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Cultivation of the type strain Azospirillum lipoferum Sp59b in the presence of the flavonoid quercetin induced modification of the structure of the bacterial lipopolysaccharide. Cultivation in the presence of the flavonoid was shown to result in altered serological characteristics of the bacteria, increased heterogeneity of the outer membrane lipopolysaccharide pool, as well as in modified composition and fatty acid ratio of lipid A. The flavonoid was shown to induce the synthesis of the O-specific polysaccharide with the repeating structure represented by a tetrasaccharide consisting of a linear trisaccharide fragment of α-L-Rhap residues in the main chain and the terminal β-D-Glcp residue. The structure of this O-specific polysaccharide was identical to the previously determined structure of the capsular polysaccharide of these bacteria grown without quercetin. Modifications in the structural composition of the capsular polysaccharide induced by cultivation in the presence of quercetin were revealed.

A rapid method for detection of the surface lipopolysaccharide antigen and the cells of the causative agent of bovine brucellosis was developed. The method represents a sandwich format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid movement along the membrane components of the test strip, immunochemical reactions, and the formation of colored bands. The novel method requires 10 minutes to determine the lipopolysaccharide antigen of the cell wall of the brucellosis causative agent at concentrations down to 10 ng/mL and the Brucella abortus cells at concentrations down to 10(6) cells/mL (5 x 10(4) cells in the sample). The specificity of the immunodetection was confirmed. The designed test system can be used for the rapid field diagnosis of brucellosis in cattle.

The interaction of sulfated polysaccharides--fucoidans from brown seaweeds Laminaria japonica, Laminaria cichorioides and Fucus evanescens with Toll-like receptors (TLRs) expressed on membranes of embryonic human kidney epithelial cells (HEK293-null, HEK293-TLR2/CD14, HEK293-hTLR4/CD14-MD2 and HEK293-hTLR2/6) was investigated. In vitro fucoidans specifically interacted with TLR-2, TLR-4, and the heterodimer TLR-2/6 resultated in activation of transcription nuclear factor NF-kappaB. Analysis of composition the hydrolyzed fucoidan from F. evanescens was carried out by gas-liquid chromatography and chromatography-mass spectrometry. Results indicated the absence of 3-3-hydroxytetradecanoic acid (3-OHC14), the basic component of lipopolysaccharides in the preparation. Thus, the obtained results suggested that fucoidans from brown seaweeds possessing immunotropic activity are independent ligands for TLRs, and are able to induce genetically determined biochemical processes of protection organisms against pathogenic microorganisms.

On the basis of synthesized negatively charged hydrosol hexacianferrate (II) ferrum (III) (HCFF) by diameter 10-20 HM are received stable conjugates with antibodies and antigens glycoprotein and lipopolysaccharide (LPS) nature. Synthesized hydrosol (HCFF) is new type of a disperse phase in the lateral flow assay. Conjugates mentioned above were applied for construction lateral flow tests-systems for revealing cholera toxin, the rabbit antibodies to recombinant glycoproteine complex from Micobacterium tuberculosis H37 Rv, human immunoglobulin, LPS antigens S. typhimurium, S. enteritidis. Developed lateral flow tests-systems had high analysis speed (5-7 min), good specificity and sensitivity: on cholera toxin of 2.0 ug/ml, on LPS antigens S. typhimurium, S. enteritidis 0.5 ug/ml.

Aim: Study of bactericidal effect of phenol on Yersinia pseudotuberculosis produced in various nutrient media., Materials and Methods: Bacteria were produced in nutrient broth (NB) and NB with glucose (NB+Glu) or galactose (NB+Gal) at 8 degrees C. Effect of phenol on bacteria was evaluated by changes in optical density of suspension and quantity of viable cells, and by staining of cells with ethidium bromide. Lipids were analyzed by thin-layer and gas-liquid chromatography, gas-liquid- chromatography--mass-spectrometry, differential scanning calorimetry; lipopolysaccharides (LPS)--by electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulfate (SDS-PAGE)., Results: Survival rate of bacteria is dependent on phenol concentration, biocide treatment time and parameters of cell cultivation. Addition of glucose or galactose into the nutrient medium increases the resistance of Yersinia against phenol. Bacterial cultures are heterogeneous in the resistance against phenol independently of the production parameters. Phenol causes damage in outer bacterial membrane, as evidenced by accumulation of lysophosphatidylethanolamine in the cell, the main product of enzyme activity of membrane-bound phospholipase A, and release into the cultural medium of part of LPS. Treatment by phenol in bactericidal concentration is accompanied by changes in phospholipidic and fatty acid composition of bacterial cell envelope., Conclusion: New data are obtained on environmental factors that contribute to the increase of resistance of bacteria against phenolic biocides.

The Mannheimia haemolytica, Mannheimia glucosida and Bibersteinia trehalosi strains and the similar fatty acid composition of cells with domination of C(16:1) and C(16:0), which were in almost equal quantities, C(14:0 and C(18:1) + C(18:2). The fatty acid composition of lipopolysaccharides (LPS) of the studied bacteria had no essential differences too. It was mainly represented by C(14:0) and 3-OH-C(14:0) which consisted of more than 80% of all LPS fatty acids. C(12:0), C(16:1) and C(16:0) were presented in LPS in small quantities. The M. haemolytica, M. glucosida and B. trehalosi strains did not differ essentially by fatty acid compositions of cells and LPS from earlier studied strains of genera Pasteurella (P. multocida), Haemophilus (H. influenzae and other species), Actinobacillus (A. pleuropneumoniae). This shows the close phylogenetic relationship of the mentioned bacteria and significance of investigated signs as chemotaxonomic markers for differentiation of taxons of the above genus level. The paper is presented in Russian.

Two strains of Legionella isolated from patient and hot water supply system during outbreak in Sverdlovsk region in 2007 were studied. Using genetic analysis methods (genes sequencing, VNTR-typing and PCR-based study of omp 28 gene), it was shown that tested strains are pathogenic but do not belong to one genetic group and epidemic cluster. Performed immunochemical analysis confirmed that both isolated strains belong to Legionella serogroup 1.

A comparative immunochemical analysis of lipopolysaccharides (LPS) in Azospirillum brasilense model strains Sp7 and Sp245 and in mutants with transformed somatic antigens has been performed. According to the results of a complex of various immunochemical methods, including studies with polyclonal antibodies against the LPS these bacteria, their LPS consist of an assembly of macromolecules with different antigenic characteristics. Two types of O-specific polysaccharides (O-PS) are present in the LPS of every strain of A. brasilense under study. The major difference between the two O-PS is the antigenic heterogeneity of one of them. This heterogeneous O-PS has been shown to possess at least two O-factors (antigenic determinants) different in their structure. Meanwhile, according to all the tests performed, the other O-PS in every strain is immunochemically homogeneous and identical to one of the determinants revealed in the more diversified O-PS. The LPS heterogeneity among the given strains may be due to the pattern of O-specific polysaccharide synthesis, one of the O-PS being an intermediate in the synthesis of the other.

Technical approaches to construction of preparations for serologic diagnostics of Legionella infection were presented in the article; antigenic- and immunoglobulin-based diagnostic kits with known characteristics were developed. Immunogenic properties of protein and lypopolysaccharide antigens, which have diagnostic value, were studied; similarity of protein antigens from 7 serogroups of L. pneumophila was demonstrated. Soluble antigen with known composition was obtained and used for the development of antigen-based polymeric kit for diagnostics of Legionella infection. On the basis of hyperimmune sera, immunoglobulin-based polymeric diagnostic kit and array of coagglutinating diagnostic kits for the mentioned 7 serogroups were developed. Antigen-based polymeric diagnostic kit was recommended for licensure.

The presence of a polysaccharide sheath on the surface of the polar flagellum of Azospirillum brasilense was revealed by immunoelectron microscopy and immunodiffusion analysis with strain-specific antibodies to lipopolysaccharides (LPS). The antigenic identity of A. brasilense Sp245 sheath material and one of the two O-specific polysaccharides of its somatic LPS was demonstrated. The screening effect of the sheath in respect to flagellin was determined by agglutination tests and by the inhibition of azospirilla motility in liquid and semisolid agarized media caused by strain-specific antibodies to LPS; no pronounced effect of genus-specific antibodies to flagellin was observed.

The composition, structure, and certain biological properties of lipopolysaccharides (LPS) isolated from six strains of bacteria Pseudomonas syringae pv. atrofaciens pathogenic for grain-crops (wheat, rye) are presented. The LPS-protein complexes were isolated by a sparing procedure (extraction from microbial cells with a weak salt solution). They reacted with the homologous O sera and contained one to three antigenic determinants. Against the cells of warm-blooded animals (mice, humans) they exhibited the biological activity typical of endotoxins (stimulation of cytokine production, mitogenetic activity, etc.). The LCD of the biovar type strain was highly toxic to mice sensitized with D-galactosamine. The structural components of LPS macromolecules obtained by mild acidic degradation were characterized: lipid A, core oligosaccharide, and O-specific polysaccharide (OPS). Fatty acids 3-HO-C10:0, C12:0, 2-HO-C12:0, 3-HO-C12:0, C16:0, C16:1, C18:0, and C18:1 were identified in lipid A of all the strains, as well as the components of the hydrophilic part: glucosamine (GlcN), ethanolamine (EtN), phosphate, and phosphoethanolamine (EtN-P). In the core LPS, glucose (Glc), rhamnose (Rha), L-glycero-D-manno-heptose (Hep), GlcN, galactosamine (GalN), 2-keto-3-deoxy-D-mannooctonic acid (KDO), alanine (Ala), and phosphate were present. The O chain of all the strains consisted of repeated elements containing a linear chain of three to four L- (two strains) or D-Rha (four strains) residues supplemented with a single residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fucp3Nac), N-acetyl-D-glucosamine (D-GlcpNAc), D-fucose (D-Fucf), or D-Rhap (strain-dependent) as a side substitute. In different strains the substitution position for Rha residues in the repeated components of the major rhamnan chain was also different. One strain exhibited a unique type of O-chain heterogeneity. Immunochemical investigation of the LPS antigenic properties revealed the absence of close serological relations between the strains of one pathovar; this finding correlates with the differences in their OPS structure. Resemblance between the investigated strains and other P. syringae strains with similar LPS structures was revealed. The results of LPS analysis indicate the absence of correlation between the OPS structure and the pathovar affiliation of the strains.

The cells of two Rhodobacter capsulatus strains, B10 and PG, and the LPS of their cell walls were studied by electrophysical and biochemical methods. Strain B10 was found to belong to the R chemotype, and strain PG, to the RS chemotype. A relation was revealed between the chemotype of the photosynthesizing bacteria Rhodobacter capsulatus and the electrophoretic properties of their cells.

The chemical composition of the lipopolysaccharide of the associative diazotrophic rhizobacterium Azospirillum brasilense Cd has been studied. Among the main components of the hydrophobic part of the lipopolysaccharide, we identified 3-hydroxytetradecanoic, hexadecenoic, 3-hydroxyhexadecanoic, hexadecanoic, octadecenoic, and nanodecanoic fatty acids; the carbohydrate part contained rhamnose, galactose, and mannose. Polyclonal antibodies against the preparation under study were raised in rabbits. Serological relations between A. brasilense Cd and other strains of Azospirillum spp. were studied using double radial immunodiffusion and enzyme-linked immunosorbent assay.

We studied closely related Azospirillum brasilense strains Sp7 and Cd. For probing of their genomes, the fragments of 85-MDa (p85) and 120-MDa (p120) from A. brasilense Sp245 plasmids were hybridized with 115-MDa (p115) and 90-Mda (p90) plasmids of strain Sp7, respectively. Strain Cd was found to lose the 115-Mda plasmid and one of the two EcoRI restriction fragments of the total DNA (localized within p115 and the chromosome) that was homologous to an EcoRI-generated p85 fragment of 2.4 kb. On the contrary, in the total DNA of strain Sp7-S, in spite of the previously established disappearance of the 115-Mda replicon, two fragments homologous to p85 were revealed, as with strain Sp7. It is suggested that the Sp7-S genome contains the total p115 DNA or at least a certain part of it. Strains Sp7 and Cd were found to differ in size and morphology of colonies on solid and semisolid media, in the levels of resistance to a cation surfactant cetavlon, and in the antigen structure of lipopolysaccharides.

A comparative study of the lipopolysaccharides (LPS) isolated from Sinorhizobium meliloti SKHM 1-188 and two its LPS-mutants (Th29 and Ts22) with sharply decreased nodulation competitiveness was conducted. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate revealed two forms of LPS in all the three strains: a higher molecular-weight LPS1, containing O-polysaccharide (O-PS), and a and lower molecular-weight LPS2 without O-PS. However, the LPS1 content in mutants was significantly smaller than in the parent strain. The LPS of the strains studied contained glucose, galactose, mannose, xylose, three nonidentified sugars--X1 (TGlc 0.53), X2 (TGlc 0.47), and X3 (TGlc 0.43), glucosamine, and ethanolamine, while the LPS of S. meliloti SKHM1-188 additionally contained galactosamine, glucuronic and galacturonic acids, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as fatty acids, such as 3-OH C14:0, 3-OH C15:0, 3-OH C16:0, 3-OH C18:0, nonidentified hydroxy X (T3-OH C14:0 1.33), C18:0, and unsaturated C18:1 fatty acids. The LPS of both mutants were similar in the component composition but differed from the LPS of the parent strain by a lower X2, X3, and 3-OH C 14:0 content and a higher KDO, C18:0, and hydroxy X content. The LPS of all the strains were subjected to mild hydrolysis with 1% acetic acid and fractionated on a column with Sephadex G-25. The higher molecular weight fractions (2500-4000 Da) contained a set of sugars typical of intact LPS and, supposedly, corresponded to the LPS polysaccharide portion (PS1). In the lower molecular weight fractions (600-770 Da, PS2), glucose and uronic acids were the major components; galactose, mannose, and X1 were present in smaller amounts. The PS1/PS2 ratio for the two mutants was significantly lower than for strain SKHM1-188. The data obtained show that the amount of O-PS-containing molecules (LPS1) in the heterogeneous lipopolysaccharide complex of the mutants was smaller than in the SKHM1-188 LPS; this increases the hydrophobicity of the cell surface of the mutant bacteria. This supposedly contributes to their nonspecific adhesion on the roots of the host plant, thus decreasing their nodulation competitiveness.

The biopolymer composition, immunotropic and immunogenic properties of the fractions of B. pseudomallei and B. mallei were under study. The first two capsular fractions of these agents were found to be similar in their biopolymer composition that was indicative of their close relations. At the same time the causative agents of glanders proved to have decreased content of high molecular glycoproteids and LPS fragments. In the causative agents of melioidosis, capsular fractions K3 and K4 were characterized by the domination of proteins with a molecular weight of 42-25 kD. Fraction K4 in B. pseudomallei and fraction K1 in B. mallei had pronounced immunosuppressing properties ensuring the protection of encapsulated microbial cells in the body. The biopolymers forming fractions K1, K2, K3 in B. pseudomallei and fraction K2 in B. mallei were characterized by immunomodulating properties.

A possibility has been demonstrated to use laser spectroscopy of bacterial glycopolymers by means of measurement of their water solutions fluorescence. Comparative investigations of native lipopolysaccharide (LPS) Ralstonia solanacearum and its structure components permits a supposition to be made that the LPS total spectrum is a result of superposition of the spectrum of O-specific polysaccharide and core oligosaccharide as well as core oligosaccharide and lipid A. The LPS spectrum maximum shift is determined by core oligosaccharide and lipid A luminescence contribution. A decrease as well as complete loss of serological activity as a result of 30 and 60 min UV irradiation of LPS has been established. It has been shown that LPS Rhizobium leguminosarum bv. viciae quenches luminescence of host-plant (pea) lectin depending on the extent of their affinity. Luminescence spectrum of glucan Sinorhizobium meliloti CXM1-188 and two its LPS-mutants differ between themselves both in luminescence intensity and in presence and expression degree of the site 2 with maximum 2.8 eV.

The antigenic identity (and close values of electrophoretic mobility) of capsular polysaccharides, exopolysaccharides, and O-specific polysaccharides was revealed in the Azospirillum brasilense strains Sp7 and Sp245 by the immunodiffusion and immunoelectrophoretic methods. Together with the literature data on the identity of the monosaccharides composition of these polymers, this gives evidence of the absence of a specific capsular antigen in the bacteria studied. Thus, extracellular Azospirillum brasilense polysaccharides are likely to represent O-antigenic lipopolysaccharide fragments excreted by the bacteria into the culture medium, and their identification as a capsule or as an exopolysaccharide depends on the strength of the attachment of these polysaccharides to the cell surface.

Pseudomonas syringae pv. maculicola dissociants producing colonies of different morphotype were found to possess similar biochemical and serological properties but different virulence to the host plant. The heterogeneous extracellular and intracellular lipopolysaccharide-protein complexes of the dissociants differed in their chemical composition and biological activity towards test plants.

Lipopolysaccharide (LPS) and its structural components: lipid A, O-specific polysaccharide (O-PS) and oligosaccharide core (OG-core) have been isolated from Cytophaga lytica. 3-Oxytetradecanoic (40.8%) and dodecanoic (28.7%) are the predominant fatty acids of lipid A; pentadecanoic (6.8%), 3-oxyhexadecanoic (6.5%) as well as hexadecanoic (5.4%) acids have been found as well. The content of the rest of fatty acids is inconsiderable (2.3 to 0.5%). OG-core contained monosaccharides both typical of the most Gram-negative bacteria (glucose, mannose, galactose, rhamnose, glucosamine) and rarely occurring one-arabinose. O-PS is represented by glucose, mannose, rhamnose, glucosamine, galactosamine as well as unidentified hexosamine.

From the biomass of five Pseudomonas fluorescens biovar I strains, including the P. fluorescens type strain IMV 4125 (ATCC 13525), lipopolysaccharides (LPS) were isolated (by extraction with a phenol-water mixture followed by repeated ultracentrifugation), as well as individual structural components of the LPS macromolecule: lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS). 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, octadecanoic, hexadecenoic, and octadecenoic fatty acids were present in lipid A of the LPS of all the strains studied. Glucosamine, ethanolamine, and phosphoethanolamine were revealed in the lipid A hydrophilic part of all of KDO, a trace amount of heptoses, ethanolamine, phosphoethanolamine, alanine, and phosphorus were identified as the main core components. Interstrain differences in the core oligosaccharide composition were revealed. Structural analysis showed that the O-PS of the type strain, as distinct from that of other strains, is heterogeneous and contains two types of repetitive units, including (1) three L-rhamnose residues (L-Rha), one 3-acetamide-3,6-dideoxy-D-galactose residue (D-Fuc3NAc) as a branching substitute of the L-rhamnan chain and (2) three L-Rha residues and two branching D-Fuc3NAc residues. The type strain is also serologically distinct from other biovar I strains due to the LPS O-chain structure, which is similar to those of the strains of the species Pseudomonas syringae, including the type strain. The data of structural analysis agree well with the results of immunochemical studies of LPS.

The effect of the ingredients of a semisynthetic culture medium on the synthesis of Y. pestis antigens (F1, LPS, "mouse" toxin) under the conditions of batch cultivation at 28 degrees C was studied. The study revealed that the amount of antigens produced by bacterial cells depended on the character of the limitation of growth.

A new method for the determination of lipopolysaccharide (LPS) admixtures in protein solutions has been developed. The method includes the periodate oxidation of LPS, biotinylation with biotin hydraside, immobilization on a nitrocellulose membrane and the development of biotinylated LPS in the streptavidin--alkaline phosphatase system. Proteins are previously removed from the solution by treatment with hot phenol. Development with the use of 5-bromoinodyl phosphate and nitrotetrazolium blue makes it possible to detect about 30 pg of LPS immobilized on the nitrocellulose membrane.

The preparations of pilopolysaccharides (LPS) of virulent and avirulent Y. pestis strains, which have a different composition, were cultivated at different temperatures. Despite the cultivation temperature of parental cells, all LPS preparation inhibited the passive hemagglutination reaction (PHR) of the red blood cells sensitized by LPS isolated from the cultures grown at 26 degrees C by using homologous antisera. In contrast, the homologous system consisting of the red blood cells sensitized by LPS from the EV NIIEG, cultured at 37 degrees C, and the antiserum to these cells proved to be more specific and it was inhibited only by the homologous LPS isolated from the strain EV NIIEG. The similar reaction of the interaction of the red blood cells sensitized by high temperature LPS agents from plasmid-free strains with the same serum was inhibited by all plague LPS preparations. The LPS preparations from the strains Y. pestis 1146 and Y. pseudotuberculosis 9532 obtained when cultivated at 37 degrees C. RHR of the red blood cells sensitized by these preparations was inhibited by homologous LPS irrespective of the temperature of cell cultivation. In all cases, the reaction was specific for Yersinia strains and it was inhibited by LPS from Ra-Rd2 cultures, variants of Salmonella and E. coli.

Lipopolysaccharide and extracellular glyco polymers were isolated from cells of phytopathogenic bacteria Ralstonia solanacearum. It was established that the O-specific polysaccharides are characterized by a linear structure, composed of tetrasaccharide repeating units containing three L-rhamnose residues and one of N-acetylglucosamine residue. Core oligosaccharide along with typical monosaccharides such as rhamnose, glucose, heptose, KDO and glucosamine included fucose, galactose and arabinose. Serological activity of lipopolysaccharide is due to both O-specific polysaccharide and lipid A component. Extracellular glyco polymers contained both neutral (GP 1) and charged (GP 2 and GP 3) components. Rhamnose and glucose (GP 1, GP 2) or galactose (GP 3) were predominant monosaccharides, GP 2 displayed a high phytotoxic action in respect to tomato plants.

K. pneumoniae lipopolysaccharide (LPS) was isolated from biomass grown under the conditions of controlled multicycle and continuous cultivation. A considerable yield of LPS containing the minimal amount of protein and nucleic acid admixtures was obtained from biomass grown by continuous cultivation with the concentration of glucose in the medium equal to 20 g/l and the content of dissolved oxygen at a level of 0% of complete saturation. Erythrocyte diagnosticum prepared on the basis of this LPS was found to have a sensitizing dose of 100 micrograms per ml of solid erythrocytic precipitate and high specificity, studied in the passive hemagglutination test with the use of rabbit sera.

Lipopolysaccharide was isolated from dry biomass of the Pseudomonas fluorescens strain IMV 472 (biovar I) by the Westphal procedure and purified by ultracentrifugation. Fractions of the structural components of the lipopolysaccharide macromolecule-lipid A, the core oligosaccharide, and the O-chain-were isolated and characterized. 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, hexadecenoic, octadecenoic, and hexadecanoic acids were identified as the main fatty acids of lipid A. The hydrophilic moiety of lipid A was found to include glucosamine, phosphoethanolamine, and phosphate. Glucose, galactose, rhamnose, glucosamine, galactosamine, alanine, phosphoethanolamine, and 2-keto-3-deoxyoctulosonic were identified in the core region of lipid A. The O-specific polysaccharide chain consisted of a repeating tetrasaccharide fragment that included two L-rhamnose residues, a D-fucose residue, and an N-acetyl-D-glucosamine residue as a side substituent. Double immunodiffusion in agar and the lipopolysaccharide precipitation reaction revealed no serological interrelation between strain IMV 472 and P. fluorescens strains studied previously.

In this investigation isogenic avirulent variants obtained from F. tularensis standard virulent strain 503 were used. The capsule-deficient variants (cap-) were shown to have no species-specific capsular antigens and to be capable of producing R-LPS having no the polysaccharide part of the molecules. The capsule-defective forms (cap +/- ) were found to synthesize capsular antigens and S-LPS whose polysaccharide part essentially differed from the O-lateral chains of LPS of the virulent strain. The study of bacterial dissemination revealed that virulent bacteria rapidly spread in the macroorganism, and their subsequent proliferation shortly led to the death of animals. Avirulent mutants (cap- and cap +/- ) appeared in the organs of animals later and proliferated slower, parasitizing in the macroorganism without fatal outcome. The cap- variants were not capable of inducing the synthesis of antitularemic antibodies and possessed no protective properties. The cap +/- mutants were capable of inducing the synthesis of antitularemic antibodies in mice. These antibodies facilitated the elimination of avirulent strains from the macroorganism, but did not ensure protection from infection with virulent strains.

A preparation containing P. aeruginosa cell-wall proteins with a mol. wt. of 9-55 kD has been obtained from P. aeruginosa by the method of extraction with the use of tris-EDTA buffer. In experiments on mice this protein preparation has shown pronounced protective properties, but, according to the data of Shwartzman's local reaction, proved to be toxic, perhaps due to the admixture of LPS. The purification of the protein preparation from the admixture of LPS will make it possible to obtain an effective vaccine against P. aeruginosa infection.

A method for analysis of phosphoethanolamine in hydrolysates of the fractions structural parts of lipopolysaccharide macromolecule on the column with cation-exchange resin Ostion LG ANB (CSFR) in the system of step gradient of sodium-citrate buffers is suggested, optimal conditions for acidic hydrolysis of polysaccharide-standard were chosen to determine phosphoethanolamine and other amino compounds contained in it. The conditions for chromatography analysis of phosphoethanolamine together with aminosugars and amino acids have been developed. Using the suggested procedure the presence of phosphoethanolamine was found in preparations of lipid A hydrolysates and core oligosaccharide isolated from some lipopolysaccharide strains of Pseudomonas fluorescens.

The preparations of lipopolysaccharide (LPS) were obtained from N.meningitidis cells grown in synthetic nutrient medium in a fermenter. Different N.meningitidis strains, groups A, B, C, were found to give different yield of the preparation of LPS: the maximum yield was obtained from N.meningitidis cells C 133 and the minimum yield, from N.meningitidis cells B 125. The comparison of group B strains 125 and 150 showed that the former gave a higher yield of LPS. The influence of the phase of growth on the yield of the preparation of LPS obtained from N.meningitidis cells 125 was shown to be manifested under the conditions of oxygen excess; in the exponential phase the highest yield of the preparation was observed. Oxygen dissolved in the medium was found to influence the yield of the preparation of LPS: with the same content of glucose in the medium the yield of LPS was higher under the conditions of the limited concentration of oxygen; with the same concentration of oxygen in the medium the yield of LPS increased with the increase of the concentration of glucose.

Fatty acid composition of lipopolysaccharides (LPS) and cytoplasmic membranes (CPM) of Yersinia pestis [correction of the plaque microbe] has been studied. Concentration of certain acids in the LPS content proved sharply different and thus the strains were separated into groups. High content of 3-oxymiristinic acid was a distinction of vaccine strains. Virulent strains of one of the groups regularly differed from the rest of virulent strains by high content of laurinic acid. Laurinic and two nonidentified acids, which were not found in the virulent cultures, were present in CMP lipids of vaccine strains.

The comparative study of the lipopolysaccharides (LPS) of virulent and avirulent strains of S. sonnei, phase I (smooth colonies), has been made. Electrophoresis of LPS and subsequent densitometry of electrophoregrams have revealed the increase of the fraction of long 0-chains with a considerable number of recurring elements in 2 out of 3 LPS preparations obtained from avirulent shigellae. In mice immunized with these LPS preparations a considerably greater number of antibody-producing cells can be detected in Jerne's test on sheep red blood cells (SRBC) sensitized with the LPS of a virulent strain than on those sensitized with the above LPS preparations. Long 0-specific chains supposedly inhibit the fixation of individual complement components on the corresponding sensitized SRBC. The LPS of the third avirulent strain of S. sonnei, phase I, with transposon integrated into its genome, which has led to the formation of the avirulent variant of a previously virulent strain, seems to contain fine structural differences from the initial virulent strain. The immunogenicity of the LPS of this avirulent strain is greatly (3-4 times) decreased, which is manifested by the number of antibody-producing cells detected in Jerne's test on SRBC sensitized with LPS preparations obtained from these two strains.

The solid-phase liposomal immunoassay procedure for the determination of Francisella tularensis lipopolysaccharide (LPS) has been developed. This assay has been made with the use of monolayer liposomes, on the average, 360 nm in diameter with their phospholipid bilayer modified with F. tularensis LPS and their internal space filled with calcein used as fluorescent marker. The assay is based on the principle of the competitive immunosorption of liposomes and antigenic LPS on the surface of polystyrene plates sensitized with specific monoclonal antibodies. The dynamic range of the method is 50-2,500 mg/ml, the variation index being 2.3-11.5%. Optimization of this method and its comparison with the enzyme immunoassay system for the given antigen-antibody pair has been made.

Lipopolysaccharides (LPS) of the representatives of strains of serogroup VI Pseudomonas syringae (P. syringae pv. atrofaciens 2399, pv. phaseolicola 120a, 7842 and P. holci 8299) possessing virulence and confinement to the host-plant are characterized by high serological activity in direct and cross reactions of the binary diffusion in agar, immunoelectrophoresis, passive hemagglutination and inhibition of passive hemagglutination. A supernatant and a sediment obtained after ultracentrifugation of LPS preparations possessed O-antigenic activity. O-specific polysaccharide (PS) is serologically less active than the LPS preparations. Problems of the intergroup and intragroup serological affinity in connection with the structure of O-specific PS. It is proved that the basic chain of O-specific polysaccharide (D-rhamnane) plays definite (but not a single) part in displaying antigenic properties of the whole LPS macromolecule.

Water extract and salt-EDTA extract of Pseudomonas syringae, pv. atrofaciens cells were fractionated by ultracentrifugation with following salting out of ultracentrifugal supernatant by ammonium sulphate at 55% saturation (pH 4.5). The composition and distribution of LPS molecular forms were studied in the obtained fractions by means of electrophoresis in 10% polyacrylamide gel with 1% sodium dodecylsulphate when staining gels by silver nitrate and cumassi. It is shown that ultracentrifugal supernatant and a sediment as well as sulphate sediment contain S-LPS and R-LPS. SR-LPS is not differentiated. Sulphate supernatant does not contain the determinable amount of S-LPS but it is enriched by the proteins with molecular weights of 65-15 kDalton. S-LPS is localized in the gel area which corresponds to mobilities of polypeptides with molecular weights 130-45 KDalton and the number of monomeric links in O-specific chains of its molecules reaches 25-30. R-LPS migrates under electrophoresis in gel to the mobility zone of polypeptides with molecular weights 14.5-16 kDalton.

The capacity of Y. pseudotuberculosis strains for disassociation with the appearance of S- and P-forms has been studied. Strains 852 and 9547 show high stability in S-forms, their conversion into R-forms occurring at 40-42 degrees C. Strain 6953 shows pronounced polymorphism and instability of its associations at different growth temperatures. Strain 9532 exists in S- and R-forms which retain their stability during numerous subculturings at different growth temperatures and prolonged storage. This strain has plasmids of 130, 72.2, 5.7 kb. All plasmids are retained in S- and R-forms, i. e. the dissociation of the strain is not accompanied by the loss of plasmids. The conversion of the strain from the S-form into the R-form leads to changes in the structure of lipopolysaccharide and the composition of low-molecular (less than 23 kD) proteins in the outer and inner membranes. In tests on guinea pigs the LD50 of the R-form of the strain is tenfold greater than that of its S-form. The dissociants of strain 9532 are transformed by plasmid DNA with equal efficiency and equally inherit them without selective pressure.

The molecular heterogeneity of S. sonnei lipopolysaccharide (LPS), reflecting the size of lateral O-specific polysaccharide chains, has been established by the method of electrophoresis in acrylamide gel in the presence of sodium dodecyl sulfate and urea. The dominating components fall into three types, viz. those with 0-3, 10-16 and 35-40 repeating structures, the remaining components being minor ones. The electrophoretic profile of S. sonnei LPS considerably differs from the profiles of Escherichia coli and S. flexneri LPS, but coincides with the LPS profiles of other strains with different virulence. The preparations of LPS obtained by extraction with trichloroacetic acid have the same electrophoretic profiles as LPS obtained by the method of aqueous phenol extraction. The domination of certain molecular variants reflects, seemingly, specific features of the biosynthesis of LPS, characteristic of a given strain. The mechanisms of the preferable synthesis of lateral O-specific chains of the definite size and the importance of the molecular parameters of lateral chains for the biological properties of LPS require further study.

Structure analysis by the methods of methylation, 1H- and 13C-n. m. r. spectroscopy has shown that O-specific polysaccharides of typical strains of Pseudomonas solanacearum (biovar I) and P. cichorii are identical by their structure and constructed of branched pentasaccharide repeating links which include three residues of rhamnose (one of them is in the branching node), one residue of beta-xylose (it occupies terminal position) and one reside of N-acetyl-beta-glucosamine. The other strain of P. solanacearum of biovar I and two strains belonging to biovars III and IV also produce structure-similar O-specific polysaccharides, constructed of linear tetrasaccharide repeating links which include three residues of alpha-L-rhamnose and one residue of N-acetyl-alpha-D-glucosamine.

Mild acid hydrolysis of the lipopolysaccharide from Yersinia kristensenii strain 103 (0:12.26) afforded teichoic acid-like polysaccharide. From the results of methylation, dephosphorylation, partial Smyth degradation, and 13C and 31P NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [formula: see text] The structure was confirmed by complete interpretation of polysaccharide 13C NMR spectrum.

The structure of the O-specific polysaccharide chain of Pseudomonas syringae pv. tabaci strain 223 (serogroup VII) lipopolysaccharide was established on the basis of one- and two-dimensional 1H NMR analysis, 13C NMR analysis and calculation of optical rotation. The structure determined by the non-destructive way was confirmed by acid hydrolysis and methylation. (Sequence: see text). O-Antigen of the strain studied is similar in structure and serological properties to O-antigens of Pseudomonas syringae strains belonging to serogroup I.

Acidic polysaccharides were isolated from the Pseudomonas aeruginosa II (Sandvik) and V (Verder-Evans) lipopolysaccharides on mild acid hydrolysis followed by gel filtration on Sephadex G-50. The Sandvik II polysaccharide consists of 2-acetamido-2-deoxy-D-galacturonic acid, 2-acetamido-2,6-dideoxy-D-glucose, and L-rhamnose in the ratio 1:1:2. The Verder-Evans V polysaccharide contained the same monosaccharides and, in addition, a D-glucose residue. On the basis of 13C NMR data, methylation analysis, Smith degradation and solvolysis with hydrogen fluoride, the following structures were determined for the repeating units of the polysaccharides: (Formula: see text).

Along with classical lipopolysaccharide (LPS), O-specific material not precipitated by ultracentrifugation has been isolated from the water-phenol extract of S. sonnei avirulent strain 9090 possessing complete antigenic properties. The purification of O-antigen contained in the supernatant fluid has been carried out by the gel filtration of the fluid, previously treated with ribonuclease, in a column packed with Sephadex G-100. The polysaccharide nature of O-antigen thus obtained, the absence of lipid A and KDO and the low content of hexoses, or core-specific saccharides of S. sonnei LPS, in this antigen make it possible to classify this material with O-components of microbial cells, described by different authors as "native protoplasmic polysaccharide" or "L-hapten" and formed by polymers of LPS O-side chains. The content of this component in S. sonnei strains under study is, on the average, 2.5% of the weight of dry microbial substance. L-hapten preparations obtained in the course of our investigations have been found to contain two O-specific antigens detected by immunoelectrophoresis and immunodiffusion, as well as by sedimentation in saccharose gradient, where they form peaks corresponding to 4.3 S and 10.8 S. This polysaccharide O-antigen is supposed to be capable of interaction with ribosomal particles and suitable for use as a component of ribosomal dysentery vaccines.

To determine the content of lipopolysaccharide (LPS) in P. aeruginosa aqueous extracts the ELISA was used. Membrane filtration and ultracentrifugation followed by precipitation with ammonium sulfate at 80% saturation and gel filtration on Sephadex G-100 have proved to be the most effective methods for purification of the aqueous extract from LPS.

The cell wall (CW), cytoplasmic (CM) and intracytoplasmic membrane (ICM) fractions were obtained by sucrose linear gradient centrifugation of membrane preparations of Methylomonas methanica. The CW fraction represented by large open fragments with 5-layer structure is enriched in lipopolysaccharides and is similar in its protein composition to the CW fractions of other gram-negative bacteria. The CM vesicles are surrounded with a unit membrane, have high activities of marker enzymes (NADH dehydrogenase, succinate dehydrogenase and ATPase), a higher phospholipid content, cytochromes of a-, b- and c-types and differ qualitatively in its protein composition from the CW fraction. The ICM fraction has much in common with the CM fraction but differs from it by a higher level of marker enzymes and cytochromes of b and c types.

The evidence for occurring lipopolysaccharide-protein complexes in the outer membrane of gram-negative bacteria has been summarized. The composition and supramolecular structure of these complexes as well as their functions in microbial envelope and substantial role in membrane organization have been discussed. The biological properties of the complexes as endotoxins and O-specific antigens have been considered.