Diglucosyl Diacylglycerol Research Articles

Knockout of ykcB, a Putative Glycosyltransferase, Leads to Reduced Susceptibility to Vancomycin in Bacillus subtilis.

Vancomycin resistance of Gram-positive bacteria poses a serious health concern around the world. In this study, we searched for vancomycin-tolerant mutants from a gene deletion library of a model Gram-positive bacterium, Bacillus subtilis, to elucidate the mechanism of vancomycin resistance. We found that knockout of ykcB, a glycosyltransferase that is expected to utilize C55-P-glucose to glycosylate cell surface components, caused reduced susceptibility to vancomycin in B. subtilis. Knockout of ykcB altered the susceptibility to multiple antibiotics, including sensitization to β-lactams and increased the pathogenicity to silkworms. Furthermore, the ykcB-knockout mutant had (i) a decreased amount of lipoteichoic acid, (ii) decreased biofilm formation, and (iii) an increased content of diglucosyl diacylglycerol, a glycolipid that shares a precursor with C55-P-glucose. These phenotypes and vancomycin tolerance were abolished by knockout of ykcC, a gene in the same operon with ykcB probably involved in C55-P-glucose synthesis. Overexpression of ykcC enhanced vancomycin tolerance in both the parent strain and the ykcB-knockout mutant. These findings suggest that ykcB deficiency induces structural changes of cell surface molecules depending on the ykcC function, leading to reduced susceptibility to vancomycin, decreased biofilm formation, and increased pathogenicity to silkworms. IMPORTANCE Although vancomycin is effective against Gram-positive bacteria, vancomycin-resistant bacteria are a major public health concern. While the vancomycin-resistance mechanisms of clinically important bacteria such as Staphylococcus aureus, Enterococcus faecium, and Streptococcus pneumoniae are well studied, they remain unclear in other Gram-positive bacteria. In the present study, we searched for vancomycin-tolerant mutants from a gene deletion library of a model Gram-positive bacterium, Bacillus subtilis, and found that knockout of a putative glycosyltransferase, ykcB, caused vancomycin tolerance in B. subtilis. Notably, unlike the previously reported vancomycin-resistant bacterial strains, ykcB-deficient B. subtilis exhibited increased virulence while maintaining its growth rate. Our results broaden the fundamental understanding of vancomycin-resistance mechanisms in Gram-positive bacteria.

A Bifunctional Glycosyltransferase from Agrobacterium tumefaciens Synthesizes Monoglucosyl and Glucuronosyl Diacylglycerol under Phosphate Deprivation

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

Proper Fatty Acid Composition Rather than an Ionizable Lipid Amine Is Required for Full Transport Function of Lactose Permease from Escherichia coli

Energy-dependent uphill transport but not energy-independent downhill transport by lactose permease (LacY) is impaired when expressed in Escherichia coli cells or reconstituted in liposomes lacking phosphatidylethanolamine (PE) and containing only anionic phospholipids. The absence of PE results in inversion of the N-terminal half and misfolding of periplasmic domain P7, which are required for uphill transport of substrates. Replacement of PE in vitro by lipids with no net charge (phosphatidylcholine (PC), monoglucosyl diacylglycerol (GlcDAG), or diglucosyl diacylglycerol (GlcGlcDAG)) supported wild type transmembrane topology of the N-terminal half of LacY. The restoration of uphill transport in vitro was dependent on LacY native topology and proper folding of P7. Support of uphill transport by net neutral lipids in vitro (PE > PC ≫ GlcDAG ≠ GlcGlcDAG provided that PE or PC contained one saturated fatty acid) paralleled the results observed previously in vivo (PE = PC > GlcDAG ≠ GlcGlcDAG). Therefore, a free amino group is not required for uphill transport as previously concluded based on the lack of in vitro uphill transport when fully unsaturated PC replaced E. coli-derived PE. A close correlation was observed in vivo and in vitro between the ability of LacY to carry out uphill transport, the native conformation of P7, and the lipid headgroup and fatty acid composition. Therefore, the headgroup and the fatty acid composition of lipids are important for defining LacY topological organization and catalytically important structural features, further illustrating the direct role of lipids, independent of other cellular factors, in defining membrane protein structure/function.

How Does a Membrane Protein Know What is In and What is Out? Lipids as Topological Determinants

Primary functions of lipids are to define barrier properties of membranes and provide a scaffold within which membrane proteins are organized. Using a genetic approach to alter the phospholipid composition of the Escherichia coli coupled with biochemical approaches to monitor topological organization of membrane proteins, dependence of lactose permease (LacY) on phosphatidylethanolamine (PE) for proper orientation with respect to the plane of the membrane was determined. Assembly of LacY in the absence of PE results in topological inversion of its N-terminal half, which is largely reversed by post-assembly synthesis of PE. Replacement of PE by the foreign lipids phosphatidylcholine, monoglucosyl diacylglycerol, or diglucosyl diacylglycerol, which exhibit similar properties to PE, restores proper topology thereby supporting common functions for lipids with diverse structures. Topology of LacY in membranes lacking PE is dependent on a connecting flexible hinge region in order for the two halves of LacY to independently respond to the lipid environment. Final topology is determined after LacY exits the translocon by long-range and short-range interactions between the net charge of extra-membrane domains and the net charge density of the phospholipid bilayer surface. PE appears to dampen the translocation potential of acidic residues in normally cytoplasmic domains in favor of the cytoplasmic retention potential of basic residues. Thus a primary role for PE is to allow the presence of acidic residues in the cytoplasmic domains of membrane proteins for functional purposes without affecting protein topological. The topologies of two amino acid permeases (PheP and GabP) unrelated to LacY are also topologically sensitive to membrane lipid composition strongly indicating that lipid environment is a significant determinant of final topological organization of multiple membrane proteins. Supported in part by NIGMS R37-GM20478.

Effect of independent variations in fatty acid structure and chain length on lipid polar headgroup composition in Acholeplasma laidlawii B membranes: regulation of lamellar/nonlamellar phase propensity.

We have studied the biosynthetic regulation of the membrane lipid polar headgroup distribution in Acholeplasma laidlawii B cells made fatty acid auxotrophic by growth in the presence of the biotin-binding agent avidin to test whether this organism has the ability to coherently regulate the lamellar/nonlamellar phase propensity of its membrane lipids. The addition of various single normal growth-supporting exogenous fatty acids to such cell cultures produces fatty acid-homogeneous cells in which the hydrocarbon chain length and structure of the fatty acyl chains of the membrane lipids can be independently varied. Moreover, in analyzing our results, we consider the fact that the individual membrane lipid classes of this organism can form either normal micellar, lamellar, or reversed cubic or hexagonal phases in isolation (Lewis, R. N. A. H., and McElhaney, R. N. (1995) Biochemistry 34, 13818-13824). When A. laidlawii cells are highly enriched in one of a homologous series of methyl isobranched, methyl anteisobranched, or omega-cyclohexyl fatty acids, neither the ratio of normal micellar/lamellar nor of inverted cubic or hexagonal/lamellar phase-forming lipids are coherently regulated, and in fact in the former case, the changes in lipid polar headgroup composition observed are generally in a direction opposite to that required to maintain the overall lamellar/nonlamellar phase preference of the total membrane lipids constant when hydrocarbon chain length is varied. Similarly, when lipid hydrocarbon structure is varied at a constant effective chain length, a similar lack of coherent regulation of membrane lipid polar headgroup distribution is also observed, although in this case a weak overall trend in the expected direction occurs. We also confirm our previous finding (Foht, P. J., Tran, Q. M., Lewis, R. N. A. H., and McElhaney, R. N. (1995) Biochemistry 34, 13811-13817) that the ratio of inverted phase-forming monoglucosyl diacylglycerol to the lamellar phase-forming glycolipid diglucosyl diacylglycerol, previously used to estimate membrane lipid phase preference in A. laidlawii A and B, is not by itself a reliable indicator of the overall lamellar/nonlamellar phase propensity of the total membrane lipids of these organisms. Our results indicate that A. laidlawii B lacks a coherent mechanism to biosynthetically regulate the polar headgroup distribution of its membrane lipids to maintain the micellar/lamellar/inverted phase propensity constant in the face of induced variations in either the chain length or the structure of its lipid hydrocarbon chains. Finally, we suggest that the lack of a coherent regulatory mechanism to regulate the overall phase-forming propensity of the total membrane lipids of this organism under these circumstances may result in part from its inability to optimize all of the biologically relevant physical properties of its membrane lipid bilayer simultaneously.

Analysis of glycolipids from black cumin (Nigella sativa L.), coriander (Coriandrum sativum L.) and niger (Guizotia abyssinica Cass.) oilseeds

Edible plant glycolipids (GL) are anticipated to play a role in human nutrition. Total glycolipids (TGL) were separated from black cumin (Nigella sativa L.), coriander (Coriandrum sativum L.) and niger (Guizotia abyssinica Cass.) seed oils by silica gel chromatography. Different GL subclasses were then identified and separated using high-performance liquid chromatography with ultraviolet adsorption (HPLC/UV). Separation was accomplished using Zorbax-Sil (5 μm) column with an isocratic elution by mixed solvents of isooctane/2-propanol (1:1, v/v) and detection at 206 nm. Methods are described for the analysis of GL constituents, sugar and sterols (ST), using gas–liquid chromatography equipped with flame ionization detector (GLC/FID). A relatively high level of TGL was found in all studied oilseeds. Six GL subclasses were detected in black cumin seed oil, wherein diglucosyldiacylglycerol (DGD) was the prevalent component, followed by glucocerebroside (CER). Among the TGL from coriander and niger oilseeds, acylated steryl glucoside (ASG), steryl glucoside (SG) and CER were detected. The fatty acid profiles of GL fractions from black cumin and niger seed oils was generally similar, wherein linoleic acid C18:2n-6 was the dominating fatty acid, followed by oleic acid C18:1n-9. Petroselinic acid C18:1n-12 was the fatty acid marker in GL subclasses obtained from coriander seed oil, followed by linoleic acid C18:2n-6. Four ST moieties were identified in black cumin and coriander SG and ASG fractions, while the fractions from niger oilseeds showed only three distinct ST peaks. As component sugar, glucose was the only sugar detected in all samples.

A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2-diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products.

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 (Freese and Fortnagel, 1967) using PCR. After cloning and expression in E. coli, SDS-PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylgl ycerol, 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl]-1,2-diacylglycerol and 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[O-beta-D-glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as 14C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-beta-D-glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyl diacylglycerol or triglucosyl diacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in beta (1-->6) linkage to the product of the preceding reaction.

Modulation of the activities of enzymes of membrane lipid metabolism by non-bilayer-forming lipids

The activities of several enzymes which catalyze the synthesis or degradation of important lipid structural components of membranes are influenced by non-bilayer-forming lipids. We present and critique the hypothesis that enzymes that catalyze interconversions between bilayer- and non-bilayer-forming lipids are regulated by the lipid packing perturbations of the non-bilayer-forming component. The consequence of this regulation is lipid polymorphic homeostasis. The enzymes which are modulated by non-bilayer components of membranes include a diglucosyl diacylglycerol synthase from mycoplasma, secretory and cell-associated phospholipase A2s, phospholipase C, phosphocholine cytidylyltransferase, phosphatidate phosphohydrolase and diacylglycerol kinase. The effect of the non-lamellar lipids, both reversed phase-forming and micellar-forming, are discused in terms of bilayer packing strain, which can effect enzyme-membrane associations, and lateral domain formation, which can modulate the effective concentration of lipid activators.

Differential scanning calorimetric study of the interaction of cholesterol with the major lipids of the Acholeplasma laidlawii B membrane.

It has been proposed that the lower levels of exogenous cholesterol incorporation into the membranes of the sterol-non-requiring as compared to the sterol-requiring mycoplasmas may be due to the much higher glycolipid content of the former and to the reduced ability of glycolipids, as opposed to phospholipids, to incorporate sterols [Efrati et al. (1986) Arch. Biochem. Biophys. 248, 282-288]. In order to test this hypothesis, we have investigated the interaction of cholesterol with the major membrane glyco- and phospholipids of the sterol-non-requiring mycoplasma Acholeplasma laidlawii B, utilizing elaidic acid-homogenous membranes in order to obviate any differences in the nature of cholesterol-lipid interactions due to variations in the fatty acid composition of the different membrane components. Specifically, we have studied the effect of increasing quantities of cholesterol on the thermotropic phase behavior of aqueous dispersions of phosphatidylglycerol, diglucosyl diacylglycerol, and monoglucosyl diacylglycerol, as well as the total membrane polar lipids of this organism, using high-sensitivity differential scanning calorimetry. We find that cholesterol is highly miscible in both the lamellar gel and liquid-crystalline states of phosphatidylglycerol but exhibits limited miscibility in the two neutral glycolipids, particularly in their lamellar gel and crystalline states. We also demonstrate that cholesterol has a limited miscibility in both the lamellar gel and liquid-crystalline states of bilayers composed of the total A. laidlawii B membrane polar lipids. These results demonstrate that the nature of cholesterol-lipid interactions depends markedly on the structure of the glycerolipid polar headgroup and suggests that the incorporation of lower levels of cholesterol into the membranes of the sterol-non-requiring mycoplasmas may indeed be due, at least in part, to their high glycolipid contents. We also show that cholesterol stabilizes the lamellar liquid-crystalline phase of the monoglucosyl diacylglycerol relative to the inverted hexagonal phase at all sterol concentrations, in contrast to the effects of cholesterol on dielaidoylphosphatidylethanolamine, which destabilizes the lamellar liquid-crystalline phase at low concentrations.

Lipoteichoic acid as a target for antimicrobial action.

Daptomycin, a lipopeptide antibiotic active against gram-positive bacteria, has been found to inhibit lipoteichoic acid (LTA) synthesis as a consequence of membrane binding in the presence of Ca2+. The present study shows that among the bacterial-membrane components, daptomycin binds the protein fraction with a noncovalent bond, as suggested by the instability of the bond in the presence of an ionic detergent such as sodium dodecyl sulfate. Analysis of membrane proteins by isoelectric focusing electrophoresis reveals that 5 bands with isoelectric points ranging from 5.9 to 6.2 bind radioactive daptomycin. These proteins are therefore called daptomycin-binding proteins. In an attempt to correlate these proteins with the main inhibition observed in LTA synthesis, two-dimensional thin-layer chromatography of lipids synthesized during daptomycin treatment was performed. A 3-fold increase in diglucosyl diacylglycerol is demonstrated, while the compounds phosphatidyl-alpha-kojibiosyldiacylglycerol, glycerophosphophosphatidyl-alpha-kojibiosyldiacyl glycerol, and glycerophosphokojibiosyldiacylglycerol, which follow diglucosyl diacylglycerol in LTA synthesis, decrease progressively with time during the course of daptomycin treatment.

Identification of daptomycin-binding proteins in the membrane of Enterococcus hirae

Daptomycin, a lipopeptide antibiotic active against gram-positive bacteria, was preliminarily shown to inhibit lipoteichoic acid (LTA) synthesis as a consequence of membrane binding in the presence of Ca2+ (P. Canepari, M. Boaretti, M. M. Lleó, and G. Satta, Antimicrob. Agents Chemother. 34:1220-1226, 1990). In the present study, it is shown that, along with binding bacterial-membrane components, daptomycin binds the protein fraction with a noncovalent bond, as suggested by the instability of the bond in the presence of ionic detergents such as sodium dodecyl sulfate. Analysis of membrane proteins by isoelectric focusing electrophoresis reveals that five bands with isoelectric points ranging from 5.9 to 6.2 bind radioactive daptomycin. These proteins are therefore called daptomycin-binding proteins. In an attempt to correlate these proteins to the main inhibition observed during LTA synthesis, two-dimensional thin-layer chromatography of lipids synthesized during daptomycin treatment was performed. A threefold increase in diglucosyl diacylglycerol is demonstrated, while the compounds phosphatidyl-alpha-kojibiosyldiacylglycerol, glycerophospho-phosphatidyl-alpha-kojibiosyldiacylglycerol, and glycerophospho-kojibiosyldiacylglycerol, which follow diglucosyl diacylglycerol in LTA synthesis, decrease progressively with time during the course of daptomycin treatment.

Excretion of extracellular lipids by Streptococcus mutans BHT and FA-1.

Streptococcus mutans BHT and FA-1, when grown to log phase on chemically defined medium containing [14C]glycerol, excreted 15% of the total biosynthesized 14C-lipid into the medium. When grown to early stationary phase, 28 to 33% of the 14C-lipid was found in the medium. The radioactive lipids of these varieties of S. mutans were identified as diacylglycerol, diglucosyl diacylglycerol (DGD), monoglucosyl diacylglycerol, diphosphatidylglycerol, phosphatidylglycerol (PG), and smaller amounts of two other lipids tentatively were identified as amino acyl-PG and glycerol phosphoryl-DGD. All lipids were found as extracellular and intracellular components from cells grown to either log or stationary phase. However, there were some shifts in the relative percentage of these lipids as the cells changed from log to stationary phase. For example, the intracellular lipid content of log-phase S. mutans BHT was composed of 49% PG and 19% DGD, but these percents shifted to 18% PG and 57% DGD when the cells were grown to stationary phase. However, the extracellular lipids of this organism contained 50 to 60% PG and 20% DGD in both log and stationary phases.

Effect of pH on the proportions of polar lipids, in chemostat cultures of Bacillus subtilis.

Significant changes in the relative proportions of the individual polar lipids of two strains of Bacillus subtilis were observed when the pH of their chemostat cultures was varied. In phosphate- and magnesium-limited cultures of B. subtilis var. niger NCIB 8058. lysylphosphatidylglycerol was present in higher proportions at low pH (5.1) than at neutral pH. With magnesium-limited cultures of this strain harvested at pH 8.0, lysylphosphatidylglycerol and phosphatidylethanolamine were not detected. Phosphate-limited cultures of B. subtilis NCIB 3610 contained no phosphatidylethanolamine or lysylphosphatidylglycerol at neutral pH, but at low pH (5.1) both these lipids were present in substantial proportions. The proportions of phosphatidylglycerol in actively dividing cells of chemostat cultures of bacilli were always greater than those of lysylphosphatidylglycerol. The reverse is commonly found in batch cultures of bacilli and staphylococci harvested at low pH. Changes in the proportions of the other polar lipids present in these bacilli (diphosphatidylglycerol and diglucosyl diacylglycerol) with pH were also noted. Certain cultures of both strains of B. subtilis contained small proportions of a peptidolipid.