Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts

We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes.     

SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.

Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids. We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin. These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase. We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity. These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure. Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.     

Anthrylvinyl-labeled phospholipids as fluorescent membrane probes. The action of melittin on mulitlipid systems

The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.     

Head group-independent interaction of phospholipids with bile salts. A fluorescence and EPR study

Bile salts are essential for phospholipid secretion into the bile. To study the relevance of the structure of phospholipids for their interaction with bile salts, we used spin-labeled or fluorescent phospholipid analogues of different head groups and acyl chain length. Those analogues form micelles in aqueous suspension. Their solubilization by bile salts resulting in the formation of mixed micelles was followed by the decrease of spin-spin interaction of spin-labeled analogues or by the relief of fluorescence self-quenching of (7-nitro-2-1,3-benzooxadiazol (NBD))-labeled analogues. Solubilization of analogue micelles occurred at and above the critical micellar concentration (CMC) of the bile salts. As revealed by stopped-flow technique, solubilization of NBD-analogues was very rapid with half times as low as 0.1 sec above the CMC of taurocholate. Both kinetics and extent of solubilization were independent of the phospholipid head group, but were significantly affected by the fatty acid chain length. Furthermore, using vesicles with varying phospholipid composition and different types of analogues in self-quenching concentrations, we could show that bile salt-mediated vesicle solubilization depended on the fatty acid chain length of phospholipids. In contrast, neither for phospholipids nor for analogues could an influence of the lipid head group on the solubilization process be observed. These findings support a head group-independent mechanism of bile salt-mediated enrichment of specific phospholipids in the bile fluid.     

Relation of Cellular Phospholipid Composition to Oligodendroglial Differentiation in C-6 Glial Cells

The relation of the polar head group composition of cellular phospholipids to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Induction of the oligodendroglial enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), was determined after alteration of the polar head group composition of phospholipids by exposure of the cells to choline analogues, especially N,N'-dimethylethanolamine. To accomplish the phospholipid alteration, cells were grown in the presence of the analogue in medium free of exogenous lipid, i.e., first for 24 h in 10% delipidated serum and then for 48 h in serum-free medium. The 48-h exposure to serum-free medium resulted in untreated C-6 cells in a several fold increase in CNP activity, but in cells treated with 2.5 mM N,N'-dimethylethanolamine, total inhibition of this induction was observed. A graded, concentration-dependent inhibitory effect of the analogue on the induction of CNP was defined. The effect of the analogue was relatively specific, e.g., the activity of another plasma membrane enzyme of C-6 cells, (Na+ + K+)-activated ATPase, was not affected. Morever, there was no evidence of a toxic effect of the analogue; thus, total protein synthesis and cell growth were not altered, and the induction of CNP in serum-free medium recurred after removal of the analogue. N,N'-Dimethylethanolamine was shown to be incorporated into cellular phospholipids, primarily at the expense of phosphatidylcholine. The data define an important role for the polar head group composition of membrane phospholipids in oligodendroglial differentiation in this model system.     

Aminophospholipid translocase of human erythrocytes: phospholipid substrate specificity and effect of cholesterol

The outside-inside translocation rate and transmembrane equilibrium distribution, at 37 degrees C, of 16 different amphiphilic spin-labeled phospholipids have been determined in human erythrocytes. The transmembrane distribution was assessed by bovine serum albumin extraction of the spin-labels present in the outer monolayer. Within 15 min, more than 90% of the phosphatidylserine analogue was found in the inner monolayer; the equilibrium distribution of phosphatidylethanolamine spin-label was approximately 85-90% inside, with a half-time for translocation of approximately 50 min. In contrast, phosphatidylcholine reached a distribution corresponding to approximately 30% of the labels inside with a half-time of approximately 8 h, and only traces of sphingomyelin were found in the inner monolayer after 16 h. Thus, the spin-label analogues distributed themselves like endogenous phospholipids in red cells with a spontaneous segregation between the amino lipids and the choline-containing phospholipids. Progressive methylation of the amine group of phosphatidylethanolamine resulted in a stepwise decrease of the specific transport; modification of the beta-carbon of the serine also decreased the efficiency of the rapid translocation without abolishing it. Phosphatidyl-propanolamine was not transported. Substitution of the glyceride group by a ceramide abolished the rapid outside-inside translocation even with a molecule bearing a serine head group. Also it was found that esterification of the sn-2 position of the glycerol component was necessary for a rapid translocation since lysophosphatidylserine was only slowly transported from outside to inside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid flop induced by transmembrane peptides in model membranes is modulated by lipid composition

Since phospholipid synthesis is generally confined to one leaflet of a membrane, membrane growth requires phospholipid translocation (flip-flop). It is generally assumed that this process is protein-mediated; however, the mechanism of flip-flop remains elusive. Previously, we have demonstrated flop of 2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl] (C6NBD) phospholipids, induced by the presence of membrane-spanning peptides in vesicles composed of an Escherichia coli phospholipid extract, supporting the hypothesis that the presence of transmembrane stretches of proteins in the bilayer is sufficient to allow phospholipid flip-flop in the inner membrane of E. coli [Kol et al. (2001) Biochemistry 40, 10500]. Here, we investigated whether the specific phospholipid composition of E. coli is a prerequisite for transmembrane helix-induced flop of phospholipids. This was tested by determining the amount of C6NBD-phospholipid that was translocated from the inner leaflet to the outer leaflet of a model membrane in time, using a dithionite reduction assay. The transmembrane peptides GWWL(AL)8WWA (WALP23) and GKKL(AL)8KKA (KALP23) induced phospholipid flop in model membranes composed of various lipid mixtures. The rate of peptide-induced flop was found to decrease with increasing dioleoylphosphatidylethanolamine (DOPE) content of vesicles composed of DOPE and dioleoylphosphatidylcholine (DOPC), and the rate of KALP23-induced flop was shown to be stimulated by higher dioleoylphosphatidylglycerol (DOPG) content in model membranes composed of DOPG and DOPC. Furthermore, the incorporation of cholesterol had an inhibitory effect on peptide-induced flop. Finally, flop efficiency was strongly dependent on the phospholipid headgroup of the NBD-phospholipid analogue. Possible implications for transmembrane helix-induced flop in biomembranes in general are discussed.     

Transmembrane transport of peptidoglycan precursors across model and bacterial membranes

Translocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced by the presence of single spanning helical transmembrane peptides that facilitate transbilayer movement of membrane phospholipids. MurG catalysed synthesis of Lipid II from Lipid I in lipid vesicles also did not result in membrane translocation of Lipid II. These findings demonstrate that a specialized protein machinery is needed for transmembrane movement of Lipid II. In line with this, we could demonstrate Lipid II translocation in isolated Escherichia coli inner membrane vesicles and this transport could be uncoupled from the synthesis of Lipid II at low temperatures. The transport process appeared to be independent from an energy source (ATP or proton motive force). Additionally, our studies indicate that translocation of Lipid II is coupled to transglycosylation activity on the periplasmic side of the inner membrane.     

Novel alk-1-enyl ether lipids isolated from Clostridium acetobutylicum

Alkenyl ether analogues of phosphatidylglycerol (plasmenylglycerol), bisphosphatidylglycerol (cardiolipin) (plasmenylglycerolphosphatidic acid), monoglycosyldiglyceride and diglycosyldiglyceride were isolated from the polar lipids of Clostridium acetobutylicum and characterized by chemical analyses and degradation. The position of the alkenyl ether bond (at C-1) and of the acyl ester bond (at C-2) as well as the configuration at C-2 of the phospholipids are the same as of the alkenyl ether phospholipids known so far. The alkenyl ether analogue of monoglycosyldiglyceride contains a galactosyl residue, that of diglycosyldiglyceride a glucosyl-galactosyl residue, glucosyl forming the terminal unit.     

Protein-mediated phospholipid translocation in the endoplasmic reticulum with a low lipid specificity

The outside-inside translocation rate of various amphiphilic spin-labeled phospholipids has been measured in rat liver endoplasmic reticulum vesicles. The eight spin-labels tested experienced a fast flip-flop rate with the same half-time of approximately 20 min at 37 degrees C. The stationary distribution of these phospholipid analogues was ca. 45% on the inner vesicular leaflet and 55% on the external one, showing that there is no net enrichment of some lipid in one layer under the experimental conditions used. The initial rate of translocation was reduced 4-fold if membranes were preincubated with N-ethylmaleimide (2 mM) and was about an order of magnitude lower in liposomes made from the extracted lipids. An apparent saturability of the transbilayer diffusion can be deduced from the variation of the initial velocity of the relocation kinetics vs the amount of analogue incorporated in the membrane. Moreover, translocation rates of two different spin-labeled phospholipids introduced simultaneously in the membrane were almost equally reduced by the presence of the other lipid. On the other hand, no competition between the water-soluble dibutyroylphosphatidylcholine and the amphiphilic spin-labeled phospholipids could be detected. Overall, these results suggest that phospholipid translocation in the endoplasmic reticulum is a protein-mediated process with a low specificity, which tends, in the absence of any other metabolic event, to equilibrate the phospholipid composition of the two membrane halves.     

Negatively charged phospholipids restore prePhoE translocation across phosphatidylglycerol-depleted Escherichia coli inner membranes

Translocation of outer membrane precursor proteins across the Escherichia coli inner membrane is severely hampered in lipid biosynthetic mutants with strongly reduced phosphatidylglycerol (PG) levels (De Vrije, T., De Swart, R. L., Dowhan, W., Tommassen, J., and De Kruijff, B. (1988) Nature 334, 173-175; Lill, R., Dowhan, W., and Wickner, W. (1990) Cell 60, 271-280). Two independent methods were used to demonstrate that anionic lipids by virtue of their negative head-group charge are involved in membrane translocation of the precursor of the pore protein PhoE. Using a lipid transfer protein-based method we show that introduction from lipid vesicles of PG and other acidic phospholipids but not of phosphatidylcholine restores efficient translocation across the membrane of PG-depleted inner membrane vesicles. Moreover, translocation was found to be proportional to the PG content in vesicles isolated from strain HDL11 in which the PG content was altered by varying the synthesis of the PG-phosphate synthase.     

Interactions of the low molecular weight group of surfactant-associated proteins (SP 5-18) with pulmonary surfactant lipids

The interaction of the low molecular weight group of surfactant-associated proteins, SP 5-18, with the major phospholipids of pulmonary surfactant was studied by fluorescence measurements of liposomal permeability and fusion, morphological studies, and surface activity measurements. The ability of SP 5-18 to increase the permeability of large unilamellar lipid vesicles was enhanced by the presence of negatively charged phospholipid. The permeability of these vesicles increased as the protein concentration was raised and the pH was lowered. SP 5-18 also induced leakage from liposomes made both from a synthetic surfactant lipid mixture and from lipids separated from SP 5-18 during its purification from canine sources. When SP 5-18 was added to egg phosphatidylglycerol liposomes, the population of liposomes which became permeable leaked all encapsulated contents, while the remaining liposomes did not leak at all. The extent of leakage was higher in the presence of 3 mM calcium. SP 5-18 also induced lipid mixing between two populations of egg phosphatidylglycerol liposomes in the presence of 3 mM calcium, as monitored by resonance energy transfer between two different fluorescent lipid probes, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine. Negative-staining electron microscopy showed that the addition of SP 5-18 and 3 mM calcium produced vesicles twice the size of control egg phosphatidylglycerol liposomes. In addition, surface balance measurements revealed that the adsorption of liposomal lipids to an air/water interface was enhanced by the presence of SP 5-18, negatively charged phospholipids, and 3 mM calcium. These observations suggest a similar lipid dependence for the interactions observed in the fluorescence and adsorption experiments.     

Phospholipid flip-out controls the cell cycle of Escherichia coli

Phospholipids are the principal constituents of biological membranes. In Escherichia coli, phospholipids are involved in the metabolism of other envelope constituents such as lipoprotein, lipopolysaccharide, certain envelope proteins and peptidoglycan. They are also involved in the regulation of the cell cycle. DNAA, the key protein in the initiation of chromosome replication, is activated by acidic phospholipids only when these are in fluid bilayers, whilst interruptions of phospholipid synthesis inhibit both the initiation of chromosome replication and cell division. The transmembrane movement or flip-flop of phospholipids from one monolayer to the other requires the passage of the polar head group through the hydrophobic core of the bilayer. Hence, in many systems, flip-flop is a slow process with half-time of days. Flip-flop accompanies the formation of non-bilayer structure. Such structures form under certain conditions of packing density and composition and have been observed both in vitro and in vivo. In bacteria, flip-flop appears to be extremely rapid, with half-times as fast as 3 min being observed. However, such rapid flip-flop may not be characteristic of all phospholipids. The asymmetrical distribution of phosphatidylethanolamine in the plasma membrane of Bacillus megaterium has been attributed to the existence of two classes of this phospholipid. In E. coli, studies of the metabolic turnover of phosphatidylserine, phosphatidylglycerol and phosphatidic acid also reveal the existence of distinct classes of these phospholipids. In this article I propose that, in E. coli, a class of phospholipids does indeed escape the rapid flip-flop mechanism; this class probably includes a subpopulation of the acidic phospholipids. Therefore during the cell cycle these phospholipids accumulate in the inner monolayer of the cytoplasmic membrane and so cause an increase in its packing density; at a critical density, phospholipids "flip out" from the inner to the outer monolayer. This flip-out occurs once per cycle and initiates cell cycle events.     

Evidence for a homogeneous lateral distribution of lipids in a bacterial membrane. A photo cross-linking approach using anthracene as a photoactivable group

A new photo cross-linking method has been developed for the study of the lateral distribution of lipids in natural membranes, which uses anthracene as a photoactivable group. This method, which rests on the potentiality of anthracene to form covalently bound dimers upon irradiation around 340-380 nm has been applied to the membrane lipids (dimannosyl diacylglycerol, phosphatidylglycerol, phosphatidylinositol) of the bacterium Micrococcus luteus. These glyco- and phospholipids were anthracene labelled by metabolically incorporating the synthetic 9-(2-anthryl)nonanoic acid. The following sequential procedure was used: dimerization of the anthracene-labelled lipids in the membrane by irradiation of the intact cells at 360 nm; extraction of the lipids and thin-layer chromatography in the first dimension to separate the various lipid dimers from the monomers; partial dedimerization of the lipid dimers by illumination of the chromatogram at around 250-280 nm; chromatography in the second dimension to separate the native lipid monomers from the corresponding residual lipid dimers. On account of the occurrence of the 3 hetero dimers phosphatidylglycerol-dimannosyl diacylglycerol, phosphatidylinositol-dimannosyl diacylglycerol and phosphatidylglycerol-phosphatidylinositol after irradiating the cells, it is concluded that in this bacterial membrane, dimannosyl diacylglycerol, phosphatidylglycerol and phosphatidylinositol are homogeneously distributed.     

Alanyl-phosphatidylglycerol synthase: mechanism of substrate recognition during tRNA-dependent lipid modification in Pseudomonas aeruginosa

Bacterial lipid homeostasis plays an important role for the adaptation to changing environments and under conditions of antimicrobial treatment. The tRNA-dependent aminoacylation of the phospholipid phosphatidylglycerol catalysed by aminoacyl-phosphatidylglycerol synthases was shown to render various organisms less susceptible to antibacterial agents. Therefore, this type of enzyme might provide a new target to potentiate the efficacy of existing antimicrobials. This study makes use of the Pseudomonas aeruginosa alanyl-phosphatidylglycerol synthase to identify the minimal core domain of this transmembrane protein, which is capable of alanyl-phosphatidylglycerol biosynthesis. Using this catalytic fragment we established a reliable activity assay that was used to study the enzymatic mechanism by analysing an overall of 33 mutant proteins in vitro. Substrate recognition was analysed by using aminoacylated microhelices as analogues of the natural tRNA substrate. The enzyme even tolerated mutated versions of this minimal substrate, which indicates that neither the intact tRNA, nor the individual sequence of the acceptor stem is a determinant for substrate recognition. Furthermore, the analysis of derivatives of phosphatidylglycerol indicated that the polar head group of the phospholipid is specifically recognized by the enzyme, whereas modification of an individual fatty acid or even the deletion of a single fatty acid did not abolish A-PG synthesis.     

Characterization of polar membrane lipids of the extremely halophilic bacterium Salinibacter ruber and possible role of cardiolipin

The lipid composition of the extremely halophilic bacterium Salinibacter ruber (Bacteroidetes) was investigated by thin layer chromatography, gas chromatography, high performance liquid chromatography and electrospray ionization-mass spectrometry. Polar lipids represent about 80% of the total lipid extract. The main polar lipids are a sulfonic acid analogue of ceramide (or capnine analogue), phosphatidylcholine, phosphatidylserine, dimethylphosphatidylethanolamine, phosphatidylglycerol, cardiolipin or bisphosphatidylglycerol, and a glycolipid. The major acyl chains in the phospholipids are C16:1 Delta9cis and C18:1 Delta11cis, while the sulfonolipid contains an amide-bound iso C15:0 fatty acid. On changing the salinity of the culture medium, no significant differences were found in the lipid profile or the unsaturation of the lipid fatty acyl chains. The structure of the cardiolipin, which represents 20% of polar lipids, has been elucidated by gas chromatography and electrospray ionization mass spectrometry analysis.     

A 2H-NMR study on the glycerol backbone of phospholipids extracted from Escherichia coli grown under high osmotic pressure: evidence for multiconformations of phosphatidylethanolamine

A glycerol-requiring auxotroph was isolated from mutagenized Escherichia coli K-12 UFAts cells. This auxotroph was used for the specific deuteration of E. coli phospholipids. The cells were grown under high osmotic pressure (in the presence of 2.0% KCl). The membrane had a highly saturated fatty acid composition (76% phosphatidylethanolamine, 20% cardiolipin and 4% phosphatidylglycerol). The deuterium magnetic resonance spectra of coarse liposomes of the extracted phospholipids with perdeuterated glycerol incorporated into them were measured. To obtain well characterized information, phospholipid mixtures reconstituted from the deuterated and nondeuterated components at the same ratios as in the case of the total extract were used. On the analysis of the spectra, the following conclusions were drawn. (1) The whole polar region of cardiolipin is dynamically symmetric and quite rigid in the presence of phosphatidylethanolamine. (2) Although the quadrupole splittings of the deuterons at the C-2 and C-3 positions of the glycerol backbone were similar to each other, those at the C-1 position for phosphatidylethanolamine and cardiolipin are different, even in the same bilayer. (3) Furthermore, each C-1 deuteron of phosphatidylethanolamine gave rise to a doublet, suggesting the presence of two backbone conformations, between which there is slow exchange. (4) The polar head group of phosphatidylethanolamine interacts with cardiolipin and phosphatidylglycerol in different ways, which could be responsible for the different osmotic properties of the vesicles composed of them.     

7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled phospholipids in lipid membranes: differences in fluorescence behavior.

Steady-state and time-resolved fluorescence properties of the 7-nitrobenz-2-oxa-1, 3-diazole-4-yl (NBD) fluorophore attached either to the sn-2 acyl chain of various phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidic acid) or to the polar headgroup of phosphatidylethanolamine were studied after insertion of these NBD-labeled lipid probes into unilamellar vesicles of phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, and phosphatidylserine. The fluorescence response of the NBD group was observed to strongly depend on the chemical structure and physical state of the host phospholipids and on the chemical structure of the lipid probe itself. Among the various fluorescence parameters studied, i.e., Stokes' shifts, lifetimes, and quantum yields, the quantum yields were by far the most affected by these structural and environmental factors, whereas the Stokes' shifts were practically unaffected. Thus, depending on the phospholipid probe and the host phospholipid, the fluorescence emission of the NBD group was found to vary by a factor of up to 5. Careful analysis of the data shows that for the various couples of probe and host lipid molecules studied, deexcitation of the fluorophore was dominated by nonradiative deactivation processes. This great sensitivity of the NBD group to environmental factors originates from its well-known solvatochromic properties, and comparison of these knr values with those obtained for n-propylamino-NBD in a set of organic solvents covering a large scale of polarity indicates that in phospholipids, the NBD fluorophore experiences a dielectric constant of around 27-41, corresponding to a medium of relatively high polarity. From these epsilon values and on the basis of models of the dielectric transition that characterizes any water-phospholipid interface, it can be inferred that for all of the phospholipid probes and host phospholipids tested, the NBD group is located in the region of the polar headgroups, near the phosphoglycerol moiety of the lipids.     

Polar lipid and isoprenoid quinone composition in the classification of Staphylococcus

Representatives of 13 species of Staphylococcus were examined using a small-scale procedure for the sequential extraction of isoprenoid quinones and polar lipids. Menaquinones were the only isoprenoid quinones found in the 77 test strains which were divided into three groups based upon the predominant isoprenologue detected: (i) S. hyicus subsp. hyicus, S. sciuri subsp. lentus and S. sciuri subsp. sciuri contained unsaturated menaquinones with six isoprene units; (ii) S. capitis, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. hyicus subsp. chromogenes, S. intermedius, S. saprophyticus, S. simulans, S. warneri and S. xylosus contained unsaturated menaquinones with seven isoprene units and (iii) S. aureus contained unsaturated menaquinones with eight isoprene units and varying amounts of the corresponding lower isoprenologue. All of the organisms contained very similar polar lipid patterns consisting of diphosphatidylglycerol, phosphatidylglycerol, beta-gentiobiosyl diacylglycerol and a number of glycolipids and phospholipids. One of the glycolipids was chromatographically indistinguishable from beta-gentiotriosyl diacylglycerol. Lysylphosphatidylglycerol was a major component in S. aureus and S. intermedius but was usually present in minor amounts in the coagulase-negative strains. The polar lipid data underline the homogeneity of the genus Staphylococcus and distinguish staphylococci from aerobic, Gram-positive cocci and from the phylogenetically related aerobic, endospore-forming bacteria. Menaquinone composition can also be used to separate staphylococci from other aerobic, Gram-positive cocci.     

Study of structure and orientation of mesentericin Y105, a bacteriocin from Gram-positive Leuconostoc mesenteroides, and its Trp-substituted analogues in phospholipid environments

Mesentericin Y105 (Mes-Y105) is a bacteriocin secreted by Leuconostoc mesenteroides which is particularly active on Listeria. It is constituted by 37 residues and reticulated by one disulfide bridge. It has two W residues, W18 and W37, which can be studied by fluorescence. Two single substituted W/F analogues were synthesized (Mes-Y105/W18 and Mes-Y105/W37) to differentiate the local environment around each W and to study their changes in the presence of lipid vesicles. Fluorescence experiments show that, for the pure Trp-analogues, W18 and W37 are fully exposed to solvent whatever pH and buffer conditions. In the presence of lipid vesicles, both became buried. Lipid affinities were estimated: they are weak for zwitterionic phospholipids but an order of magnitude higher for negatively charged phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids. On negatively charged PG lipids, Mes-Y105 and Mes-Y105/W37 display comparable lipid affinities. A decrease in lipid affinity is observed for Mes-Y105/W18 compared to Mes-Y105, which means that W37 would seem to be required for increased lipid selectivity. In the lipid-bound state W18 is strongly dehydrated, probably embedded into the acyl chains, while W37 stands more at the interface. Mes-Y105 was also studied by polarization modulation infrared reflection absorption spectroscopy (PMIRRAS), alone and in various phospholipid environments, to obtain structural information and to assess lipid perturbations. At nanomolar concentrations close to those required for anti-Listeria activity, Mes-Y105 forms films at the air/water interface and inserts into negatively charged lipid monolayers. In situ infrared data show that Mes-Y105 binding only affects the polar head group vibrations while the lipid order of the acyl chains remains unaffected. The PMIRRAS show that Mes-Y105 folds into an N-terminal antiparallel beta-sheet followed by an alpha-helix, both structures being tilted (40 degrees) compared to the normal at the interface, which is in agreement with the thickness estimated by Brewster angle microscopy (BAM). All these data support the proposal of a new model for Mes-Y105 at the membrane interface.