Key role of the diglucosyldiacylglycerol synthase for the nonbilayer-bilayer lipid balance of Acholeplasma laidlawii membranes

In the single membrane of Acholeplasma laidlawii, a specific glucosyltransferase (DGlcDAG synthase) synthesizes the major, bilayer-forming lipid diglucosyldiacylglycerol (DGlcDAG) from the preceding major, nonbilayer-prone monoglucosyldiacylglycerol (MGlcDAG). This is crucial for the maintenance of phase equilibria close to a potential bilayer-nonbilayer transition and a nearly constant spontaneous curvature for the membrane bilayer lipid mixture. The glucolipid pathway is also balanced against the phosphatidylglycerol (PG) pathway to maintain a certain lipid surface charge density. The DGlcDAG synthase was purified approximately 5000-fold by three chromatographic techniques and identified as a minor 40 kDa membrane protein. In CHAPS mixed micelles, a cooperative dependence on anionic lipid activators was confirmed, with PG as the best. The dependence of the enzyme on the soluble UDP-glucose substrate followed Michaelis-Menten kinetics, while the kinetics for the other (lipid) substrate MGlcDAG exhibited cooperativity, with Hill coefficients in the range of 3-5. Vmax and the Hill coefficient, but not Km, for the MGlcDAG substrate were increased by increased PG concentrations, but above 3 mol % MGlcDAG, the rate of synthesis was constant. Hence, the DGlcDAG synthase is more affected by the lipid activator than by the lipid substrate at physiological lipid concentrations. The enzyme was shown to be sensitive to curvature "stress" changes, i.e., was stimulated by various nonbilayer lipids but inhibited by certain others. Certain phosphates were also stimulatory. With the two purified MGlcDAG and DGlcDAG synthases reconstituted together in the presence of a potent nonbilayer lipid, the strong responses in the amounts of MGlcDAG and DGlcDAG synthesized mimicked the responses in vivo. This supports the important regulatory functions of these enzymes.     

The enzymatic synthesis of membrane glucolipids in Acholeplasma laidlawii.

In membranes of the prokaryote Acholeplasma laidlawii, the physiological regulation of the two major membrane lipids, monoglucosyldiacylglycerol (MGlcDAG) and diglucosyldiacylglycerol (DGlcDAG), is governed by factors affecting the equilibria between lamellar and non-lamellar phases of the membrane lipids. The synthesis of the glucolipids is considered to be a two-step glucosylation: (i) DAG+UDP-Glc----MGlcDAG+UDP; and (ii) MGlcDAG+UDP-Glc----DGlcDAG+UPD. This was corroborated by in vivo pulse labelling experiments showing turnover of MGlcDAG but not DGlcDAG. The enzymatic synthesis of MGlcDAG was localized to fresh or freeze-dried membranes in vitro. Synthesis of DGlcDAG was minor in such membranes but of substantial magnitude in intact cells. Synthesis of MGlcDAG was stimulated by small amounts of SDS but completely inhibited upon solubilization of the membranes by a variety of detergents. The inhibitory effect of several UDP-Glc analogs on glucolipid synthesis demonstrated the importance of UDP-Glc as the sugar donor. Synthesis of both glucolipids was lost in freeze-dried plus lipid-extracted cells but restored when lipids were transferred back to the extracted cell membrane. By selectively adding specific lipids, a strong dependence on the acceptor lipid DAG, as well as the need for general matrix lipids for enzyme activity, was established. In addition, the anionic phosphatidylglycerol (PG), but not the other phospholipids, had a strong stimulatory effect. The presence of different phosphorylating agents stimulated the synthesis of DGlcDAG and partially inhibited that of MGlcDAG. This, together with the lipid dependency, may constitute mechanisms for the regulation of the enzyme activities in vivo.     

alpha-methylene ordering of acyl chains differs in glucolipids and phosphatidylglycerol from Acholeplasma laidlawii membranes: (2)H-NMR quadrupole splittings from individual lipids in mixed bilayers

A Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with alpha-deuterated oleic acid. Phosphatidylglycerol (PG), the glucolipids monoglucosyldiacylglycerol (MGlcDAG), diglucosyldiacylglycerol (DGlcDAG) and monoacyldiglucosyldiacylglycerol, and the phosphoglucolipid glycerophosphoryldiglucosyldiacylglycerol (GPDGlcDAG) were purified, and the phase behaviour and molecular ordering for the individual lipids, as well as for mixtures of the lipids, were studied by (2)H-, (31)P-NMR and X-ray scattering methods. The chemical structure of all the A. laidlawii lipids, except PG, has been determined and verified previously; here also the chemical structure of PG was verified, utilising mass spectrometry and (1)H and (13)C high resolution NMR spectroscopy. For the first time, lipid dimers were found in the mass spectrometry measurements. The major findings in this work are: (1) addition of 50 mol% of PG to the non-lamellar-forming lipid MGlcDAG does not significantly alter the transition temperature between lamellar and non-lamellar phases; (2) the (2)H-NMR quadrupole splitting patterns obtained from the lamellar liquid crystalline phase are markedly different for PG on one hand, and DGlcDAG and GPDGlcDAG on the other hand; and (3) mixtures of PG and DGlcDAG or MGlcDAG give rise to (2)H-NMR spectra consisting of a superposition of splitting patterns of the individual lipids. These remarkable features show that the local ordering of the alpha-carbon of the acyl chains is different for PG than for MGlcDAG and DGlcDAG, and that this difference is preserved when PG is mixed with the glucolipids. The results obtained are interpreted in terms of differences in molecular shape and hydrophilicity of the different polar headgroups.     

Lateral organization in Acholeplasma laidlawii lipid bilayer models containing endogenous pyrene probes.

In membranes of the small prokaryote Acholeplasma laidlawii bilayer- and nonbilayer-prone glycolipids are major species, similar to chloroplast membranes. Enzymes of the glucolipid pathway keep certain important packing properties of the bilayer in vivo, visualized especially as a monolayer curvature stress ('spontaneous curvature'). Two key enzymes depend in a cooperative fashion on substantial amounts of the endogenous anionic lipid phosphatidylglycerol (PG) for activity. The lateral organization of five unsaturated A. laidlawii lipids was analyzed in liposome model bilayers with the use of endogenously produced pyrene-lipid probes, and extensive experimental designs. Of all lipids analyzed, PG especially promoted interactions with the precursor diacylglycerol (DAG), as revealed from pyrene excimer ratio (Ie/Im) responses. Significant interactions were also recorded within the major nonbilayer-prone monoglucosylDAG (MGlcDAG) lipids. The anionic precursor phosphatidic acid (PA) was without effects. Hence, a heterogeneous lateral lipid organization was present in these liquid-crystalline bilayers. The MGlcDAG synthase when binding at the PG bilayer interface, decreased acyl chain ordering (increase of membrane free volume) according to a bis-pyrene-lipid probe, but the enzyme did not influence the bulk lateral lipid organization as recorded from DAG or PG probes. It is concluded that the concentration of the substrate DAG by PG is beneficial for the MGlcDAG synthase, but that binding in a proper orientation/conformation seems most important for activity.     

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.     

Monoglucosyldiacylglycerol, a foreign lipid, can substitute for phosphatidylethanolamine in essential membrane-associated functions in Escherichia coli

The mechanisms by which lipid bilayer properties govern or influence membrane protein functions are little understood, but a liquid-crystalline state and the presence of anionic and nonbilayer (NB)-prone lipids seem important. An Escherichia coli mutant lacking the major membrane lipid phosphatidylethanolamine (NB-prone) requires divalent cations for viability and cell integrity and is impaired in several membrane functions that are corrected by introduction of the "foreign" NB-prone neutral glycolipid alpha-monoglucosyldiacylglycerol (MGlcDAG) synthesized by the MGlcDAG synthase from Acholeplasma laidlawii. Dependence on Mg(2+) was reduced, and cellular yields and division malfunction were greatly improved. The increased passive membrane permeability of the mutant was not abolished, but protein-mediated osmotic stress adaptation to salts and sucrose was recovered by the presence of MGlcDAG. MGlcDAG also restored tryptophan prototrophy and active transport function of lactose permease, both critically dependent on phosphatidylethanolamine. Three mechanisms can explain the observed effects: NB-prone MGlcDAG improves the quenched lateral pressure profile across the bilayer; neutral MGlcDAG dilutes the high anionic lipid surface charge; MGlcDAG provides a neutral lipid that can hydrogen bond and/or partially ionize. The reduced dependence on Mg(2+) and lack of correction by high monovalent salts strongly support the essential nature of the NB properties of MGlcDAG.     

Irreversible binding and activity control of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii at an anionic lipid bilayer surface

1,2-Diacylglycerol 3-glucosyltransferase is associated with the membrane surface catalyzing the synthesis of the major nonbilayer-prone lipid alpha-monoglucosyl diacylglycerol (MGlcDAG) from 1,2-DAG in the cell wall-less Acholeplasma laidlawii. Phosphatidylglycerol (PG), but not neutral or zwitterionic lipids, seems to be essential for an active conformation and function of the enzyme. Surface plasmon resonance analysis was employed to study association of the enzyme with lipid bilayers. Binding kinetics could be well fitted only to a two-state model, implying also a (second) conformational step. The enzyme bound less efficiently to liposomes containing only zwitterionic lipids, whereas increasing molar fractions of the anionic PG or cardiolipin (CL) strongly promoted binding by improved association (k(a1)), and especially a decreased rate of return (k(d2)) from the second state. This yielded a very low overall dissociation constant (K(D)), corresponding to an essentially irreversible membrane association. Both liposome binding and consecutive activity of the enzyme correlated with the PG concentration. The importance of the electrostatic interactions with anionic lipids was shown by quenching of both binding and activity with increasing NaCl concentrations, and corroborated in vivo for an active enzyme-green fluorescent protein hybrid in Escherichia coli. Nonbilayer-prone lipids substantially enhanced enzyme-liposome binding by promoting a changed conformation (decreasing k(d2)), similar to the anionic lipids, indicating the importance of hydrophobic interactions and a curvature packing stress. For CL and the nonbilayer lipids, effects on enzyme binding and consecutive activity were not correlated, suggesting a separate lipid control of activity. Similar features were recorded with polylysine (cationic) and polyglutamate (anionic) peptides present, but here probably dependent on the selective charge interactions with the enzyme N- and C-domains, respectively. A lipid-dependent conformational change and PG association of the enzyme were verified by circular dichroism, intrinsic tryptophan, and pyrene-probe fluorescence analyses, respectively. It is concluded that an electrostatic association of the enzyme with the membrane surface is accompanied by hydrophobic interactions and a conformational change. However, specific lipids, the curvature packing stress, and proteins or small molecules bound to the enzyme can modulate the activity of the bound A. laidlawii MGlcDAG synthase.     

Phosphatidylethanolamine and monoglucosyldiacylglycerol are interchangeable in supporting topogenesis and function of the polytopic membrane protein lactose permease

To determine the specific role lipids play in membrane protein topogenesis in vivo, the orientation with respect to the membrane bilayer of Escherichia coli lactose permease (LacY) transmembrane (TM) domains and their flanking extramembrane domains was compared after assembly in native membranes and membranes with genetically modified lipid content using the substituted cysteine accessibility method for determining TM domain mapping. LacY assembled in the absence of the major membrane lipid phosphatidylethanolamine (PE) does not carry out uphill transport of substrate and displays an inverted orientation for the N-terminal six-TM domain helical bundle (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107-2116). Strikingly, the replacement of PE in vivo by the foreign lipid monoglucosyldiacylglycerol (MGlcDAG), synthesized by the Acholeplasma laidlawii MGlcDAG synthase, restored uphill transport and supported the wild type TM topology of the N-terminal helical bundle of LacY. An interchangeable role in defining membrane protein TM domain orientation and supporting function is played by the two most abundant lipids, PE and MGlcDAG, in gram-negative and gram-positive bacteria, respectively. Therefore, these structurally diverse lipids endow the membrane with similar properties necessary for the proper organization of protein domains in LacY that are highly sensitive to lipids as topological determinants.     

Role of Glycolipids in the Pathogenesis of Enterococcus faecalis Urinary Tract Infection

Background

After uropathogenic Escherichia coli (UPEC), Enterococcus faecalis is the second most common pathogen causing urinary tract infections. Monoglucosyl-diacylglycerol (MGlcDAG) and diglucosyl-diacylglycerol (DGlcDAG) are the main glycolipids of the E. faecalis cell membrane. Examination of two mutants in genes bgsB and bgsA (both glycosyltransferases) showed that these genes are involved in cell membrane glycolipid biosynthesis, and that their inactivation leads to loss of glycolipids DGlcDAG (bgsA) or both MGlcDAG and DGlcDAG (bgsB). Here we investigate the function of bgsB and bgsA regarding their role in the pathogenesis in a mouse model of urinary tract infection and in bacterial adhesion to T24 bladder epithelial cells.

Results

In a mouse model of urinary tract infection, we showed that E. faecalis 12030ΔbgsB and E. faecalis 12030ΔbgsA mutants, colonize uroepithelial surfaces more efficiently than wild-type bacteria. We also demonstrated that these mutants showed a more than three-fold increased binding to human bladder carcinoma cells line T24 compared to the wild-type strain. Bacterial binding could be specifically inhibited by purified glycolipids. Lipoteichoic acid (LTA), wall-teichoic acid (WTA), and glycosaminoglycans (GAGs) were not significantly involved in binding of E. faecalis to the bladder epithelial cell line.

Conclusions

Our data show that the deletion of bgsB and bgsA and the absence of the major glycolipid diglucosyl-diacylglycerol increases colonization and binding to uroepithelial cells. We hypothesize that secreted diglucosyl-diacylglycerol blocks host binding sites, thereby preventing bacterial adhesion. Further experiments will be needed to clarify the exact mechanism underlying the adhesion through glycolipids and their cognate receptors.     

Changing the phospholipid composition of Staphylococcus aureus causes distinct changes in membrane proteome and membrane‐sensory regulators

The dynamic lipid composition of bacterial cytoplasmic membranes has a profound impact on vital bacterial fitness and susceptibility to membrane‐damaging agents, temperature, or osmotic stress. However, it has remained largely unknown how changes in lipid patterns affect the abundance and expression of membrane proteins. Using recently developed gel‐free proteomics technology, we explored the membrane proteome of the important human pathogen Staphylococcus aureus in the presence or absence of the cationic phospholipid lysyl‐phosphatidylglycerol (Lys‐PG). We were able to detect almost half of all theoretical integral membrane proteins and could reliably quantify more than 35% of them. It is worth noting that the deletion of the Lys‐PG synthase MprF did not lead to a massive alteration but a very distinct up‐ or down‐regulation of only 1.5 or 3.5% of the quantified proteins. Lys‐PG deficiency had no major impact on the abundance of lipid‐biosynthetic enzymes but significantly affected the amounts of the cell envelope stress‐sensing regulatory proteins such as SaeS and MsrR, and of the SaeS‐regulated proteins Sbi, Efb, and SaeP. These data indicate very critical interactions of membrane‐sensory proteins with phospholipids and they demonstrate the power of membrane proteomics for the characterization of bacterial physiology and pathogenicity.     

Sequence properties of the 1,2-diacylglycerol 3-glucosyltransferase from Acholeplasma laidlawii membranes. Recognition of a large group of lipid glycosyltransferases in eubacteria and archaea

Synthesis of the nonbilayer-prone alpha-monoglucosyldiacylglycerol (MGlcDAG) is crucial for bilayer packing properties and the lipid surface charge density in the membrane of Acholeplasma laidlawii. The gene for the responsible, membrane-bound glucosyltransferase (alMGS) (EC ) was sequenced and functionally cloned in Escherichia coli, yielding MGlcDAG in the recombinants. Similar amino acid sequences were encoded in the genomes of several Gram-positive bacteria (especially pathogens), thermophiles, archaea, and a few eukaryotes. All of these contained the typical EX(7)E catalytic motif of the CAZy family 4 of alpha-glycosyltransferases. The synthesis of MGlcDAG by a close sequence analog from Streptococcus pneumoniae (spMGS) was verified by polymerase chain reaction cloning, corroborating a connection between sequence and functional similarity for these proteins. However, alMGS and spMGS varied in dependence on anionic phospholipid activators phosphatidylglycerol and cardiolipin, suggesting certain regulatory differences. Fold predictions strongly indicated a similarity for alMGS (and spMGS) with the two-domain structure of the E. coli MurG cell envelope glycosyltransferase and several amphipathic membrane-binding segments in various proteins. On the basis of this structure, the alMGS sequence charge distribution, and anionic phospholipid dependence, a model for the bilayer surface binding and activity is proposed for this regulatory enzyme.     

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.     

Ceramide/sphingosine/sphingosine 1-phosphate metabolism on the cell surface and in the extracellular space

Sphingolipid metabolites, ceramide, sphingosine, and sphingosine 1-phosphate, have emerged as a new class of lipid biomodulators of various cell functions. These metabolites are known to function not only as intracellular second messengers, but also in the extracellular space. Sphingosine 1-phosphate especially has numerous functions as an important extracellular mediator that binds to cell surface S1P receptors. Recent studies have also shown that sphingolipid-metabolizing enzymes function not only in intracellular organelles but also in the extracellular spaces, including the outer leaflet of the plasma membrane. This review focuses on the metabolic enzymes (acid and alkaline sphingomyelinases, neutral ceramidase, and sphingosine kinase) that are involved in the production of the sphingolipid metabolites in these extracellular spaces, and on the metabolic pathway itself.     

CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum

Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. In this study, we investigated the membrane organization of CDP-diacylglycerol synthesis. Separation of mildly disrupted A431 cell membranes on sucrose density gradients revealed cofractionation of CDS and PI synthase activities with cholesterol-poor, endoplasmic reticulum (ER) membranes and partial overlap with plasma membrane caveolae. Cofractionation of CDS activity with caveolae was also observed when low-buoyant density caveolin-enriched membranes were prepared using a carbonate-based method. However, immunoisolation studies determined that CDS activity localized to ER membrane fragments containing calnexin and type III inositol (1,4,5)-trisphosphate receptors but not to caveolae. Membrane fragmentation in neutral pH buffer established that CDP-diacylglycerol and PI syntheses were restricted to a subfraction of the calnexin-positive ER. In contrast to lipid rafts enriched for caveolin, cholesterol, and GM1 glycosphingolipids, the CDS-containing ER membranes were detergent soluble. In cell imaging studies, CDS and calnexin colocalized in microdomain-sized patches of the ER and also unexpectedly at the plasma membrane. These results demonstrate that key components of the PI pathway localize to nonraft, phospholipid-synthesizing microdomains of the ER that are also enriched for calnexin.     

Quantitative Studies of White Matter : I. Enzymes involved in glucose-6-phosphate metabolism

Total lipid and six enzymes closely related to the metabolism of glucose-6-phosphate have been measured in ten tracts of the rabbit. Lipid content appears to be a valid indicator of the degree of myelination. Heavily myelinated tracts have much larger amounts of glucose-6-phosphate dehydrogenase than lightly myelinated ones but there is no corresponding difference in 6-phosphogluconate dehydrogenase. In fact the ratios between the two enzymes were found to vary over a ninefold range. Hexokinase is found in largest amounts in tracts with relatively little lipid, and this tends to be true for phosphofructokinase as well. The fibrillar layer of olfactory bulb is exceptional with regard to both enzymes, and to glucose-6-phosphate dehydrogenase. The enzymes are present in amounts which are more than adequate to support glucose metabolism at a rate commensurate with the known rates of O₂ uptake by various tracts. The distribution of some of the enzymes is compatible with the notion that the nodes of Ranvier are regions of high metabolic activity. A simple algebraic relationship is found to hold fairly well for the distribution of four of the enzymes among the tracts.     

On the role of anionic lipids in charged protein interactions with membranes

We investigate the role of anionic lipids in the binding to, and subsequent movement of charged protein groups in lipid membranes, to help understand the role of membrane composition in all membrane-active protein sequences. We demonstrate a small effect of phosphatidylglycerol (PG) lipids on the ability of an arginine (Arg) side chain to bind to, and cross a lipid membrane, despite possessing a neutralizing charge. We observe similar membrane deformations in lipid bilayers composed of phosphatidylcholine (PC) and PC/PG mixtures, with comparable numbers of water and lipid head groups pulled into the bilayer hydrocarbon core, and prohibitively large ~20 kcal/mol barriers for Arg transfer across each bilayer, dropping by just 2-3 kcal/mol due to the binding of PG lipids. We explore the causes of this small effect of introducing PG lipids and offer an explanation in terms of the limited membrane interaction for the choline groups of PC lipids bound to the translocating ion. Our calculations reveal a surprising lack of preference for Arg binding to PG lipids themselves, but a small increase in interfacial binding affinity for lipid bilayers containing PG lipids. These results help to explain the nature of competitive lipid binding to charged protein sequences, with implications for a wide range of membrane binding domains and cell perturbing peptides.     

The membrane phospholipid cardiolipin plays a pivotal role in bile acid adaptation by Lactobacillus gasseri JCM1131T

Bile acids exhibit strong antimicrobial activity as natural detergents, and are involved in lipid digestion and absorption. We investigated the mechanism of bile acid adaptation in Lactobacillus gasseri JCM1131^T. Exposure to sublethal concentrations of cholic acid (CA), a major bile acid in humans, resulted in development of resistance to otherwise-lethal concentrations of CA by this intestinal lactic acid bacterium. As this adaptation was accompanied by decreased cell-membrane damage, we analyzed the membrane lipid composition of L. gasseri. Although there was no difference in the proportions of glycolipids (~70%) and phospholipids (~20%), adaptation resulted in an increased abundance of long-sugar-chain glycolipids and a 100% increase in cardiolipin (CL) content (to ~50% of phospholipids) at the expense of phosphatidylglycerol (PG). In model vesicles, the resistance of PG vesicles to solubilization by CA increased with increasing CL/PG ratio. Deletion of the two putative CL synthase genes, the products of which are responsible for CL synthesis from PG, decreased the CL content of the mutants, but did not affect their ability to adapt to CA. Exposure to CA restored the CL content of the two single-deletion mutants, likely due to the activities of the remaining CL synthase. In contrast, the CL content of the double-deletion mutant was not restored, and the lipid composition was modified such that PG predominated (~45% of total lipids) at the expense of glycolipids. Therefore, CL plays important roles in bile acid resistance and maintenance of the membrane lipid composition in L. gasseri.     

Phosphatidylglycerol biosynthesis in Bacillus licheniformis Resolution of membrane-bound enzymes by affinity chromatography on cytidinediphospho-sn-1,2-diacylglycerol Sepharose.

Cytidinediphospho-sn-1,2-diaclglycerol (CDP-diglyceride) has been covalently linked to Sephrose 4B via adipic acid dihydrazide spacer arm forming an effective affinity chromatography column. This liponucleo-tide ligand and sn-glycero-3-phosphate are subtracts for the formation of 3-sn-phoshatidyl-1'-sn-glycero-3'-phosphate (PGP) catalyzed in both eukaryotic and prokaryotic organisms by sn-glycero-3-phosphate: CMP phosphatidlytranferase (PGP synthetase). Using this CDP-diglyceride Sephrose affinity column we were able to resolve the membrane associated 3-sn-phosphatidyl'1-sn-glycerol (PG) synthesizing system present in Bacillus licheniformis into two activities. A PGP synthetase activity was adsorbed to the affinity column and was eluted using buffer containg CDP-diglyceride; a PGP phosphatease acactivity had no affinity for the column. Both PGP synthase and PGP phosphatase of B. licheniformis were associated with a membrane component of the cell as evidenced by sucrose gradient centrifugation, differential centrifugation, and solubilization by buffers containing detergent...     

Occurrence of corrinoid-containing membrane proteins in anaerobic bacteria

In Methanobacterium thermoautotrophicum a corrinoid-carrying membrane protein complex has been found, to which a tentative role in methane formation has been ascribed. To test this hypothesis representatives from different orders of methanogenic bacteria were examined for membrane-bound cobamides. These species differed in cell carbon precursor, in methane precursor, in occurrence of cytochromes and of the enzyme CO dehydrogenase, and in the systematic position (Methanobacteriales, Methanomicrobiales). All methanogenic bacteria contained cobamides in the membranes in amounts of about 60 nmol/g cell dry weight, in addition to different amounts of cobamides in the soluble cell fraction. The only central metabolic reaction obviously common to all of these methanogens was methyl coenzyme M reduction to CH₄. It is concluded that the membrane corrinoid participates in this energy-conserving reaction.Sulfate-reducing and acetogenic bacteria were included in this survey. They contained different amounts of cobamides in the soluble cell fraction but not in the membrane, a possible exception being Acetobacterium woodii.     

The lipid dependence of diadinoxanthin de-epoxidation presents new evidence for a macrodomain organization of the diatom thylakoid membrane

The present study shows that thylakoid membranes of the diatom Cyclotella meneghiniana contain much higher amounts of negatively charged lipids than higher plant or green algal thylakoids. Based on these findings, we examined the influence of SQDG on the de-epoxidation reaction of the diadinoxanthin cycle and compared it with results from the second negatively charged thylakoid lipid PG. SQDG and PG exhibited a lower capacity for the solubilization of the hydrophobic xanthophyll cycle pigment diadinoxanthin than the main membrane lipid MGDG. Although complete pigment solubilization took place at higher concentrations of the negatively charged lipids, SQDG and PG strongly suppressed the de-epoxidation of diadinoxanthin in artificial membrane systems. In in vitro assays employing the isolated diadinoxanthin cycle enzyme diadinoxanthin de-epoxidase, no or only a very weak de-epoxidation reaction was observed in the presence of SQDG or PG, respectively. In binary mixtures of the inverted hexagonal phase forming lipid MGDG with the negatively charged bilayer lipids, comparable suppression took place. This is in contrast to binary mixtures of MGDG with the neutral bilayer lipids DGDG and PC, where rapid and efficient de-epoxidation was observed. In complex lipid mixtures resembling the lipid composition of the native diatom thylakoid membrane, we again found strong suppression of diadinoxanthin de-epoxidation due to the presence of SQDG or PG. We conclude that, in the native thylakoids of diatoms, a strict separation of the MGDG and SQDG domains must occur; otherwise, the rapid diadinoxanthin de-epoxidation observed in intact cells upon illumination would not be possible.