Total lipids with short and long acyl chains from Acholeplasma form nonlamellar phases.

The cell-wall-less bacterium Acholeplasma laidlawii A-EF22 synthesizes eight glycerolipids. Some of them form lamellar phases, whereas others are able to form normal or reversed nonlamellar phases. In this study we examined the phase properties of total lipid extracts with limiting average acyl chain lengths of 15 and 19 carbon atoms. The temperature at which these extracts formed reversed hexagonal (HII) phases differed by 5-10 degreesC when the water contents were 20-30 wt%. Thus the cells adjust the ratio between lamellar-forming and nonlamellar-forming lipids to the acyl chain lengths. Because short acyl chains generally increase the potential of lipids to form bilayers, it was judged interesting to determine which of the A. laidlawii A lipids are able to form reversed nonlamellar phases with short acyl chains. The two candidates with this ability are monoacyldiglucosyldiacylglycerol (MADGlcDAG) and monoglucosyldiacylglycerol. The average acyl chain lengths were 14.7 and 15.1 carbon atoms, and the degrees of acyl chain unsaturation were 32 and 46 mol%, respectively. The only liquid crystalline phase formed by MADGlcDAG is an HII phase. Monoglucosyldiacylglycerol forms reversed cubic (Ia3d) and HII phases at high temperatures. Thus, even when the organism is grown with short fatty acids, it synthesizes two lipids that have the capacity to maintain the nonlamellar tendency of the lipid bilayer. MADGlcDAG in particular contributes very powerfully to this tendency.     

Isolation and characterization of a novel monoacylated glucopyranosyl neutral lipid from the plasma membrane of Acholeplasma laidlawii B

The level of a normally minor component of the membrane polar lipids of Acholeplasma laidlawii B was significantly increased when the glucose supplement in the growth medium was reduced. Under such glucose-limiting conditions the proportion of this component was found to be dependent upon the fatty acid supplement and could approach 55-60% of the total polar lipids when palmitic acid was used to supplement the growth medium. A number of physical measurements, along with specific chemical and enzymic degradation studies followed by a careful analysis of the degradation products, enabled us to tentatively identify this lipid as a polyprenyl-alpha-D-glucoside with a long-chain fatty acid esterified to the 2-hydroxyl group of the sugar moiety. This lipid exhibited some unusual thermotropic phase properties and our observations suggest that it may not be easily miscible with the other membrane lipid components. The structure and physical properties of this unusual glycolipid also suggest that it may be capable of forming non-bilayer phases under physiologically relevant conditions.     

The structure of a glycerylphosphoryldiglucosyl diglyceride from the lipids of Acholeplasma laidlawii strain B

1. The phosphatidylglucose structure proposed previously (Smith & Henrikson, 1965) for the glucose-containing phospholipid from Acholeplasma laidlawii is incorrect. 2. The structure now proposed is 3-(sn-glycerol-3-phosphoryl-6'-[O-alpha-d-glucopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl])- sn-1,2-diglyceride, a new type of bacterial lipid. 3. Deacylation of the lipid gave a single water-soluble phosphate ester which could be distinguished on chromatography from synthetic samples of glucosylphosphorylglycerols. 4. Hydrolysis of the lipid with alkali gave a mixture of fatty acids, glycerol 2-phosphate, sn-glycerol 3-phosphate and O-alpha-d-glucopyranosyl-(1-->2)-O-alpha- d-glucopyranosyl-(1-->1)-d-glycerol. 5. The lipid was unaffected on incubation with phospholipases A, C and D. 6. Diglucosyl diglyceride was isolated after treatment of the lipid with 60% HF, establishing the location of the fatty acid residues. 7. Periodate oxidation studies showed that the sn-glycerol 3-phosphate was esterified to the 6-hydroxyl group of one of the glucose residues in diglucosyl diglyceride.     

Selective acylation of membrane proteins in Acholeplasma laidlawii

In membranes of the cell-wall-less prokaryote Acholeplasma laidlawii most proteins are of the integral type. A substantial fraction of these proteins are enriched in hydrophilic amino acid residues. Approximately 20 different major as well as minor proteins were found to be covalently modified with acyl chains. The same set of proteins are acylated when cells are grown in different fatty-acid-supplemented media. In individual proteins the ratio of palmitoyl/oleoyl acyl chains was 12-14 times larger than the acyl chain ratio in polar membrane lipids. The transmembrane protein D12 has close to two acyl chains per molecule. Proteins T2 and T4a, localized in the outer and inner leaflet of the membrane, respectively, occur each as pairs with a difference in relative molecular mass within each pair of approximately 2000. Each of these proteins as well as the other acyl proteins, except the light form of T4a, has close to one acyl chain per molecule. The extent of acylation was increased for certain proteins and decreased for others by treatment with globomycin or phenethylalcohol. The relative amounts of the T2 and T4a pairs were affected by these drugs. It is concluded that the mechanism of acylation is different from that in Escherichia coli lipoprotein and Bacillus penicillinase. The mean hydrophobicity [Kyte & Doolittle (1982) J. Mol. Biol. 157, 105-132] of the A. laidlawii acyl proteins are similar to those of other bacterial acyl proteins but significantly lower than for non-acylated integral membrane proteins, supporting an anchoring function of the acyl chains. The number of membrane acyl proteins in A. laidlawii and two other mycoplasmas are at least twice that in other bacteria.     

Radiation studies of Acholeplasma laidlawii: the role of membrane composition

Acholeplasma laidlawii, a mycoplasma, is unable to synthesize unsaturated fatty acids but it will incorporate them into its plasma membrane if they are supplied exogeneously. Thus the fatty acid composition of the cell membrane can be defined by growing the organism in media containing specific fatty acids. We obtained cells with predominantly one type of unsaturated fatty acid (either oleic, linoleic or linolenic acid) or cells with only saturated fatty acid in the cell membrane. The cells were irradiated with 7 MeV electrons and the effect of membrane fatty acid composition on cell survival was examined. At 200 Gy/min and 0.5 degrees C (melting ice) there was little difference in the radiation sensitivities of the cells grown in unsaturated fatty acids either in aerated or anoxic radiation conditions. However, the cells containing saturated fatty acids irradiated in anoxic conditions were markedly more sensitive than the cells containing unsaturated fatty acids. At 200 Gy/min and 37 degrees C the two types of cells were of similar sensitivity both in aerated and anoxic radiation conditions. At 5 Gy/min at 0.5 degrees C the cells containing linolenic acid (18:3) were less sensitive than those containing solely saturated fatty acids. However, at 5 Gy/min at 37 degrees C there was no difference in sensitivity between these two types of cell. Our results strongly argue against the involvement of lipid peroxidation as a molecular change leading to cell death.     

Osmotic behaviour of Acholeplasma laidlawii B cells with membrane lipids in liquid-crystalline and gel state.

The osmotic behaviour of Acholeplasma laidlawii B cells was investigated with combined spectrophotometric and enzymatic measurements. The conclusion could be drawn that this osmotic behaviour depends largely on the physical state of the membrane lipids. When part of the membrane lipids is in the liquid-crystalline phase the cell is able to swell and behaves as a good osmometer. However, when the membrane lipid is in the gel phase, the cell is unable to swell and the change in absorbance of the cell suspension is then completely due to lysis.     

Glucose transport in Acholeplasma laidlawii B: dependence on the fluidity and physical state of membrane lipids.

The uptake of D-glucose by Acholeplasma laidlawii B occurs via a mediated transport process, as shown by the following observations: (i) glucose permeates A. laidlawii B cells at a rate at least 100 times greater than would be expected if its entry occurred only by simple passive diffusion; (ii) the apparent activation energy for glucose uptake in A. laidlawii is significantly lower than that expected and observed for the passive permeation of this sugar; (iii) glucose uptake appears to be a saturable process; (iv) glucose uptake can be completely inhibited by low concentrations of phloretin and phlorizin; and (v) glucose uptake is markedly inhibited at temperatures above 45 C, whereas the passive entry of erythritol continues to increase logarithmically until at least 60 C. The metabolism of D-glucose by this organism is rapid and, at low glucose concentrations, the intracellular radioactivity derived from D-[14-C]glucose is at any given time a reflection of the net effect of glucose transport, glucose metabolism, and loss from the cell of radioactive metabolic products. Care must thus be taken when attempting to determine the rate of glucose transport by measuring the accumulation by the cells of the total radioactivity derived from D-[14-C]glucose. The rate of uptake of D-glucose by A. laidlawii B cells is markedly dependent on the fatty acid composition and cholesterol content of the plasma membrane and exhibits a direct dependence on the fluidity of the membrane lipids as measured by their reversible, thermotropic gel to liquie-crystalline phase transition temperatures. In contrast to the transport rates, the apparent activation energy for glucose uptake above the phase transition temperature is not dependent on membrane lipid composition. At the temperature range within the membrane lipid phase transition region, the apparent activation energy of glucose uptake is different from the activation energy observed at temperatures above the phase transition. This may reflect the superimposed operation within the phase transition region of more than one temperature-dependent process.     

Transmembrane electrical potential affects the lipid composition of Acholeplasma laidlawii

In membranes of Acholeplasma laidlawii, lipid composition is regulated as a function of several stimuli affecting the volume and length of the hydrocarbon chains and the hydrocarbon-water interfacial area. This regulation is vizualized as changes in the relative amounts of the major polar lipids monoglucosyl diglyceride and diglucosyl diglyceride. These lipids form reversed hexagonal and lamellar phases with water, respectively. However, mixtures of the two lipids, in the molar proportions found in the A. laidlawii membrane, form a lamellar phase. By adjustment of the glycolipid ratio as a response to environmental stimuli, a certain stability of the lamellar membrane is maintained. In growing cells with oleoyl membrane lipids, a transmembrane electrical potential of approximately -50 mV (inside negative), but no transmembrane pH difference, was found. Addition of the K+ ionophore valinomycin caused a rapid and dose-dependent hyperpolarization remaining for at least 7 h. Simultaneously, a rapid and lasting metabolic decrease in the ratio monoglucosyl diglyceride/diglucosyl diglyceride occurred. The increase in potential and the decrease in the lipid ratio were both reversed in a dose-dependent manner by extracellular KCl. Likewise, the lipophilic cation tetraphenylphosphonium caused a dose-dependent decrease in membrane potential and an increase in the monoglucosyl diglyceride/diglucosyl diglyceride ratio, respectively. The ionophores monensin and particularly nigericin had similar but less pronounced effects on the potential and lipid ratios as valinomycin. The uncoupler carbonyl cyanide m-chlorophenylhydrazone had no effect on cell growth, membrane potential, or lipid regulation at 10 microM. These dissimilar structures and the low concentrations used make a direct disturbance of drug molecules on lipid packing in membranes less likely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural features of glycosyltransferases synthesizing major bilayer and nonbilayer-prone membrane lipids in Acholeplasma laidlawii and Streptococcus pneumoniae

In membranes of Acholeplasma laidlawii two consecutively acting glucosyltransferases, the (i) alpha-monoglucosyldiacylglycerol (MGlcDAG) synthase (alMGS) (EC ) and the (ii) alpha-diglucosyl-DAG (DGlcDAG) synthase (alDGS) (EC ), are involved in maintaining (i) a certain anionic lipid surface charge density and (ii) constant nonbilayer/bilayer conditions (curvature packing stress), respectively. Cloning of the alDGS gene revealed related uncharacterized sequence analogs especially in several Gram-positive pathogens, thermophiles and archaea, where the encoded enzyme function of a potential Streptococcus pneumoniae DGS gene (cpoA) was verified. A strong stimulation of alDGS by phosphatidylglycerol (PG), cardiolipin, or nonbilayer-prone 1,3-DAG was observed, while only PG stimulated CpoA. Several secondary structure prediction and fold recognition methods were used together with SWISS-MODEL to build three-dimensional model structures for three MGS and two DGS lipid glycosyltransferases. Two Escherichia coli proteins with known structures were identified as the best templates, the membrane surface-associated two-domain glycosyltransferase MurG and the soluble GlcNAc epimerase. Differences in electrostatic surface potential between the different models and their individual domains suggest that electrostatic interactions play a role for the association to membranes. Further support for this was obtained when hybrids of the N- and C-domain, and full size alMGS with green fluorescent protein were localized to different regions of the E. coli inner membrane and cytoplasm in vivo. In conclusion, it is proposed that the varying abilities to bind, and sense lipid charge and curvature stress, are governed by typical differences in charge (pI values), amphiphilicity, and hydrophobicity for the N- and (catalytic) C-domains of these structurally similar membrane-associated enzymes.     

Phase equilibria of model milk membrane lipid systems

The phase behaviour of mixtures of recombined milk membrane lipids dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), dioleoylphosphatidylethanolamine (DOPE), phosphatidylinositol (PI) and dioleoylphosphatidylserine (DOPS) in 60% water was examined as a function of temperature between 5 and 90 degrees C. The aim was to examine under which lipid composition the average properties turn from balanced over to hydrophobic. The phase boundaries were determined by small angle X-ray diffraction (SAXD) and differential scanning calorimetry (DSC). The lamellar phase was dominating in the DOPC/SM/DOPE system. The phase boundary for the reversed hexagonal phase was only observed at high DOPE content within the examined temperature interval. The anionic phospholipids PI and DOPS induced a swollen lamellar phase, but no significant change of the transition between the lamellar phase and the reversed hexagonal phase was observed.     

Adsorption of mycoplasmavirus MV-L2 to Acholeplasma laidlawii: effects of changes in the acyl-chain composition of membrane lipids

Avian erythroblastosis virus (AEV) is an oncogenic retrovirus of birds. The AEV-encoded erbB polypeptide, a transmembrane glycoprotein bearing an N-terminal domain exposed on the surface of virally transformed cells, plays a crucial role in AEV-mediated oncogenesis. We report here a characterization of a mutated form of the AEV erbB protein which lacks over two-thirds of the extracellular region of this oncogenic protein. This mutant v-erbB protein, although lacking the three possible extracellular sites of N-linked protein glycosylation, appears unimpaired in the ability to transform cells to an oncogenic phenotype.     

The effects of ionizing radiation on the peroxide content of a pure polyunsaturated lipid dispersion and of lipids and membranes derived from Acholeplasma laidlawii

Dispersions of a pure unsaturated phospholipid, dilinoleoylphosphatidyl choline, formed conjugated diene hydroperoxides when irradiated in air with 7 MeV electrons (150 Gy and 300 Gy). Peroxide formation was optimized when the dispersions were irradiated in air at 37 degrees C at a dose rate of 5 Gy/min. No significant loss of linoleic acid from the irradiated phospholipid dispersions was observed after doses of 150 or 300 Gy. Small amounts of thiobarbituric acid-reactive material were formed in irradiated unsaturated phospholipid dispersions. However, lipids or membranes isolated from 48 hour cultures of Acholeplasma laidlawii grown in media supplemented with either linoleic or linolenic acid did not appear to be peroxidized by irradiation under the same conditions.