Characterization Of Lipopolysaccharide Research Articles

Top-Down Characterization of Bacterial Lipopolysaccharides and Lipooligosaccharides Using Activated-Electron Photodetachment Mass Spectrometry.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are located in the outer membrane of Gram-negative bacteria and are comprised of three distinctive parts: lipid A, core oligosaccharide (OS), and O-antigen. The structure of each region influences bacterial stability, toxicity, and pathogenesis. Here, we highlight the use of targeted activated-electron photodetachment (a-EPD) tandem mass spectrometry to characterize LPS and LOS from two crucial players in the human gut microbiota, Escherichia coli Nissle and Bacteroides fragilis. a-EPD is a hybrid activation method that uses ultraviolet photoirradiation to generate charge-reduced radical ions followed by collisional activation to produce informative fragmentation patterns. We benchmark the a-EPD method for top-down characterization of triacyl LOS from E. coli R2, then focus on characterization of LPS from E. coli Nissle and B. fragilis. Notably, a-EPD affords extensive fragmentation throughout the backbone of the core OS and O-antigen regions of LPS from E. coli Nissle. This hybrid approach facilitated the elucidation of structural details for LPS from B. fragilis, revealing a putative hexuronic acid (HexA) conjugated to lipid A.

Synthesis and characterization of lipopolysaccharide (LPS) anchored polystyrene microparticles as a synthetic model system for attachment studies

Outer membrane lipopolysaccharides (LPS) play a crucial role in determining attachment behavior and pathogenicity of bacteria. The aim of this study was to develop a simple procedure for anchoring bacterial lipopolysaccharides to polystyrene (PS) microparticles as a model system for in situ attachment studies. By using a swell-capture methodology, commercially available LPS of Pseudomonas aeruginosa (strain ATCC 27316 serotype 10.22) was anchored onto PS microparticles in a proof-of-concept study. A detailed chemical and morphological characterization has proven the success of LPS incorporation. It was shown that the coverage and structure of the LPS film was concentration dependent. The procedure can easily be adapted to LPS of other bacterial strains to generate a synthetic model toolkit for attachment studies.

A short history of innate immunity

Innate immunity refers to the mechanisms responsible for the first line of defense against pathogens, cancer cells and toxins. The innate immune system is also responsible for the initial activation of the body's specific immune response (adaptive immunity). Innate immunity was studied and further developed in parallel with adaptive immunity beginning in the first half of the 19th century and has been gaining increasing importance to our understanding of health and disease. In the present overview, we describe the main findings and ideas that contributed to the development of innate immunity as a continually expanding branch of modern immunology. We start with the toxicological studies by Von Haller and Magendie, in the late 18th and early 19th centuries, and continue with the discoveries in invertebrate immunity that supported the discovery and characterization of lipopolysaccharide (LPS) and pattern recognition receptors that led to the development of the pattern recognition and danger theory.

Extraction and Characterization of Lipopolysaccharide from Salmonella typhi

There is no standard process available for high purity LPS isolation, and it may be appropriate to mix two purification steps. For Salmonella typhi LPS that were subjected to normal microbiological & biochemical screening protocols, updated phenol-water extraction protocol & non-phenolic extraction methods were also used in present research. Pellet of crude LPS obtained with wet weight was 2.0 gm. yield of LPS was more by hot phenol method 1.94 mg/ml as compared to non-phenolic method 0.40 mg/ml. relative purity of LPS obtained by hot phenol method was more as compared to non-phenolic method, as protein content was 23.60 mg/ml in LPS extracted from hot phenol method & 26.62 mg/ ml in LPS extracted from non-phenolic method. However, Nucleic acid contain was comparable in LPS extracted from both methods. Qualitative analysis showed ladder like bands of LPS extracted by hot phenol method as compared to single band obtained for LPS extracted by non-phenolic method. Findings of silver staining clearly revealed ladder pattern of multi-rung bands that are characteristics of smooth form of gram negative bacteria due to difference in length of carbohydrate chain of O-antigen component. In order to clarify diseased conditions & better work on LPS profiling, this research may be crucial to lead to more successful diagnosis and care.

Evaluation of a modified method of extraction, purification, and characterization of lipopolysaccharide (O antigen) from Salmonella Typhimurium.

Background and Aim:Lipopolysaccharide (LPS) is an integral part of the outer cell membrane complex of Gram-negative bacteria. It plays an important role in the induction and stimulation of the immune system. Various LPS purification protocols have been developed. However, analysis of their efficacy is limited by contamination during downstream applications or the public health hazard of LPS. The aim of this study was to evaluate a modified method for extracting LPS as well as assess the purity of the extracted LPS by high-performance liquid chromatography (HPLC) analysis. Further, we evaluated its immunopotentiating function by measuring the relative RNA expression levels of splenic immune-related genes such as interleukin 1β (IL-1β) and interferon-γ (IFN-γ), after intramuscular injection of increasing concentrations of the extracted LPS in specific pathogen-free (SPF) chick.Materials and Methods:Isolation, identification, and serotyping of Salmonella Typhimurium were performed using chicken flocks. We then performed molecular typing of Salmonella isolates using conventional polymerase chain reaction (PCR). A new protocol for purification of LPS from Salmonella isolate (S. Typhimurium) was conducted. HPLC analysis of the extracted LPS in the current study was compared to existing methods. An in vivo study was performed to evaluate the ability of LPS to induce an immune response by measuring relative IFN-γ and IL-1β gene expression after injecting increasing concentrations of the extracted LPS into SPF chicks.Results:Isolation and serotyping revealed that Salmonella enterica was of the serovar Typhimurium. Confirmation was conducted by molecular typing through conventional PCR. Fractionation of the LPS extract by HPLC revealed a high degree of purity comparable with standard commercial LPS. These results demonstrate the high purity of extracted LPS based on our modified method using propanol and sodium hydroxide mixture. Intramuscular injection of the extracted LPS in 22 day-old SPF chicks, compared to the negative control, revealed significant upregulation of IFN-γ and slight downregulation of IL-1β.Conclusion:The new modified method can be used for high purity LPS extraction and demonstrates effective immunopotentiating activity.

Neisseria meningitidis: analysis of pili and LPS in emerging Brazilian strains.

Background:Neisseria meningitidis is the main cause of bacterial meningitis in Brazil, where the main serogroups isolated are B and C; however, the serogroup W has recently emerged. LPS and type IV pili are important virulence factors that increase meningococci pathogenicity.Methods:The characterization of Lipopolysaccharide (LPS) and type IV pili in 19 meningococci strains of serogroup B, 21 of serogroup C, 45 of serogroup W and 28 of serogroup Y, isolated in Brazil between 2011 and 2017, was conducted using the Enzyme-linked Immunosorbent Assay (Dot- ELISA) technique and monoclonal antibodies.Results:We would like to emphasize the importance of characterizing relevant antigens, such as pili and LPS, the use of monoclonal antibodies to support it, and how such studies improve vaccine development and monitoring. Most of the strains studied presented L3,7,9 LPS and type IV pili; both antigens are associated with the capacity to cause invasive disease.Conclusion:Due to the impact of meningococcal disease, it is important to maintain and improve vaccine studies. Epitopes characterization provides data about the virulence of circulating strains. The use of monoclonal antibodies and serological techniques are relevant and support vaccine development.

Preparation and characterization of lipopolysaccharide (LPS) monolayers for investigating their role in bacterial adhesion

This study was conducted to determine the role played by the length variations and heterogeneous spatial distribution of lipopolysaccharide (LPS) molecules, in gram-negative cells, in mediating adhesion to surfaces. We used extracted LPS molecules from four representative bacterial strains, prepared single molecule thick LPS monolayers using Langmuir-Blodgettry on hydrophobic substrates and used them as model surfaces for AFM studies using nano probes. The prepared LPS monolayers were uniformly distributed over the substrates and enabled preliminary force investigations. Such model monolayers are better alternative to whole-cell analysis and are expected to enable the formulation of an interfacial model for bacterial adhesion.

Disruption of the E. coli LptC dimerization interface and characterization of lipopolysaccharide and LptA binding to monomeric LptC.

Lipopolysaccharide (LPS) is an essential element of nearly all Gram-negative bacterial outer membranes and serves to protect the cell from adverse environmental stresses. Seven members of the lipopolysaccharide transport (Lpt) protein family function together to transport LPS from the inner membrane (IM) to the outer leaflet of the outer membrane of bacteria such as Escherichia coli. Each of these proteins has a solved crystal structure, including LptC, which is a largely periplasmic protein that is associated with the IM LptB2 FG complex and anchored to the membrane by an N-terminal helix. LptC directly binds LPS and is hypothesized to be involved in the transfer of LPS to another periplasmic protein, LptA. Purified and in solution, LptC forms a dimer. Here, point mutations designed to disrupt formation of the dimer are characterized using site-directed spin labeling double electron electron resonance (DEER) spectroscopy, light scattering, circular dichroism, and computational modeling. The computational studies reveal the molecular interactions that drive dimerization of LptC and elucidate how the disruptive mutations change this interaction, while the DEER and light scattering studies identify which mutants disrupt the dimer. And, using electron paramagnetic resonance spectroscopy and comparing the results to the previous quantitative characterization of the interactions between dimeric LptC and LPS and LptA, the functional consequences of monomeric LptC were also determined. These results indicate that disruption of the dimer does not affect LPS or LptA binding and that monomeric LptC binds LPS and LptA at levels similar to dimeric LptC.

Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

A prerequisite to the discovery and characterization of lipopolysaccharide (LPS) interaction with specific receptors and the resulting pathophysiological effects is the comprehensive structural analysis of LPS species. This brief review is aimed to summarize the use of gel electrophoresis linked to other biochemical technologies for detecting and characterizing LPSs. Lipopolysaccharide aggregates alone or mixtures containing LPSs and proteins/peptides can be separated by native agarose gel electrophoresis (NAGE), after which LPSs are detected with imidazole and zinc salts. A double-staining process with Coomassie brilliant blue R-250 enables the use of NAGE for detecting and studying protein-LPS interactions. For compositional analysis, the LPS aggregates are separated, with high resolution, by surfactant-polyacrylamide gel electrophoresis. After reverse staining with zinc-imidazole and elution from gel microparticles, glycoform-specific LPSs are ready for structural and biological analysis. For sequence analysis based on tandem electrospray ionization mass spectrometry (ESI-MS/MS) of oligosaccharides, LPSs are subjected to mild acid hydrolysis, dephosphorylation and permethylation. Also, O-deacylated LPS forms can be analyzed by matrix-assisted laser desorption/ionization-time of flight-MS. By comparison to spectra of unpurified LPSs, mass spectra of the micropurified LPSs show reduce heterogeneity and increased signal-to-noise ratios. Furthermore, the micropurification of LPSs prior to MS allows a higher sensitivity of detection for less abundant LPS glycoforms. The micropurified LPS fractions can be used to form self-assembled nanoaggregates which may be detected by dynamic light scattering. The effect of the O-side chain length on the Z-potential of LPS aggregates may be estimated by measurements based on laser Doppler electrophoresis. The thus obtained glycoform-specific LPSs are not only intact chemically but also biologically active as tested by e.g. Limulus amoebocyte lysate test, TNF-α assay and agonistic effect on human Toll-like receptor 4.

Characterization of the lipopolysaccharide induced model of Parkinson's disease: Role of oxidative stress and neuroinflammation

Abstract Introduction Primary pathology underlying Parkinson's disease (PD) is the loss of dopaminergic neurons in the substantia nigra (SN). A variety of genetic and environmental factors underlie this loss of dopaminergic neurons. However, recent studies have highlighted the role of elevated oxidative stress and the pro-inflammatory responses contributing to or exacerbating the nigrostriatal degeneration. Methods With the establishment of neuroinflammation as an important process involved in the PD pathogenesis, in the present study this pathogenic feature was replicated in animals using lipopolysaccharide (LPS) (5 ug/5 ul PBS) infused stereotaxically into the SN of rats. Results LPS injected into the SN successfully replicated the pathogenic features of PD in rats as it elicited an inflammatory response via action of microglia. LPS infusion resulted in glial cell activation as depicted from immunohistochemistry (IHC) analysis of GFAP and Iba-1. Also, a significant increase in the mRNA expression of proinflammatory cytokines, i.e. TNF-α and IL-1β, was observed after 7 days of LPS infusion whereas the alterations in the oxidative stress markers, i.e ROS, lipid peroxidation, NO formation, NADPH oxidase activity, glutathione system, SOD and catalase, became highly significant after 14 days of infusion. As a consequence, after 21 days of LPS infusion we observed activation of apoptotic pathway indicated by increased expression of caspases 3 and caspase 9. This was followed by a significant decline in the expression of tyrosine hyroxylase (TH) as revealed by IHC. Further, there was a marked decrease in the level of dopamine and its metabolites enough for the production of behavioral abnormality in rats. Conclusion Hence, the present study provides extensive characterization of LPS induced model of PD. Study also confirms the co-existence and complex interplay between inflammation and oxidative stress contributing equally to the dopaminergic neuronal degeneration process in PD.

Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli

Certain Shiga toxin-producing Escherichia coli (STEC) are virulent human pathogens that are most often acquired through contaminated food. The United States Department of Agriculture, Food Safety and Inspection Service has declared several serogroups of STEC as adulterants in non-intact raw beef products. Hence, sensitive and specific tests for the detection of these STEC are a necessity for implementation in food safety programs. E. coli serogroups are identified by their respective O-antigen moiety on the lipopolysaccharide (LPS) macromolecule. We propose that the development of O-antigen-specific immunological assays can facilitate simple and rapid discriminatory detection of STEC in beef. However, the resources (antigens and antibodies) required for such development are not readily available. To overcome this, we extracted and characterized LPS and O-antigen from six STEC strains. Using hot phenol extraction, we isolated the LPS component from each strain and purified it using a series of steps to eliminate proteins, nucleic acids, and lipid A antigens. Antigens and crude LPS extracts were characterized using gel electrophoresis, immunoblotting, and modified Western blotting with commercially available antibodies, thus assessing the serogroup specificity and sensitivity of available ligands as well. The results indicate that, while many commercially available antibodies bind LPS, their activities and specificities are highly variable, and often not as specific as those required for serogroup discrimination. This variability could be minimized by the production of antibodies specific for the O-antigen. Additionally, the antigens generated from this study provide a source of characterized LPS and O-antigen standards for six serogroups of STEC.

The isolation and characterization of lipopolysaccharides from Microcystis aeruginosa, a prominent toxic water bloom forming cyanobacteria

Massive toxic blooms of cyanobacteria represent a major threat to water supplies worldwide, yet serious gaps exist in understanding their complex toxic effects, including the role of lipopolysaccharides (LPS). The present comparative study focused on the levels and biological activities of LPS isolated from Microcystis aeruginosa, which is one of the most globally distributed toxic species. Using hot phenol extraction, LPS was isolated from 3 laboratory cultures and 11 natural water blooms. It formed 0.2–0.7% of the original dry biomass of the cyanobacteria, based on gravimetry. Additional analyses by commercial anti-LPS ELISA were correlated with gravimetry but showed concentrations that were about 7-times lower, which indicated either impurities in isolated LPS or the poor cross-reactivity of the antibodies used. LPS isolates from M. aeruginosa were potent pyrogens in the traditional Limulus amebocyte lysate (LAL)-test, but comparison with the PyroGene test demonstrated the limited selectivity of LAL with several interferences. The determined pyrogenicity (endotoxin units, EU) ranged from very low values in laboratory cultures (less than 0.003 up to 0.008–EU per 100 pg LPS) to higher values in complex bloom samples (0.01–0.078 EU per 100 pg of LPS), which suggested the role of bloom-associated bacteria in the overall effects. Potent pro-inflammatory effects of the studied LPS from both cultures and bloom samples were observed in a highly-relevant ex vivo human blood model by studying reactive oxygen species production in phagocytes as well as increased productions of interleukin 8, IL-8, and tumor necrosis factor α, TNF-α. LPS from M. aeruginosa seem to modulate several pathways involved in the regulation of both innate immunity and specific responses. In comparison to the standard pathogenic bacterial LPS (World Health Organization Escherichia coli O113:10 endotoxin; activity 1 EU per 100 pg), the studied cyanobacterial samples had pyrogenicity potencies that were at least 12-times lower. However, the health risks associated with LPS from M. aeruginosa should not be underestimated, especially with respect to diverse biological effects observed ex vivo and in the case of massive blooms in drinking water reservoirs, where the estimated pyrogenicity can reach up to 46,000 EU per mL of water.

Molecular cloning and characterization of lipopolysaccharide- and β-1,3-glucan-binding protein from the giant freshwater prawn Macrobrachium rosenbergii and its transcription in relation to foreign material injection and the molt stage

Lipopolysaccharide (LPS)- and β-1,3-glucan-binding protein (LGBP) complementary (c)DNA was cloned from the hepatopancreas of the giant freshwater prawn Macrobrachium rosenbergii using oligonucleotide primers and a reverse-transcription polymerase chain reaction (RT-PCR). Both 3′- and 5′-regions were isolated by the rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1389 bp encoding a protein of 378 amino acids (aa) including a 15-aa signal peptide. The calculated molecular mass of the mature protein (363 aa) was 41.2 kDa with an estimated p I of 4.73. The M. rosenbergii LGBP sequence contains (1) three putative integrin-binding motifs, (2) a glucanase motif, (3) a putative N-glycosylation site, (4) four protein kinase C phosphorylation sites, (5) four casein kinase II phosphorylation sites, and (6) a putative recognition motif. Sequence comparison showed that the deduced amino acids of LGBP of M. rosenbergii had overall similarities of 60–71% to those of known crustacean LGBPs and β-1,3-glucan-binding proteins (BGBPs). The LGBP of M. rosenbergii was mainly expressed in the hepatopancreas. The LGBP transcript of M. rosenbergii was downregulated in haemocytes, but was upregulated in the hepatopancreas when injected with LPS and poly:IC after 12 h. The LGBP messenger (m)RNA expression of prawns in the postmolt stage was significantly upregulated in haemocytes, but downregulated in the hepatopancreas, which revealed a complementary relationship between haemocytes and the hepatopancreas in the molt cycle.

Isolation and characterization of lipopolysaccharides from different rhizobial isolates

Formation of nodules on roots or in stems (in some cases) of leguminous plants is the unique ability of gram-negative bacteria, Rhizobia, which converts atmospheric nitrogen into usable forms by the host plant. Lipopolysaccharide (LPS) is the outer membrane component of the gram-negative bacteria, known to be an essential factor in host recognition, specificity, and initial infection processes. In the present study, we extracted lipopolysaccharides from different rhizobial isolates by a modified phenol-water method and partially characterized by polyacrylamide gel electrophoresis with silver staining. The results showed two separate banding regions, LPS-I and LPS-II. The high molecular weight and electrophoretic mobility of LPS-I region resembles that of lysozyme, used as a standard marker. The LPS-II region has a low molecular weight and electrophoretic mobility greater than that of lysozyme. The LPS-II region was due to incomplete LPS, which either lacks the entire O-antigen repeating unit or contains only one or two repeating units. The banding patterns of LPS vary among the different rhizobial isolates. Results revealed that the type of LPS structure and banding regions greatly facilitate the further characterization of the LPS modifications required for symbiosis.

Characterization of Lipopolysaccharides from Pseudomonas fluorescens IMB 2108 (Biovar II) and IMB 2111 (Biovar IV) with O-Chains Represented by α-Glucan

Results of studies of the structurally unique O-chains of lipopolysaccharides, which were isolated from the dry biomass of Pseudomonas fluorescens IMB 2108 (biovar II) and IMB 2111 (biovar IV) by the Westphal technique and purified by repeated ultracentrifugation, are reported. The bulk of the lipopolysaccharide preparations contained S- and R-molecules at an average molar ratio of 1: 2. The main components of the hydrophobic moiety of lipid A were 3-hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, and octadecanoic acids, as well as hexadecenoic and octadecenoic acids. Glucosamine and phosphoethanolamine were identified as components of the hydrophilic moiety of lipid A. The degree of lipid A phosphorylation amounted to 3-4%. Fractions of the core oligosaccharide contained glucose, galactose, mannose, rhamnose, arabinose, glucosamine (only in strain IMB 2108), alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulosonic acid (KDO). Heptose was present in trace amounts. O-specific polysaccharide chains were represented by a linear polymer of D-glucose units, which were linked together via alpha-(1,4) glycoside bonds. The existence of P. fluorescens strains that have alpha-1,4-glucan as the O-chain of their lipopolysaccharides has not been described before.

Chemical and biological characterization of lipopolysaccharides from the Pseudomonas syringae pv. maculicola IMV 381 collection culture and its dissociants

Lipopolysaccharides (LPS) were isolated from the crude bacterial mass of the Pseudomonas syringae pv. maculicola IMV 381 collection culture and its virulent and avirulent subcultures isolated earlier from the heterogeneous collection culture due to its natural variability during long-term storage. The composition, immunochemical properties, and certain parameters of the biological activity of the LPS preparations obtained were studied. The structural parts of the LPS macromolecule--lipid A, the core oligosaccharide, and O-specific polysaccharide (OPS)--were isolated and characterized. The following fatty acids were identified in the lipid A composition of all cultures: 3-OH-C10:0, C12:0, 2-OH-C12:0, 3-OH-C12:0, C16:1, C16:0, C18:1, and C18:0. Glucosamine (GlcN), ethanolamine (EtN), phosphoethanolamine (EtN-P), and phosphorus (P) were revealed in the hydrophilic portion of the macromolecule. In the core portion of the LPS macromolecule, glucose (Glc), rhamnose (Rha), GlcN, galactosamine (GalN), 2-keto-3-deoxyoctulosonic acid (KDO), alanine (Ala), and P were found. The peculiarities of the structure of LPS isolated from the stable collection culture (LPS(stab)) and its virulent (LPS(vir)) and avirulent (LPS(air)) subcultures were studied. LPS(vir) and LPS(avir) were identical in the monosaccharide composition and contained as the main components L-rhamnose (L-Rha) and 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc), like LPS(stab) studied earlier. The NMR spectra of LPS(vir) were identical to the spectra of LPS(stab), whose O-chain repeating unit structure was studied by us earlier, whereas LPS(avir) differed from LPS(vir) in the NMR spectrum and was identified by us as the SR form. LPS(avir) was serologically identical to LP(stab) and LPS(vir). Hence, the degree of polymerism of the LPS O-chain of P. syringae pv. maculicola IMV 381 is the main virulence factor in the infected model plants. Serological relationships were studied between P. syringae pv. maculicola IMV 381 and the strains of other pathovars with structurally similar LPS.

Extraction and Characterization of Lipopolysaccharide from Aeromonas hydrophila and Its Effects on Survival and Hematology of the Carp, Cyprinus carpio

Extraction and Characterization of Lipopolysaccharide from Aeromonas hydrophila and Its Effects on Survival and Hematology of the Carp, Cyprinus carpio

Chemical and Immunobiological Characterization of Lipopolysaccharides from Prevotella intermedia and Prevotella nigrescens

Chemical and Immunobiological Characterization of Lipopolysaccharides from <i>Prevotella intermedia</i> and <i>Prevotella nigrescens</i>

Characterization of lipopolysaccharides from Ralstonia solanacearum

Lipopolysaccharides (LPSs) from four strains of Ralstonia solanacearum belonging to biovar I (ICMP 6524, 8115, 5712, and 8169) were isolated and investigated. The structural components of the LPS molecule, such as lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS), were obtained after mild acid hydrolysis of the LPS preparations. In lipid A from all the LPS samples studied, 3-hydroxyhexadecanoic, 2-hydroxyhexadecanoic, tetradecanoic, and hexadecanoic fatty acids prevailed. The dominant monosaccharides of the core oligosaccharides of all of the strains studied were rhamnose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and heptose. However, individual strains varied in the content of galactose, ribose, xylose, and arabinose. Three types of the O-PS structure were established, which differed in their configuration (alpha or beta), as well as in the type of the bond between glucosamine and rhamnose residues (1-->2 or 1-->3).

Biochemical characterization of lipopolysaccharides extracted from a hydrophobic strain of Pasteurella multocida.

Lipopolysaccharides were extracted from freeze-dried cells of Pasteurella multocida strain P-1581 (serotype 8) by the phenol-chloroform-petroleum ether method and biochemically analysed using standard procedures. The primary neutral sugars were glucose, galactose and heptose. No deoxy sugars were detected. Amino sugars included galactosamine, glucosamine and glucosamine-6-phosphate. 3-Deoxy-D-manno-2-octulosonic acid was present at a relatively low concentration. The analyses included identification and quantification of phosphate and alanine. The primary fatty acids and their approximate relative ratios were 3-hydroxytetradecanoate and tetradecanoate 2:1. Tetradecanoic acid was bound almost exclusively by ester linkages. 3-Hydroxytetradecanoic acid was bound primarily by amide linkages, although significant numbers of ester-bound residues were noted. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses indicated that the lipopolysaccharides were of low molecular weight.