and characterized from the cell membrane of Mycobacterium phlei. The purified cls obtained from C-18 RP-HPLC column had a molecular weight of 58 kDa with an isoelectric point of 4.5. The enzyme activity (11.5+0.15 µM of CL phosphorous. ml-1 minute-1 for PG as substrate and 14+0.35µM of CL phosphorous. ml-1 minute-1 for CDP-DG as substrate) was optimal at pH 4.8 and showed K_M values of 55+0.05µM and 2.56+0.04µM for phosphatidyl glycerol and CDP-diacylglycerol, respectively, with an absolute requirement of Mg²⁺ and Mn²⁺ ions for its activity however, Ca²⁺ ions inhibited the activity of the cls. The partial amino acid sequence of cls showed significant homology with pgsA3 gene of M. tuberculosis and in this organism the CL biosynthesis is very high having three genes coding for PLs biosynthesis therefore, enzymes involved in CL biosynthesis may be an attractive drug target in the development of new antimycobacterial drugs.     

Creation of a fully functional cysteine-less variant of osmosensor and proton-osmoprotectant symporter ProP from Escherichia coli and its application to assess the transporter's membrane orientation

Transporter ProP of Escherichia coli is an osmosensor and an osmoprotectant transporter. Previous results suggest that medium osmolality determines the proportions of ProP in active and inactive conformations. A cysteine-less (Cys-less) variant was created and characterized as a basis for structural and functional analyses based on site-directed Cys substitution and chemical labeling of ProP. Parameters describing the osmosensory and osmoprotectant transport activities of Cys-less ProP-(His)(6) variants were examined, including the threshold for osmotic activation and the absolute transporter activity at high osmolality (in both cells and proteoliposomes), the dependence of K(M) and V(max) for proline uptake on osmolality, and the rate constant for transporter activation in response to an osmotic upshift (in cells only). Variant ProP-(His)(6)-C112A-C133A-C264V-C367A (designated ProP) retained similar activities to ProP-(His)(6) in both cells and proteoliposomes. The bulky Val residue was favored over Ala or Ser at position 264, whereas Val strongly impaired function when placed at position 367, highlighting the importance of residues at those positions for osmosensing. In the ProP* background, variants with a single Cys residue at positions 112, 133, 241, 264, 293, or 367 retained full function. The native Cys at positions 112, 133, 264, and 367, predicted to be within transmembrane segments of ProP, were poorly reactive with membrane-impermeant thiol reagents. The reactivities of Cys at positions 241 and 293 were consistent with exposure of those residues on the cytoplasmic and periplasmic surfaces of the cytoplasmic membrane, respectively. These observations are consistent with the topology and orientation of ProP predicted by hydropathy analysis.     

The role of the carboxyl terminal alpha-helical coiled-coil domain in osmosensing by transporter ProP of Escherichia coli

Concentrative uptake of osmoprotectants via transporter ProP contributes to the rehydration of Escherichia coli cells that encounter high osmolality media. A member of the major facilitator superfamily, ProP is activated by osmotic upshifts in whole bacteria, in cytoplasmic membrane vesicles and in proteoliposomes prepared with the purified protein. Soluble protein ProQ is also required for full osmotic activation of ProP in vivo. ProP is differentiated from structural and functional homologues by its osmotic activation and its C-terminal extension, which is predicted to form an alpha-helical coiled-coil. A synthetic polypeptide corresponding to the C-terminus of ProP (ProP-p) formed a dimeric alpha-helical coiled-coil. A derivative of transporter ProP lacking 26 C-terminal amino acids was expressed but inactive. A derivative harbouring amino acid changes K460I, Y467I and H495I (each at the core, coiled-coil 'a' position) required a larger osmotic upshift for activation than did the wild type transporter. The same changes extended, stabilized and altered the oligomeric state of the coiled-coil formed by ProP-p. Amino acid change R488I (also at the 'a' position) further increased the magnitude of the osmotic upshift required to activate ProP, reduced the activity attained and rendered ProP activation transient. Unexpectedly, replacement R488I destabilized the coiled-coil formed by ProP-p. The activity and osmotic activation of ProP were even more strongly attenuated by helix-destabilizing change I474P. These data demonstrate that the carboxyl terminal domain of ProP can form a homodimeric alpha-helical coiled-coil with unusual properties. They implicate the C-terminal domain in the osmotic activation of ProP.     

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.     

Genetic regulation of cardiolipin synthase in Escherichia coli.

Recombinant DNA and genetic techniques were used to construct Escherichia coli strains SOH92 [phi(cls-lacZ+)] and SOH93 [phi(cls-'lacZ)hyb]. beta-Galactosidase (116 kDa) synthesized by strain SOH92 was primarily present in the particulate fraction. Strain SOH92 produced about 20-fold more beta-galactosidase activity than strain SOH93. Expression of clsphilacZ in both SOH92 and SOH93 was influenced by the terminal electron acceptor (increasing in the order oxygen, nitrate, fumarate) when the cells were cultured in minimal medium with glycerol as the sole carbon source. As strains SOH92 and SOH93 progressed from early to late log growth phase under aerobic conditions in LB broth, clsphilacZ expression increased about 2.5-fold. Fusion strains containing a pss-1 allele had an increased cardiolipin (CL) level, but no corresponding increase in clsphilacZ expression was observed. A cls::Tn10dTet null mutation was introduced into SOH92 and SOH93. The strains produced less CL, but no corresponding changes in clsphilacZ expression were observed. A high copy number plasmid bearing the cls gene had no effect on clsphilacZ expression. Taken together, these results indicate that cls is not subject to autogenous regulation.