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.     

Nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides ATCC 17023.

Chemical analysis of the lipopolysaccharide from Rhodopseudomonas sphaeroides ATCC 17023, isolated by the phenol-chloroform-petroleum ether method, revealed the presence of glucuronic acid, 2-keto-3-deoxyoctonate, threonine, and phosphorus in the polysaccharide moiety. The lipid A component contained glucosamine, glucosamine phosphate, amide-bound 3-oxotetradecanoic acid and 3-hydroxytetradecanoic acid, and ester-bound 3-hydroxydecanoic acid and 7-tetradecenoic acid. Structural similarity of the lipid A from R. sphaeroides ATCC 17023 to enterobacterial lipid A is indicated by the existence of a serological cross-reaction occurring between the lipid A from R. sphaeroides ATCC 17023 and that from Salmonella minnesota R595. The lipopolysaccharide and lipid A of R. sphaeroides, however, were found to be neither toxic in mice nor pyrogenic in rabbits.     

Location of fatty acids in lipid A obtained from lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023

Monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 was initially purified by silicic acid column chromatography to yield a single major pentaacyl MLA fraction. This fraction was methylated and further purified by reverse-phase high performance liquid chromatography to yield three prominent peak fractions. Laser desorption mass spectrometry of these three fractions allowed us to complete the important structural analysis of lipid A from this source. Three structurally distinct forms of dimethyl MLA were identified where Mr = 1447, 1449, and 1451 atomic mass units. These forms differed only by the presence or absence of unsaturation and keto group in the fatty acids. We established that the acyloxyacyl group (either delta 7-tetradecenoyloxytetradecanoate or tetradecanoyloxytetradecanoate) and the 3-ketotetradecanoate or hydroxytetradecanoate occupied the 2'- and 2-positions of the glucosamine disaccharide, respectively. Analysis of several minor fractions suggests that there is considerable structural heterogeneity in the MLA. With this new knowledge, the study of the structure-to-function relationship of the reported lack of toxicity of lipopolysaccharide from R. sphaeroides can be completed.     

Role of fatty acid structure in the reversible activation of phosphatidylcholine synthesis in lymphocytes

Fatty acids rapidly accelerate (1.5-7.0-fold) the incorporation of [methyl-3H]choline chloride into the phosphatidylcholine fraction of bovine lymphocyte lipids. This ability of fatty acids to activate choline phospholipid synthesis has been correlated with certain structural features of fatty acids. Mono- and polyenoic unsaturated fatty acids of 18 and 20 carbons in length are highly active, whereas their saturated analogues are nearly inactive. Among the unsaturated fatty acids, the cis-isomers are active, while the trans-isomers are relatively ineffective. The delayed addition of bovine serum albumin (5 mg/ml) and other lipid-binding proteins to activated cells rapidly counteracts the lipid effects. The activated state of the cell membrane thus appears to be a dynamic one, requiring the continued interaction of the fatty acid with a lipid-sensitive target molecule of the cell surface that in turn appears to coordinate the enzymatic components of this pathway.     

Leukocytes utilize myeloperoxidase-generated nitrating intermediates as physiological catalysts for the generation of biologically active oxidized lipids and sterols in serum

The initiation of lipid peroxidation and the concomitant formation of biologically active oxidized lipids and sterols is believed to play a central role in the pathogenesis of inflammatory and vascular disorders. Here we explore the role of neutrophil- and myeloperoxidase (MPO)-generated nitrating intermediates as a physiological catalyst for the initiation of lipid peroxidation and the formation of biologically active oxidized lipids and sterols. Activation of human neutrophils in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide (nitrogen monoxide, NO) metabolism, generated an oxidant capable of initiating peroxidation of lipids. Formation of hydroxy- and hydroperoxyoctadecadienoic acids [H(P)ODEs], hydroxy- and hydroperoxyeicosatetraenoic acids [H(P)ETEs], F(2)-isoprostanes, and a variety of oxysterols was confirmed using on-line reverse phase HPLC tandem mass spectrometry (LC/MS/MS). Lipid oxidation by neutrophils required cell activation and NO(2)(-), occurred in the presence of metal chelators and superoxide dismutase, and was inhibited by catalase, heme poisons, and free radical scavengers. LC/MS/MS studies demonstrated formation of additional biologically active lipid and sterol oxidation products known to be enriched in vascular lesions, such as 1-hexadecanoyl-2-oxovalaryl-sn-glycero-3-phosphocholine, which induces upregulation of endothelial cell adhesion and chemoattractant proteins, and 5-cholesten-3beta-ol 7beta-hydroperoxide, a potent cytotoxic oxysterol. In contrast to the oxidant formed during free metal ion-catalyzed reactions, the oxidant formed during MPO-catalyzed oxidation of NO(2)(-) readily promoted lipid peroxidation in the presence of serum constituents. Collectively, these results suggest that phagocytes may employ MPO-generated reactive nitrogen intermediates as a physiological pathway for initiating lipid peroxidation and forming biologically active lipid and sterol oxidation products in vivo.     

Acyl chain specificity of the acyltransferases LpxA and LpxD and substrate availability contribute to lipid A fatty acid heterogeneity in Porphyromonas gingivalis

Porphyromonas gingivalis lipid A is heterogeneous with regard to the number, type, and placement of fatty acids. Analysis of lipid A by matrix-assisted laser desorption ionization-time of flight mass spectrometry reveals clusters of peaks differing by 14 mass units indicative of an altered distribution of the fatty acids generating different lipid A structures. To examine whether the transfer of hydroxy fatty acids with different chain lengths could account for the clustering of lipid A structures, P. gingivalis lpxA (lpxA(Pg)) and lpxD(Pg) were cloned and expressed in Escherichia coli strains in which the homologous gene was mutated. Lipid A from strains expressing either of the P. gingivalis transferases was found to contain 16-carbon hydroxy fatty acids in addition to the normal E. coli 14-carbon hydroxy fatty acids, demonstrating that these acyltransferases display a relaxed acyl chain length specificity. Both LpxA and LpxD, from either E. coli or P. gingivalis, were also able to incorporate odd-chain fatty acids into lipid A when grown in the presence of 1% propionic acid. This indicates that E. coli lipid A acyltransferases do not have an absolute specificity for 14-carbon hydroxy fatty acids but can transfer fatty acids differing by one carbon unit if the fatty acid substrates are available. We conclude that the relaxed specificity of the P. gingivalis lipid A acyltransferases and the substrate availability account for the lipid A structural clusters that differ by 14 mass units observed in P. gingivalis lipopolysaccharide preparations.     

Identification of a chemotaxis operon with two cheY genes in Rhodobacter sphaeroides

A large chemotaxis operon was identified in Rhodobacter sphaeroides WS8-N using a probe based on the 3' terminal portion of the Rhizobium meliloti cheA gene. Two genes homologous to the enteric cheY were identified in an operon also containing cheA , cheW , and cheR homologues. The deduced protein sequences of che gene products were aligned with those from Escherichia coli and shown to be highly conserved. A mutant with an interrupted copy of cheA showed normal patterns of swimming, unlike the equivalent mutants in E. coli which are smooth swimming. Tethered cheA mutant cells showed normal responses to changes in organic acids, but increased, inverted responses to sugars. The unusual behaviour of the cheA mutant and the identification of two homologues of cheY suggests that R. sphaeroides has at least two pathways controlling motor activity. To identify functional similarity between the newly identified R. sphaeroides Che pathway and the methyl-accepting chemotaxis protein (MCP)-dependent pathway in enteric bacteria, the R. sphaeroides cheW gene was expressed in a cheW mutant strain of E. coli and found to complement, causing a partial return to a swarming phenotype. In addition, expression of the R. sphaeroides gene in wild-type E. coli resulted in the same increased tumbling and reduced swarming as seen when the native gene is over-expressed in E. coli . The identification of che homologues in R. sphaeroides and complementation by cheW suggests the presence of MCPs in an organism previously considered to use only MCP-independent sensing. The MCP-dependent pathway, appears conserved. In R. sphaeroides this pathway may mediate responses to sugars, while responses to organic acids may in involve a second system, possibly using the second CheY protein identified in this study.     

Inhibition of type 1 and type 2 5alpha-reductase activity by free fatty acids,active ingredients of Permixon

In different cell systems, the lipido-sterolic extract of Serenoa repens (LSESr, Permixon inhibits both type 1 and type 2 5alpha-reductase activity (5alphaR1 and 5alphaR2). LSESr is mainly constituted of fatty acids (90+/-5%) essentially as free fatty acids (80%). Among these free fatty acids, the main components are oleic and lauric acids which represent 65% and linoleic and myristic acids 15%.To evaluate the inhibitory effect of the different components of LSESr on 5alphaR1 or 5alphaR2 activity, the corresponding type 1 and type 2 human genes have been cloned and expressed in the baculovirus-directed insect cell expression system Sf9. The cells were incubated at pH 5.5 (5alphaR2) and pH 7.4 (5alphaR1) with 1 or 3nM testosterone in presence or absence of various concentrations of LSESr or of its different components. Dihydrotestosterone formation was measured with an automatic system combining HPLC and an on-line radiodetector.The inhibition of 5alphaR1 and 5alphaR2 activity was only observed with free fatty acids: esterified fatty acids, alcohols as well as sterols assayed were inactive. A specificity of the fatty acids in 5alphaR1 or 5alphaR2 inhibition has been found. Long unsaturated chains (oleic and linolenic) were active (IC(50)=4+/-2 and 13+/-3 microg/ml, respectively) on 5alphaR1 but to a much lesser extent (IC(50)>100 and 35+/-21 microg/ml, respectively) on 5alphaR2. Palmitic and stearic acids were inactive on the two isoforms. Lauric acid was active on 5alphaR1 (IC(50)=17+/-3 microg/ml) and 5alphaR2 (IC(50)=19+/-9 microg/ml). The inhibitory activity of myristic acid was evaluated on 5alphaR2 only and found active on this isoform (IC(50)=4+/-2 microg/ml).The dual inhibitory activity of LSESr on 5alpha-reductase type 1 and type 2 can be attributed to its high content in free fatty acids.     

Cerulenin-induced changes in the lipopolysaccharide content and phospholipid composition of Proteus mirabilis.

Inhibition of Proteus mirabilis growth by cerulenin, a specific inhibitor of fatty acid biosynthesis, was reversed by exogenously supplied fatty acid mixtures containing oleic acid and palmitic or pentadecanoic acids. The growth rate of the cells treated with cerulenin in the presence of the fatty acid mixtures was slower, however, than that of untreated cells, and their lipopolysaccharide content was decreased by 30-50%, resulting in an increased sensitivity of the organisms to rifamycin and vancomycin. Polyacrylamide gel electrophoresis of the lipopolysaccharide fraction from cerulenin-treated cells revealed that of the two P. mirabilis lipopolysaccharide types, the relative amount of the higher molecular weight lipopolysaccharide was reduced from 50% to 30% of the total lipopolysaccharide. Fatty acid analysis of the phospholipid and lipopolysaccharide fractions from cells grown with cerulenin, pentadecanoate, and oleate revealed that over 60% of the native even-numbered fatty acids of the phospholipid fraction was substituted by the odd-numbered fatty acid, while no incorporation of either the pentadecanoate or oleate could be demonstrated in the lipid A moiety of the lipopolysaccharide. The only change in the lipid A observed was an increase in the content of 3-hydroxymyristic acid accompanied by a decrease in the nonhydroxylated fatty acids, supporting the highly conserved nature of this molecule.     

Partial characterization of lipid A of intraperiplasmically grown Bdellovibrio bacteriovorus.

The lipid A components of substrate cell origin incorporated by Bdellovibrio bacteriovorus during intraperiplasmic growth (D. R. Nelson and S. C. Rittenberg, J. Bacteriol. 147:860-868, 1981) were shown to be integrated into its lipopolysaccharide structure. Lipid A isolated from bdellovibrios grown on Escherichia coli was resolved into two fractions by thin-layer chromatography. Fraction 2 had the same Rf as the single lipid A fraction of axenicaly grown bdellovibrios, and both stained identically with aniline-diphenylamine reagent. Fraction 1 resembled, in Rf and staining reaction, the slower migrating of two lipid A fractions obtained from the E.coli used as the substrate cell. Both fractions 1 and 2 contained glucosamine, a substrate cell-derived compound. Greater than 65% of the fatty acids in fraction 1 were derived from the substrate cell, whereas more than 60% of the fatty acids of fraction 2 were synthesized by the bdellovibrio. Nevertheless, each fraction contained significant amounts of fatty acid of both origins. The substrate cell-derived fatty acids had the same distribution of N-acyl and O-acyl linkages as in E. coli lipid A. The data indicate that the two lipid A moieties in lipopolysaccharide of intraperiplasmically grown bdellovibrios are hybrids of substrate cell-derived and bdellovibrio-synthesized components. The data also suggest that disaccharide units and N- and O-acyl linkages preexisting in the substrate cell lipid A may be conserved. A possible explanation for the unequal distribution of substrate cell-derived material in the two lipid A fractions of the bdellovibrio is suggested.     

Reconstitution of the lipid-depleted pyruvate oxidase system of Escherichia coli: the palmitic acid effect

Previous work has shown that the coupling of the soluble Escherichia coli pyruvate oxidase to a lipid-depleted membrane terminal electron transport system requires the addition of ubiquinone and a neutral lipid fraction (C. Cunningham and L. P. Hager (1975) J. Biol. Chem. 250, 7139-7146). The active factor present in the neutral lipid fraction has now been isolated and characterized. NMR, uv, and mass spectroscopic analysis identifies palmitic acid as the active component. A comparison of palmitic acid with other fatty acids of varying chain lengths indicates that most fatty acids having chain lengths in the range C12 to C20 have comparable activity to palmitic acid. Exceptions are stearic and arachidic acid which have greatly reduced activity. Fatty acids of C6 to C10 chain length showed about one third the activity of palmitic acid. Fatty acids having chain lengths of 2 to 5 carbon atoms are essentially inactive. The carboxyl function of the fatty acid is required for activity. Derivatives of fatty acids in which the carboxyl group had been modified to an alcohol, aldehyde, or methyl ester function show greatly diminished activity. Both the cis and trans forms of unsaturated long-chain fatty acids are active. The stimulation of the electron transfer reaction by fatty acids occurs at the ubiquinone level of the electron transport chain. Ubiquinone-30 is rapidly reduced by pyruvate oxidase only in the presence of palmitic acid.     

27-Hydroxyoctacosanoic acid is a major structural fatty acyl component of the lipopolysaccharide of Rhizobium trifolii ANU 843

A new hydroxylated, very long-chain fatty acid has been isolated and characterized from the lipopolysaccharide (LPS) of Rhizobium trifolii ANU 843. The lipid A of the organism was degraded by mild alkali and borohydride and the products methylated, peracetylated, and fractionated on a C18 reverse-phase column. The major lipid fraction was reduced with lithium triethylborohydride, methylated, peracetylated, and subjected to thin layer chromatography. The methylated peracetylated acid and the reduced diacetylated diol (1,27-dihydroxyoctacosane diacetate) were isolated and characterized by mass spectrometry and 1H NMR spectroscopy using homonuclear decoupling. The identity and linkage of the new fatty acid in the lipopolysaccharide was confirmed by 1H NMR spectroscopy of purified lipid A fractions and similar NMR studies of lipid A after acylation by phenylisocyanate. In the native LPS, the 27-hydroxy C-28 fatty acid is acylated at the 27-hydroxy position by other 3-hydroxy fatty acids. About 50% of the total fatty acid content of the LPS of R. trifolii ANU 843 is 27-hydroxyoctacosanoic acid. This oxyacyloxy structure involving 27-hydroxyoctacosanoic appears to be the major structural feature of the lipid A of this organism.     

Nature and location of amide-bound (R)-3-acyloxyacyl groups in lipid A of lipopolysaccharides from various gram-negative bacteria

It has previously been demonstrated [Eur. J. Biochem. 124, 191-198 (1982) and 137, 15-22 (1983)] that the lipid A component of Salmonella and Proteus lipopolysaccharides contains amide-linked (R)-3-acyloxyacyl residues. In the present study lipid A of other gram-negative bacteria was analysed for the presence of amide-bound 3-acyloxyacyl residues. It was found that such residues are constituents of all lipid A tested (Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeruginosa, Xanthomonas sinensis, Bacteroides fragilis, Vibrio cholerae, Fusobacterium nucleatum, Rhodospirillum tenue, Acinetobacter calcoaceticus, and Escherichia coli). Amide-linked (R)-3-acyloxyacyl groups, therefore, represent common and ubiquitous structural elements of bacterial lipid A. The composition of 3-acyloxyacyl groups differed considerably among different bacteria. As amide-bound (R)-3-hydroxy fatty acids straight chain and isobranched acyl groups with 10-17 carbon atoms were identified. The most frequently encountered fatty acids, substituting the 3-hydroxyl group of 3-hydroxy fatty acids, were nonhydroxylated straight chain and isobranched acyl residues with 10-17 carbon atoms as well as (S)-2-hydroxy fatty acids with 12 carbon atoms. In some cases, using laser desorption mass spectrometry, the distribution of 3-acyloxyacyl residues over the two available glucosamine amino groups of the lipid A backbone was investigated.     

Structural analysis of the lipopolysaccharide from Chlamydophila psittaci strain 6BC.

The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3-deoxy-Dmanno-oct-2-ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of approximately 3. 3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion-exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcpN -(1 -->6)-alpha-D-GlcpN 1,4'-bisphosphate and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)]-alpha-Kdo-(2-->4)-alpha-Kdo-(2- ->6)-beta-D-GlcpN-(1-->6)-alpha-D-GlcpN 1,4'-bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry experiments on lipid A and de-O-acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long-chain and branched (R)-configured 3-hydroxy fatty acids, the latter being exclusively present in an amide linkage.     

Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein.

One of the earliest steps in the development of the atherosclerotic lesion is the accumulation of monocyte/macrophages within the vessel wall. Oxidized lipids present in minimally modified-low density lipoproteins (MM-LDL) contribute to this process by activating endothelial cells to express monocyte-specific adhesion molecules and chemoattractant factors. A major focus of our group has been the isolation and characterization of the biologically active oxidized lipids in MM-LDL. We have previously characterized three oxidized phospholipids present in MM-LDL, atherosclerotic lesions of fat fed rabbits, and autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) that induced human aortic endothelial cells to adhere human monocytes in vitro. We have used sequential normal and reverse phase-high performance liquid chromatography to isolate various isomers of an oxidized phospholipid from autoxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. The fatty acid in the sn-2 position of this biologically active isomer and its dehydration product was released by phospholipase A(2) and characterized. Hydrogenation with platinum(IV) oxide/hydrogen suggested a cyclic moiety, and reduction with sodium borohydride suggested two reducible oxygen-containing groups in the molecule. The fragmentation pattern produced by electrospray ionization-collision induced dissociation-tandem mass spectrometry was consistent with a molecule resembling an E-ring prostaglandin with an epoxide at the 5,6 position. The structure of this lipid was confirmed by proton nuclear magnetic resonance spectroscopy analysis of the free fatty acid isolated from the dehydration product of m/z 828.5. Based on these studies, we arrived at the structure of the biologically active oxidized phospholipids as 1-palmitoyl-2-(5, 6-epoxyisoprostane E(2))-sn-glycero-3-phosphocholine. The identification of this molecule adds epoxyisoprostanes to the growing list of biologically active isoprostanes.     

Structural characterization of the lipid A component of pathogenic Neisseria meningitidis.

The lipid A component of meningococcal lipopolysaccharide was structurally characterized by using chemical modification methods, methylation analysis, 31P nuclear magnetic resonance, and laser desorption mass spectroscopy. It was shown that Neisseria meningitidis lipid A consists of a 1,4'-bisphosphorylated beta(1'----6)-linked D-glucosamine disaccharide (lipid A backbone), both phosphate groups being largely replaced by O-phosphorylethanolamine. This disaccharide harbors two nonsubstituted hydroxyl groups at positions 4 and 6', the latter representing the attachment site of the oligosaccharide portion in lipopolysaccharide. In addition, it is substituted by up to six fatty acid residues. In the major lipid A component, representing a hexaacyl species, the hydroxyl groups at positions 3 and 3' carry (R)-3-hydroxydodecanoic acid [12:0(3-OH)], whereas the amino groups at positions 2 and 2' are substituted by (R)-3-(dodecanoyloxy)tetradecanoic acid [3-O(12:0)-14:0]. A minor portion was present as a tetraacyl lipid A component lacking either dodecanoic acid (12:0) or 12:0 and 12:0(3-OH). N. meningitidis lipid A, therefore, significantly differs from Escherichia coli lipid A by the nature and locations of fatty acids and the substitution of O-phosphorylethanolamine for the nonglycosyl (4'-P) and glycosyl phosphate.     

Expression of the gltP gene of Escherichia coli in a glutamate transport-deficient mutant of Rhodobacter sphaeroides restores chemotaxis to glutamate

Rhodobacter sphaeroides is chemotactic to glutamate and most other amino acids. In Escherichia coli , chemotaxis involves a membrane-bound sensor that either binds the amino acid directly or interacts with the binding protein loaded with the amino acid. In R. sphaeroides , chemotaxis is thought to require both the uptake and the metabolism of the amino acid. Glutamate is accumulated by the cells via a binding protein-dependent system. To determine the role of the binding protein and transport in glutamate taxis, mutants were created by Tn 5 insertion mutagenesis and selected for growth in the presence of the toxic glutamine analogue γ-glutamyl-hydrazide. One of the mutants, R. sphaeroides MJ7, was defective in glutamate uptake but showed wild-type levels of binding protein. The mutant showed no chemotactic response to glutamate. Both glutamate uptake and chemotaxis were recovered when the gltP gene, coding for the H⁺-linked glutamate carrier of E. coli , was expressed in R. sphaeroides MJ7. It is concluded that the chemotactic response to glutamate strictly requires uptake of glutamate, supporting the view that intracellular metabolism is needed for chemotaxis in R. sphaeroides .     

Short-term n-3 fatty acid supplementation but not aspirin increases plasma proresolving mediators of inflammation

Resolution of inflammation is an active process involving specialized proresolving mediators (SPM) formed from the n-3 fatty acids. This study examined the effect of n-3 fatty acid supplementation and aspirin on plasma SPMs in healthy humans. Healthy volunteers (n = 21) were supplemented with n-3 fatty acids (2.4g/day) for 7 days with random assignment to take aspirin (300 mg/day) or placebo from day 5 to day 7. Blood was collected at baseline (day 0), day 5, and day 7. Plasma 18R/S-HEPE, E-series resolvins, 17R/S-HDHA, D-series resolvins, 14R/S-HDHA, and MaR-1 were measured by LC/MS/MS. At baseline concentrations of E- and D- series resolvins and the upstream precursors 18R/S-HEPE, 17R/S-HDHA ranged from 0.1nM to 0.2nM. 14R/S-HDHA was 3-fold higher than the other SPMs at baseline but MaR-1 was below the limit of detection. Supplementation with n-3 fatty acids significantly increased RvE1, 18R/S-HEPE, 17R/S-HDHA, and 14R/S-HDHA but not other SPMs. The addition of aspirin after 5 days of n-3 fatty acids did not affect concentrations of any SPM. N-3 fatty acid supplementation for 5 days results in concentrations of SPMs that are biologically active in healthy humans. Aspirin administered after n-3 fatty acids did not offer any additional benefit in elevating the levels of SPMs.     

Mutations in firA, encoding the second acyltransferase in lipopolysaccharide biosynthesis, affect multiple steps in lipopolysaccharide biosynthesis.

The product of the firA (ssc) gene is essential for growth and for the integrity of the outer membrane of Escherichia coli and Salmonella typhimurium. Recently, Kelly and coworkers (T. M. Kelly, S. A. Stachula, C. R. H. Raetz, and M. S. Anderson, J. Biol. Chem., 268:19866-19874, 1993) identified firA as the gene encoding UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase, the third step in lipid A biosynthesis. We studied the effects of six different mutations in firA on lipopolysaccharide synthesis. All of the firA mutants of both E. coli and S. typhimurium examined had a decreased lipopolysaccharide synthesis rate. E. coli and S. typhimurium strains defective in firA produced a lipid A that contains a seventh fatty acid, a hexadecanoic acid, when grown at the nonpermissive temperature. Analysis of the enzymatic activity of other enzymes involved in lipid A biosynthesis revealed that the firA mutations pleiotropically affect lipopolysaccharide biosynthesis. In addition to that of UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase, the enzymatic activity of the lipid A 4' kinase (the sixth step of lipid A biosynthesis) was decreased in strains with each of the firA mutations examined. However, overproduction of FirA was not accompanied by overexpression of the lipid A 4' kinase.     

Analysis of the motA flagellar motor gene from Rhodobacter sphaeroides a bacterium with a unidirectional, stop-start flagellum

Rhodobacter sphaeroides swims by unidirectional rotation of a single medial flagellum, re-orienting randomly by Brownian motion when flagellar rotation tops and restarts. Previously we identified a mutant with a paralysed flagellum, which was complemented by a Rhodobacter gene that had homology to motB of Escherichia coli , a bacterium with bidirectional flagella. In the current work, interposon mutagenesis upstream of the Rhodobacter motB gene gave rise to another paralysed mutant, RED5. DNA sequence analysis of this upstream region showed one open reading frame, the predicted polypeptide sequence of which shows homology to the MotA protein of E. coli . MotA is thought to be a proton 'pore' involved in converting proton-motive force into flagellar rotation. Several potential proton-binding amino acids were conserved between putative membrane-spanning regions of R. sphaeroides and E. coli MotA sequences, along with a highly charged cytoplasmic linker region. Complementation studies with mutant RED5 showed the presence of an active promoter upstream from motA which was found to be necessary for expression of both motA and motB , Examination of the upstream DNA sequence showed only one putative promoter-like sequence which resembled a σ⁵⁴- type promoter, including a potential enhancer binding site. The overall similarities between the R. sphaeroides MotA protein and those from other bacteria suggest that, despite the novel unidirectional rotation of he R. sphaeroides flagellum, the function of the MotA protein is similar to that in bacteria with bidirectional flagella.