Study of immunochemical heterogeneity of Azospirillum brasilense lipopolysaccharides

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.     

Detection of a sheath on <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> polar flagellum

The presence of a polysaccharide sheath on the surface of the polar flagellum of Azospirillum brasilense was revelted 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.     

Immunoelectron microscopy investigation of the cell surface of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains

The genus-specific surface protein antigens of Azospirillum brasilense strains were visualized immunochemically. The procedure used for cell sample preparation was optimized to ensure that the surface protein structures were detected on cells in situ. Gold and gold-silver nanoparticles were conjugated to antibodies raised against the flagellin of A. brasilense type strain Sp7, against the lipopolysaccharide of A. brasilense Sp245, and against the genus-specific protein determinants of A. brasilense Sp7. Electron microscopic analysis using nanoparticle-labeled antibodies revealed antigenic determinants of the polar flagellum on the A. brasilense Sp245 cell surface, which in these bacteria are normally screened from the surroundings by a lipopolysaccharide sheath. Pili-like structures were detected on the Sp245 wild-type strain and on its Fla^– Swa^– Omegon-Km mutant SK048, which are presumably involved in microcolonial spreading in these bacteria.     

Composition,structure, and biological properties of lipopolysaccharides from different strains of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">atrofaciens</Emphasis>

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-C_10:0, C_12:0, 2-HO-C_12:0, 3-HO-C_12:0, C_16:0, C_16:1, C_18:0, and C_18:1 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-mannooctonoic 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 substituent. 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.     

Phenol Oxidase Activity of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 Mutants with Modified Motility and <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 Phase Variants with Different Plasmid Composition

Herein, we reveal the alteration in phenol oxidase enzymes complex production from Azospirillum brasilense Sp245 omegon mutants with polar and lateral flagella dysfunction and from A. brasilense Sp7 phase variants with different plasmid composition. The enzymatic activities for various laccases, tyrosinases, Mnperoxidases, and lignin peroxidases as well as the isomorphic composition of intracellular laccases and tyrosinases were estimated for the studied variants and the parent strains. It was noted that various genetic events correlating with phenotypic heterogeneity in A. brasilense populations affect their phenol oxidase activity level.     

Detection of putative polysaccharide biosynthesis genes in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains from serogroups I and II

It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.     

Characterization of the lipopolysaccharide of <Emphasis Type="Italic">Escherichia coli</Emphasis> 126

The lipopolysaccharide (LPS) of Escherichia coli 126 was isolated and studied. The lipid A fatty acid composition of the investigated LPS was similar to that of other members of the family Enterobacteriaceae. The E. coli 126 LPS was more toxic than the LPSs of previously studied E. coli strains and of other members of the Enterobacteriaceae (Budvicia aquatica and Pragia fontium), and was less pyrogenic than pyrogenal. SDS-PAG electrophoresis showed a bimodal distribution typical of S-form LPSs. The LPS of E. coli 126 decreased the adhesive index indicating a possible competition between LPS molecules of E. coli 126 and adhesins of E. coli F-50 on rabbit erythrocytes. The LPS of E. coli 126 in a homologous system showed antigenic activity in the reactions of double immunodiffusion in agar by Ouchterlony. No serological cross-reaction of the LPS of other E. coli strains, as well as of that of the B. aquatica type strain, with the antiserum to E. coli 126 was observed. The structural components of the lipopolysaccharide obtained by mild acid hydrolysis were lipid A, the core oligosaccharide, and the O-specific polysaccharide. Based on the data of monosaccharide analysis and ¹H and ¹³C NMR spectroscopy it was found that the O-specific polysaccharide had the structure characteristic of the representatives of E. coli serogroup O15.     

Changes in cell surface properties and biofilm formation efficiency in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 mutants in the putative genes of lipid metabolism <Emphasis Type="Italic">mmsB1</Emphasis> and <Emphasis Type="Italic">fabG1</Emphasis>

The previously obtained insertion mutants of Azospirillum brasilense Sp245 in the genes mmsB1 and fabG1 (strains SK039 and Sp245.1610, respectively) were characterized by impaired flagellation and motility. The putative products of expression of these genes are 3-hydroxyisobutyrate dehydrogenase and 3-oxoacyl-[acyl-carrier protein] reductase, respectively. In the present work, A. brasilense strains Sp245, SK039, and Sp245.1610 were found to have differences in the content of 3-hydroxyhexadecanoic, hexadecanoic, 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, and nonadecanoic acids in their lipopolysaccharide preparations, as well as in cell hydrophobicity and hemagglutination activity and dynamics of cell aggregation, in biomass amount, and in the relative content of lipopolysaccharide antigens in mature biofilms formed on hydrophilic or hydrophobic surfaces.     

Structural properties of capsular and O-specific polysaccharides of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 under varying cultivation conditions

Effect of the carbon source in the culture medium and of the growth phase on the composition and structure of the capsular polysaccharides (CPSs) and lipopolysaccharides (LPSs) of the bacterium Azospirillum brasilense Sp245 was studied. Growth with fructose resulted in an increased carbohydrate content in the CPSs, while long-term cultivation resulted in an increased content of phosphorus in both CPSs and LPSs. The LPSs produced on the medium with fructose (regardless of the cultivation duration) and the LPSs of the bacteria grown with sodium malate until the stationary phase were characterized by higher levels of unsaturated fatty acids than the LPSs of the bacteria grown with sodium malate to the late exponential phase. The structures of the polysaccharides from the isolated glycopolymers were established using monosaccharide analysis, including determination of the absolute configurations and 1D and 2D NMR spectroscopy. This study is the first to report that the CPS of A. brasilense Sp245 grown with sodium malate to the end of the exponential phase is structurally identical to the O-polysaccharide from the LPS of this bacterium and that the LPS and CPS of A. brasilense Sp245 grown with fructose contain an additional homoglucan of the following structure: [→3)-α-D-Glcp-(1→] _n .     

The effect of mutations affecting synthesis of lipopolysaccharides and calcofluor-binding polysaccharides on biofilm formation by <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

The thickness and antigenic properties of biofilms produced by Azospirillum brasilense Sp245 and its mutants deficient in the synthesis of lipopolysaccharides (Lps) and calcofluor-binding polysaccharides (CBPS) at the interface between water and hydrophilic or hydrophobic solid surfaces were compared. The mutants deficient in acidic LpsI synthesis produce thicker biofilms on hydrophilic surfaces. Biofilms produced on hydrophobic surfaces by bacteria that are unable to synthesize CBPS are less pronounced. Defects in CBPS production in Azospirillum mutants with impaired flagellar motility can cause adverse effects on the cell ability to attach to hydrophobic and hydrophilic surfaces. The loss of the neutral LpsII antigen by the mutants capable of producing CBPS does not affect their behavior on hydrophobic surfaces, which is probably due to the compensatory increase in the total polysaccharide production. The fundamental change in the Lps structure correlates with the activation of biofilm formation by the relevant mutants on hydrophilic and hydrophobic surfaces.     

Changes in the Antigenic Properties of Azospirillum brasilense Lipopolysaccharide,Induced by Addition of Tris-(hydroxymethyl)-aminomethane into the Culture Medium

Addition of tris-(hydroxymethyl)-aminomethane (Tris) into the culture medium of Azospirillum brasilense sp245 changes the antigenic properties of the lipopolysaccharide (LPS) isolated from the external membrane of the bacteria. LPS preparations from the bacteria grown in the presence of Tris have been analyzed by immunodiffusion, using monospecific antibodies. The disappearance of the precipitation band corresponding to one of the two O-specific polysaccharides of the LPS (O-PS) and changes in the electrophoretic profile have been revealed. However, only minor differences in absorption spectra of products of O-PS1 reaction with phenol/sulfuric acid have been demonstrated between the bacteria grown under standard conditions and in the presence of Tris.     

Early plant growth and biochemical responses induced by <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 lipopolysaccharides in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) seedlings are attenuated by procyanidin B2

This study analyzes the effects of procyanidin B2 on early wheat plant growth and plant biochemical responses promoted by lipopolysaccharides (LPS) derived from the rhizobacteria Azospirillum brasilense Sp245. Measurements of leaf, root length, fresh weight, and dry weight showed in vitro plant growth stimulation 4 days after treatment with A. brasilense as well as LPS. Superoxide anion (O₂ ^·?) and hydrogen peroxide (H₂O₂) levels increased in seedling roots treated with LPS (100 μg mL^?1). The chlorophyll content in leaf decreased while the starch content increased 24 h after treatment in seedling roots. The LPS treatment induced a high increase in total peroxidase (POX) (EC 1.11.1.7) activity and ionically bound cell wall POX content in roots, when compared to respective controls. Early plant growth and biochemical responses observed in wheat seedlings treated with LPS were inhibited by the addition of procyanidin B2 (5 μg mL^?1), a B type proanthocyanidin (PAC), plant-derived polyphenolic compound with binding properties of LPS. All results suggest first that the ionically bound cell wall POX enzymes could be a molecular target of A. brasilense LPS, and second that the recognition or association of LPS by plant cells is required to activate plant responses. This last event could play a critical role during plant growth regulation by A. brasilense LPS.     

A method of acoustic analysis for detection of bacteriophage-infected microbial cells

The dependence of the changes of physical parameters of the suspension of Azospirillum brasilense Sp7 cells infected by FAb-Sp7 bacteriophage on their number and exposure time was studied using a biological sensor based on a piezoelectric resonator with a lateral electric field. The change in the value of the analytical signal was recorded at 1 minute from the beginning of the infection of the cells by bacteriophage. The selectivity of the action of the FAb-Sp7 bacteriophage was studied for Azospirillum brasilense (strains Cd, Sp107, Sp245, Jm6B2, Br14, KR77, S17, S27, SR55, and SR75), A. lipoferum (strains Sp59b, SR65, and RG20a), A. halopraeferans Au4, Nitrospirillum amazonense Am14, Niveispirillum irakense (strains KBC1 and KA3) bacteria, as well as for heterologous bacteria of the genera Escherichia coli (strains XL-1 and B-878), Pseudomonas putida (strains C-11 and BA-11), and Acinetobacter calcoaceticum A-122. The limit of the reliable determination of the concentration of microbial cells during bacteriophage infection process was found: ~10⁴ cells/mL. At the same time, the presence of heterologous cell cultures (E. coli XL-1 cells) did not complicate the detection. It was shown that the method of electroacoustical analysis of cell suspensions can be used for the detection of microbial cells of Azospirillum infected by the FAb-Sp7 bacteriophage. The results are promising for the development of methods for determining and controlling the number of soil microorganisms.     

Stabilizing effect of <Emphasis Type="Italic">Azospirillum</Emphasis> lectins on β-glucosidase activity

Lectins from the surface of Azospirillum brasilense Sp7 and Azospirillum brasilense Sp7.2.3 (a mutant with impaired lectin activity) were shown to induce a stabilizing effect on the activity of almond β-glucosidase under conditions of thermoinactivation and proteolytic enzyme treatment. Differences were revealed in the influence of lectins with various antigenic properties. Our results indicate that the effects of lectins on the catalytic activity of the enzyme are mainly associated with conformational changes in lectin molecules during mutagenesis, but not with carbohydrate specificity (general property). These data should be taken into account in evaluating the role of lectins in the formation of nitrogen-fixing associations.     

Chromosomal <Emphasis Type="Italic">flhB1</Emphasis> gene of the alphaproteobacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 is essential for correct assembly of both constitutive polar flagellum and inducible lateral flagella

Azospirillum brasilense has the ability of swimming and swarming motility owing to the work of a constitutive polar flagellum and inducible lateral flagella, respectively. The interplay between these flagellar systems is poorly understood. One of the key elements of the flagellar export apparatus is the protein FlhB. Two predicted flhB genes are present in the genome of A. brasilense Sp245 (accession nos. HE577327–HE577333). Experimental evidence obtained here indicates that the chromosomal coding sequence (CDS) AZOBR_150177 (flhB1) of Sp245 is essential for the production of both types of flagella. In an flhB1::?Omegon-Km mutant, Sp245.1063, defects in polar and lateral flagellar assembly and motility were complemented by expressing the wild-type flhB1 gene from plasmid pRK415. It was found that Sp245.1063 lost the capacity for slight but statistically significant decrease in mean cell length in response to transfer from solid to liquid media, and vice versa; in the complemented mutant, this capacity was restored. It was also shown that after the acquisition of the pRK415-harbored downstream CDS AZOBR_150176, cells of Sp245 and Sp245.1063 ceased to elongate on solid media. These initial data suggest that the AZOBR_150176-encoded putative multisensory hybrid sensor histidine kinase–response regulator, in concert with FlhB1, plays a role in morphological response of azospirilla to changes in the hardness of a milieu.     

Effect of bacterial lipopolysaccharides on morphogenetic activity in wheat somatic calluses

We evaluated the effect of lipopolysaccharides from the plant-growth-promoting associative bacterium Azospirillum brasilense Sp245 and from the enteric bacterium Escherichia coli K12 on the morphogenic potential of in vitro-growing somatic calluses of soft spring wheat (Triticum aestivum L. cv. Saratovskaya 29). A genetic model was used that included two near-isogenic lines of T. aestivum L. cv. Saratovskaya 29 with different embryogenic capacities; one of these lines carries the Rht-B1 dwarfing gene, whereas the other lacks it. When added to the nutrient medium, the lipopolysaccharide of A. brasilense Sp245 promoted the formation of calluses with meristematic centers and stimulated the regeneration ability of the cultured tissues in both lines. By contrast, the lipopolysaccharide of the enteric bacterium E. coli K12 barely affected the morphogenetic activity of callus cells and the yield of morphogenic calluses and regenerated plants. These findings indicate that the lipopolysaccharide of the plant-growth-promoting associative bacterium A. brasilense Sp245 specifically enhances the morphogenetic activity of wheat somatic tissues, which increases the efficacy of culturing of genotypes with a relatively low morphogenic potential. The results of the study may contribute to the improvement of the efficacy of plant cell selection and gene engineering and to a better understanding of the mechanisms responsible for plant recognition of lipopolysaccharides of associative bacteria.     

Heterologous expression of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> serotype 1 O-antigen analog in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 W3110 by transferring a minimum number of genes involved in O-polysaccharide biosynthesis

Objective

To heterologously produce the Shigella dysenteriae serotype 1 O-polysaccharide (O-PS, O-antigen) in Escherichia coli by transferring the minimum number of genes instead of the entire O-PS gene cluster.

Results

The three glycosyltransferase genes (rfbR, rfbQ and rfp) responsible for the formation of the O-repeat unit were introduced into E. coli K-12 W3110 to synthesize S. dysenteriae 1 O-PS. The specific O-antigen ladder type with different chain lengths of O-repeat units was observed in the recombinant E. coli strain by SDS-PAGE silver staining and western blotting using S. dysenteriae 1 lipopolysaccharide antiserum. Analysis by mass spectrometry and ion chromatography suggested generation of the specific S. dysenteriae 1 O-repeat unit structure with an extra glucose residue attached.

Conclusions

Recombinant E. coli expressing specific glycosyltransferase genes can generate the O-PS of S. dysenteriae 1 and might be able to synthesize heterologous O-antigens of various pathogenic bacteria for vaccine preparation.

     

Analysis of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> plasmid loci coding for (Lipo)polysaccharides synthesis enzymes

Lipopolysaccharides LpsI and LpsII containing the same O-specific polysaccharide (OPS), yet different in antigenic structure and charge, have been revealed in the rhizobacterium Azospirillum brasilense Sp245. In the present work, four putative glycosyltransferase genes were identified in a 14-kbp fragment of a 120-MDa plasmid of Sp245, p120-lpsKM348X. Insertional mutagenesis of one of them, encoding the predicted ADP-heptose:LPS-heptosyltransferase, resulted in LpsI loss. By means of DNA hybridizations and PCR with primers specific towards several sites of p120-lpsKM348X, it was demonstrated that homologous segments of 120-MDa plasmids of A. brasilense strains Sp245 and Sp107, which are characterized by identical structures of the OPS repeating units, are practically identically organized. In an 85-MDa plasmid of Sp245, a locus was identified with high homology to the plasmid genes of glycosyltransferases and conserved membrane-bound proteins from a wide range of soil bacteria.     

Dynamics of the changes of electrophysical properties of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 cells at their binding with wheat germ agglutinin

The electrooptical properties of Azospirillum brasilense Sp7 cell suspensions, have been studied at a specific interaction with wheat germ agglutinin (WGA), using the dependences between the changes of optical densities of cell suspensions at the electric orientation of cells and the orienting field frequencies of 740, 1000, 1450, 2000, and 2800 kHz. It was shown that the electrooptical (EO) properties of cell suspensions changed at the interaction of A. brasilense Sp7 cells with WGA and that the EO signal value changed irrespective of the cultivation conditions. At the same time, the dynamics of the changes of the EO properties of microbial suspensions was different for microbial cells grown under different conditions. It may be evidence of the differences in the cell surface properties of microbial cells, and of the dependence, between bacterial response to lectin and growth conditions. The possibility of using the EO analysis of bacterial suspensions for the study of the high-specific binding of polypeptide molecular signals with the bacterial target cells and for assessment of the dynamics of this process has been demonstrated.     

Relations between the chemotype of <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> strains and the cell electrophoretic properties

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.