Lipopolysaccharides of Thiocystis violacea, Thiocapsa pfennigii, and Chromatium tepidum, species of the family Chromatiaceae.

The lipopolysaccharides (LPS) of three species of purple sulfur bacteria (Chromatiaceae), Thiocystis violacea, Thiocapsa pfennigii, and the moderately thermophilic bacterium Chromatium tepidum, were isolated. The LPS of Thiocystis violacea and Chromatium tepidum contained typical O-specific sugars, indicating O-chains. Long O-chains were confirmed for these species by sodium deoxycholate gel electrophoresis of their LPS. Thiocapsa pfennigii, however, had short or no O-chains. The core region of the LPS of all three species comprised D-glycero-D-mannoheptose as the only heptose and 2-keto-3-deoxyoctonate. The lipid A, obtained from the LPS by mild acid hydrolysis, contained glucosamine as the main amino sugar. Amide-bound 3-hydroxymyristic acid was the only hydroxy fatty acid. The main ester-bound fatty acid in all lipid A fractions was 12:0. Mannose and small amounts of 2,3-diamino-2,3-dideoxy-D-glucose were common constituents of the lipid A of the three Chromatiaceae species investigated. All lipid A fractions were essentially free of phosphate.     

Structural studies on the lipid A component of enterobacterial lipopolysaccharides by laser desorption mass spectrometry. Location of acyl groups at the lipid A backbone

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Escherichia coli and Proteus mirabilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-O-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN II) of the backbone carries, in S. minnesota and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible. It was found that: (a) in all three lipids A GlcN II is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. minnesota and P. mirabilis. In lipids A which were obtained from the P. mirabilis Re mutant grown at low temperature (12 degrees C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetra-decanoic acid at GlcN II is positionally replaced by delta 9-hexadecenoic acid (palmitoleic acid). It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to GlcN II are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to GlcN I are free or only partially substituted.     

Structural analysis of Francisella tularensis lipopolysaccharide.

The structure of the lipid A and core region of the lipopolysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a beta-GlcN-(1-6)-GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O-1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O-3 and O-4 of beta-GlcN, and no substituent at O-4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH4 reduced LPS had the following structure: where Delta-GalNA-(1-3)-beta-QuiNAc represents a modified fragment of the O-chain repeating unit. Two shorter oligosaccharides lacking the O-chain fragment were also identified. A minor amount of the disaccharide beta-GlcN-(1-6)-alpha-GlcN-1-P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end. The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2-N-(3-O-acyl4-acyl2)-beta-GlcN-(1-6)-2-N-acyl1-3-O-acyl3-GlcN where acyl1, acyl2 and acyl3 are 3-hydroxyhexadecanoic or 3-hydroxyoctadecanoic acids, acyl4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH-1 of the reducing end glucosamine, and OH-3 and OH-4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure.     

Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota strain R7 (chemotype Rd1P−)

Abstract Rabbit polyclonal antibodies against the lipopolysaccharide (LPS) of the Rd₁P⁻ mutant strain R7 of Salmonella minnesota were serologically characterized using R7 LPS, dephosphorylated LPS, deacylated LPS, deacylated, dephosphorylated and reduced LPS, and synthetic partial structures. The latter comprised partial structures of the core region of Rd₁P⁻ LPS bound to the β 1 → 6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or artificial glycoconjugates comprised of the synthetic oligosaccharides coupled to bovine serum albumin. Using a passive hemolysis and an enzyme immunoassay, absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core region and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The majority of phosphate-independent antibodies was directed against the trisaccharide l -glycero-α- d -manno-heptopyranose(1 → 3)- l -glycero-α- d -manno-heptopyranose(1 → 5)3-deoxy- d -manno-octulosonic acid. Antibodies against the 1 → 3- and 1 → 7-linked heptose disaccharides and against a single heptose were also detected, however, with low titers. No antibodies were found which required the presence of fatty acids.     

Isolation and structural analysis of two lipid A precursors from a KDO deficient mutant of Salmonella typhimurium differing in their hexadecanoic acid content

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-d-manno-octulosonic acid region of lipopolysaccharides.Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue.The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-d-manno-octulosonic acid and/or other polar substituents.Abbreviations LDMS laser desorption mass spectrometry - KDO 3-Deoxy-d-manno-octulosonic acid - Ts5 Salmonella typhimurium mutant Ts5 - PCP phenol/chloroform/petroleum ether - H₂F₂ hydrogen fluoride This work is dedicated to Prof. Dr. Drews, Freiburg, on the occasion of his 60th birthday     

Characterization of the lipopolysaccharide and beta-glucan of the fish pathogen Francisella victoria

Lipopolysaccharide (LPS) and beta-glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods. The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3-aminobutyric acid, and a novel branched amino acid, thus confirming F. victoria as a unique species. The structure identified composes the largest oligosaccharide elucidated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, including the first application of H2BC sequence to oligosaccharides. The non-phosphorylated lipid A region of the LPS was identical to that of other Francisellae, although one of the lipid A components has not been found in Francisella novicida. The heptoseless core-lipid A region of the LPS contained a linear pentasaccharide fragment identical to the corresponding part of F. tularensis and F. novicida LPSs, differing in side-chain substituents. The linkage region of the O-chain also closely resembled that of other Francisella. LPS preparation contained two characteristic glucans, previously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual beta-(1-6)-glucan with (glycerol)2phosphate at the reducing end.     

Correlation of the biologic responses of C3H/HEJ mice to endotoxin with the chemical and structural properties of the lipopolysaccharides from Pseudomonas aeruginosa and Escherichia coli

The basis of the biologic responses of C3H/HeJ mice to endotoxin administration in relation to the structural linkages in the lipid A portion of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa and Escherichia coli were investigated. P. aeruginosa LPS was found to be immunogenic, mitogenic, and toxic, but not lethal, in C3H/HeJ mice. The observed mitogenicity in spleen cells was directed toward immunoglobulin- (Ig) bearing cells, was present in response to isolated and solubilized lipid A, and was inhibitable by polymixin B. The P. aeruginosa LPS was chemically analyzed in order to define its composition and exclude the presence of contaminating proteins being responsible for the biologic responses of C3H/HeJ mice that were observed. Structural analysis of the linkages of the fatty acids to the glucosamine backbone in the lipid A of P. aeruginosa and E. coli revealed similarities in terms of the ratio of hydroxy fatty acids to straight chain fatty acids and the way in which these 2 types of fatty acids were linked to the backbone. Differences were seen in the carbon chain length of the fatty acid substituents, and the substituent on the hydroxy fatty acid that is directly ester linked to the glucosamine backbone. These data indicate that the refractivity of C3H/HeJ mice to the biologic effects after the administration of Gram-negative endotoxins may be limited to enterobacterial LPS. Those differences we found in the chain length and/or linkages of the fatty acid substituents in the lipid A portion of the LPS between P. aeruginosa and E. coli may be sufficient to render C3H/HeJ mice responsive to the biologic effects of nonenterobacterial endotoxins.     

Incorporation of Substrate Cell Lipid A Components into the Lipopolysaccharide of Intraperiplasmically Grown Bdellovibrio bacteriovorus

The composition of Bdellovibrio bacteriovorus lipopolysaccharide (LPS) was determined for cells grown axenically and intraperiplasmically on Escherichia coli or Pseudomonas putida. The LPS of axenically grown bdellovibrios contained glucose and fucosamine as the only detectable neutral sugar and amino sugar, and nonadecenoic acid (19:1) as the predominant fatty acid. Additional fatty acids, heptose, ketodeoxyoctoic acid, and phosphate were also detected. LPS from bdellovibrios grown intraperiplasmically contained components characteristic of both axenically grown bdellovibrios and the substrate cells. Substrate cell-derived LPS fatty acids made up the majority of the bdellovibrio LPS fatty acids and were present in about the same proportions as in the substrate cell LPS. Glucosamine derived from E. coli LPS amounted to about one-third of the hexosamine residues in intraperiplasmically grown bdellovibrio LPS. However, galactose, characteristic of the E. coli outer core and O antigen, was not detected in the bdellovibrio LPS, suggesting that only lipid A components of the substrate cell were incorporated. Substrate cell-derived and bdellovibrio-synthesized LPS materials were conserved in the B. bacteriovorus outer membrane for at least two cycles of intraperiplasmic growth. When bdellovibrios were grown on two different substrate cells successively, lipid A components were taken up from the second while the components incorporated from the lipid A of the first were conserved in the bdellovibrio LPS. The data show that substrate cell lipid A components were incorporated into B. bacteriovorus lipid A during intraperiplasmic growth with little or no change, and that these components, fatty acids and hexosamines, comprised a substantial portion of bdellovibrio lipid A.     

Chemical analysis of and degradation studies on the cell wall lipopolysaccharide of Rhodopseudomonas capsulata

A lipopolysaccharide (LPS) has been isolated from the gram-negative photosynthetic bacterium Rhodopseudomonas capsulata. Chemical analysis revealed the presence of d-glucose, d-galactose, l-rhamnose, 3-O-methyl-l-rhamnose (l-acofriose), d-glucosamine, 2-keto-3-deoxyoctonate, and neuraminic acid. The LPS does not contain l-glycero-d-mannoheptose, a typical component of the LPS of enteric bacteria. Fatty acid analysis showed that, apart from lauric acid, two hydroxy fatty acids (hydroxycaproic and hydroxymyristic acids) are the main components. By hydrolysis in weak acid, the LPS has been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Presumably the lipid A contains a glucosamine backbone. Whereas the OH-groups of glucosamine are esterified with lauric and hydroxycaproic acids, hydroxymyristic acid is linked to the amino group of the sugar. By separation of the degraded polysaccharide by gel filtration, a fraction has been isolated which inhibited hemagglutination in a system containing antiserum, obtained by immunization of rabbits with whole cells, and isolated LPS. This fraction, which includes the determinant group, contains the sugars glucose, rhamnose, and acofriose. A second fraction obtained in this way was found to be serologically inactive and is composed of glucose, galactose, neuraminic acid, and phosphate.     

Structure and serological characterization of the O-antigen of Proteus mirabilis O18 with a phosphocholine-containing oligosaccharide phosphate repeating unit

A phosphorylated, choline-containing polysaccharide was obtained by O-deacylation of the lipopolysaccharide (LPS) of Proteus mirabilis O18 by treatment with aqueous 12% ammonia, whereas hydrolysis with dilute acetic acid resulted in depolymerisation of the polysaccharide chain by the glycosyl phosphate linkage. Treatment of the O-deacylated LPS with aqueous 48% hydrofluoric acid cleaved the glycosyl phosphate group but, unexpectedly, did not affect the choline phosphate group. The polysaccharide and the derived oligosaccharides were studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the pentasaccharide phosphate repeating unit was established: [carbohydrate structure in text] Where ChoP=Phosphocoline Immunochemical studies of the LPS, O-deacylated LPS and partially dephosphorylated pentasaccharide using rabbit polyclonal anti-P. mirabilis O18 serum showed the importance of the glycosyl phosphate group in manifesting the serological specificity of the O18-antigen.     

Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides.

The chemical structure of free lipid A isolated from rough- and smooth-form lipopolysaccharides (R-LPS and S-LPS, respectively) of the human gastroduodenal pathogen Helicobacter pylori was elucidated by compositional and degradative analysis, nuclear magnetic resonance spectroscopy, and mass spectrometry. The predominant molecular species in both lipid A components are identical and tetraacylated, but a second molecular species which is hexaacylated is also present in lipid A from S-LPS. Despite differences in substitution by acyl chains, the hydrophilic backbone of the molecules consisted of beta(1,6)-linked D-glucosamine (GlcN) disaccharide 1-phosphate. Because of microheterogeneity, nonstoichiometric amounts of ethanolamine-phosphate were also linked to the glycosidic hydroxyl group. In S-LPS, but not in R-LPS, the hydroxyl group at position 4' was partially substituted by another phosphate group. Considerable variation in the distribution of fatty acids on the lipid A backbone was revealed by laser desorption mass spectrometry. In tetraacyl lipid A, the amino group of the reducing GlcN carried (R)-3-hydroxyoctadecanoic acid (position 2), that of the nonreducing GlcN carried (R)-3-(octadecanoyloxy)octadecanoic acid (position 2'), and ester-bound (R)-3-hydroxyhexadecanoic acid was attached at position 3. Hexaacyl lipid A had a similar substitution by fatty acids, but in addition, ester-bound (R)-3-(dodecanoyloxy)hexadecanoic acid or (R)-3(tetradecanoyloxy)hexadecanoic acid was attached at position 3'. The predominant absence of ester-bound 4'-phosphate and the presence of tetraacyl lipid A with fatty acids of 16 to 18 carbons in length differentiate H. pylori lipid A from that of other bacterial species and help explain the low endotoxic and biological activities of H. pylori LPS.     

Isolation and characterization of lipopolysaccharides from cell walls of blue-green algae of the genus Phormidium.

Lipopolysaccharide (LPS) fractions were isolated from three species of blue-green algae of the genus Phormidium, namely, P. africanum, P. laminosum, and P. uncinatum, by using a phenol-water procedure followed by exhaustive extraction with ammonium oxalate. The materials obtained were shown to be closely related biochemically. Nearly 60% of the LPS consisted of the polysaccharides galactose, glucose, mannose, xylose, arabinose, and rhamnose and an unidentified, fast-moving sugar residue. In addition, glucosamine, galactosamine, and 2-keto-3-deoxyoctonate were detected. Oleic, stearic, and palmitic acids were found in the hydrolysate of the lipid component, which averaged 1.5% of the LPS. Concomitantly, the protein component (7 to 20%) was shown to contain the following amino acids: aspartic acid, threonine, serine glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, and arginine. Whole cells, as well as the LPS, of Phormidium possessed antigenic properties.     

Protective effects of several amino acid-nutrients on gastric hemorrhagic erosions in acid-irrigated stomachs of septic rats

Our previous report demonstrates that severe gastric mucosal damage is produced in lipopolysaccharide (LPS)-intoxicated rats. In the present study, we examined protective effects of several amino acids including taurine, phenylalanine and L-Arginine on gastric hemorrhagic erosions in acid-irrigated stomachs of LPS rats. The animals were deprived of food for 24 hr. Intravenous LPS (3 mg/kg) was challenged 12 hr after withdrawal of food. Gastric vagotomy was performed, followed by irrigation the stomachs for 3 hr with a physiological acid solution containing 100 mM HCl and 54 mM NaCl. The ulcerogenic parameters including increased gastric acid back-diffusion, mucosal histamine concentrations, lipid peroxide productions, luminal hemoglobin contents, stomach erosions and the lowered glutathione levels were markedly enhanced in LPS rat stomachs irrigated with acid solution. Both phenylalanine and taurine caused dose-dependent attenuations of these ulcerogenic parameters in LPS rats. L-arginine also was effective in inhibition. The inhibitory effect was restored by pretreatment of nitric oxide synthase inhibitors, such as N(G)-nitro-L-arginine-methyl ester or L-N(G)-(1-iminoethyl)-lysine. Furthermore, marked amelioration of hemorrhagic erosions in LPS rats was observed when a combination of these amino acid nutrients was used. The results provide evidence that these amino acid nutrients may ameliorate gastric hemorrhagic erosion via GSH synthesis stimulation, histamine cell membrane stabilization and antioxidant actions in LPS rat stomachs.     

Structural study on lipid A and the O-specific polysaccharide of the lipopolysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn's disease.

Bacteroides vulgatus has been shown to be involved in the aggravation of colitis. Previously, we separated two potent virulence factors, capsular polysaccharide (CPS) and lipopolysaccharide (LPS), from a clinical isolate of B. vulgatus and characterized the structure of CPS. In this study, we elucidated the structures of O-antigen polysaccharide (OPS) and lipid A in the LPS. LPS was subjected to weak acid hydrolysis to produce the lipid A fraction and polysaccharide fraction. Lipid A was isolated by preparative TLC, and its structure determined by MS and NMR to be similar to that of Bacteroides fragilis except for the number of fatty acids. The polysaccharide fraction was subjected to gel-filtration chromatography to give an OPS-rich fraction. The structure of OPS was determined by chemical analysis and NMR spectroscopy to be a polysaccharide composed of the following repeating unit: [-->4)alpha-L-Rhap(1-->3)beta-D-Manp(1-->].     

Free amino acid and protein titers inAnthonomus grandis larvae venomized byBracon mellitor

Changes in the free amino acid levels of extracts from body whole homogenates of 3rd instar boll weevilAnthonomus grandis Boheman (Curculionidae) larvae which had been stung once by females of the ectoparasitoidBracon mellitor Say (Braconidae) were determined. Stung larvae exhibited an initial rise in free amino acid levels within 6 min after the venom was injected followed by a decline within 2h. A second free amino acid peak occurred 4 days after initial sting. Observed increases in free amino acid levels were always followed by synchronized decreases in total soluble proteins. The possible nutritional regulatory effect of ectoparasitoid venom on the physiology of the boll weevil is discussed.     

Incorporation of ethanolamine into insulin-sensitive glycosylated phosphatidylinositol of chick embryo fibroblasts

Insulin sensitive glycosylated phosphatidylinositol (GPI) from chick embryo fibroblasts was isolated and partially characterized. [(3)H]Ethanolamine was incorporated into lipids different from phosphatidylethanolamine, as shown by two sequential thin layer chromatographies (TLC) using an acidic solvent system followed by a basic solvent system. Other isotopes, myo-[(3)H]inositol, [(3)H]glucosamine, [(3)H]galactose, and [(3)H]palmitic acid were also incorporated into these lipids. These lipids were separated into two peaks on the second basic TLC, designated as peaks I and II from the origin. Insulin stimulation of cells caused a rapid breakdown of these two lipids. These two lipids were treated by nitrous acid and phosphatidylinositol-specific phospholipase C (PI-PLC). The radioactivity of peak I lipid was decreased by both treatments, and that of peak II lipid was also decreased by PI-PLC treatment but not significantly by nitrous acid treatment. Peak II lipid did not fulfill the criteria for GPI. Tritium released by the treatment of PI-PLC of peak I lipid was recovered in the aqueous phase. [(3)H]Ethanolamine-labeled peak I lipid was hydrolyzed by acid treatment and the hydrolysis products were analyzed by TLC and high performance liquid chromatography (HPLC). Tritium label was recovered as native label at the rate of 95%. [(3)H]Ethanolamine of peak I lipid was reductively methylated completely with formaldehyde and cyanoborohydride, as shown by HPLC analysis. The results indicate that peak I lipid contains primary ethanolamine as a glycan component and is insulin-sensitive free GPI.     

Absence of a characteristic cell wall lipopolysaccharide in the phototrophic bacterium Chloroflexus aurantiacus.

Two strains of the gliding phototrophic bacterium Chloroflexus aurantiacus were investigated for the presence of lipopolysaccharide (LPS). With both strains, all fractions of hot phenol-water extracts and the extracted cell residues from whole cells or cell homogenates were found to be free from characteristic LPS constituents, such as 3-hydroxy fatty acids, 2-keto-3-deoxyoctonate, heptoses, or O-chain sugars. Phenolchloroform-petroleum ether extracts were also free from precipitable LPS. A lipid A fraction could not be obtained, and there was no hint for glucosamine as a possible lipid A backbone amino sugar. Absence of LPS was confirmed by sodium deoxycholate gel electrophoresis.     

Dephosphorylated NPr of the nitrogen PTS regulates lipid A biosynthesis by direct interaction with LpxD

Bacterial phosphoenolpyruvate-dependent phosphotransferase systems (PTS) play multiple roles in addition to sugar transport. Recent studies revealed that enzyme IIA^Ntr of the nitrogen PTS regulates the intracellular concentration of K⁺ by direct interaction with TrkA and KdpD. In this study, we show that dephosphorylated NPr of the nitrogen PTS interacts with Escherichia coli LpxD which catalyzes biosynthesis of lipid A of the lipopolysaccharide (LPS) layer. Mutations in lipid A biosynthetic genes such as lpxD are known to confer hypersensitivity to hydrophobic antibiotics such as rifampin; a ptsO (encoding NPr) deletion mutant showed increased resistance to rifampin and increased LPS biosynthesis. Taken together, our data suggest that unphosphorylated NPr decreases lipid A biosynthesis by inhibiting LpxD activity.     

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.     

Identification of phosphorylated 3-deoxy-manno-octulosonic acid as a component of Haemophilus influenzae lipopolysaccharide.

A phosphorylated 3-deoxy-manno-octulosonic acid (KDO) was released from the lipopolysaccharide (LPS) of the deep rough mutant (Rb+169) of Haemophilus influenzae by acid hydrolysis. Both phosphorylated and dephosphorylated KDO, produced by treatment with alkaline phosphatase, were identified by gas chromatography-mass spectrometry after trimethylsilylation. This technique provides a rapid and reliable method for the identification of phosphorylated KDO in LPS.