A Comparison of the Lipopolysaccharides and O-Specific Polysaccharides of Azospirillum brasilense Sp245 and Its Omegon-Km Mutants KM018 and KM252

The lipopolysaccharides (LPSs) extracted from the outer membrane of Azospirillum brasilense Sp245 and its Omegon-Km mutants KM018 and KM252 with a hot aqueous solution of phenol were found to differ in the content of carbohydrates, glucosamine, and total phosphorus and in the proportion of octadecenoic and hexadecanoic acids in the lipid moieties of the LPSs. The carbohydrate moieties of the LPSs were heterogeneous in charge. The analysis of the O-specific polysaccharides (O-PSs) of the mutants KM018 and KM252 by gas–liquid chromatography, IR spectroscopy, and NMR spectroscopy showed that they are composed of the same linear pentasugar repeating units 2)--D-Rhap-(1 3)--D-Rhap-(1 3)--D-Rhap-(1 2)--D-Rhap-(1 2)--D-Rhap-(1 as the O-PSs of the parent strain Sp245. The reported differences in the biological activity of the LPSs of the parent and mutant strains can be due to their different chemical composition.     

Participation of azospirillium polysaccharides in interaction with wheat root surface]

The present study was undertaken to comparatively investigate the attachment capacities of Azospirillum brasilense Sp245 and its lipopolysaccharide-defective Omegon-Km mutants KM018 and KM252, as well as their activities with respect to the alteration of the morphology of wheat seedling root hairs. The adsorption dynamics of the parent Sp245 and mutant KM252 strains of azospirilla on the seedling roots of the soft spring wheat cv. Saratovskaya 29 were similar; however, the attachment capacity of the mutant KM252 was lower than that of the parent strain throughout the incubation period (15 min to 48 h). The mutation led to a considerable decrease in the hydrophobicity of the Azospirillum cell surface. The lipopolysaccharides extracted from the outer membrane of A. brasilense Sp245 and mutant cells with hot phenol and purified by chromatographic methods were found to induce the deformation of the wheat seedling root hairs, the lipopolysaccharide of the parent strain being the most active in this respect. The role of the carbohydrate moiety of lipopolysaccharides in the interaction of Azospirillum cells with plants is discussed.     

Involvement of the Lipopolysaccharides of Azospirilla in the Interaction with Wheat Seedling Roots

The present study was undertaken to comparatively investigate the attachment capacities of Azospirillum brasilenseSp245 and its lipopolysaccharide-defective Omegon-Km mutants KM018 and KM252, as well as their activities with respect to the alteration of the morphology of wheat seedling root hairs. The adsorption dynamics of the parent Sp245 and mutant KM252 strains of azospirilla on the seedling roots of the soft spring wheat cv. Saratovskaya 29 were similar; however, the attachment capacity of the mutant KM252 was lower than that of the parent strain throughout the incubation period (15 min to 48 h). The mutation led to a considerable decrease in the hydrophobicity of the Azospirillumcell surface. The lipopolysaccharides extracted from the outer membrane of A. brasilenseSp245 and mutant cells with hot phenol and purified by chromatographic methods were found to induce the deformation of the wheat seedling root hairs, the lipopolysaccharide of the parent strain being the most active in this respect. The role of the carbohydrate moiety of lipopolysaccharides in the interaction of Azospirillumcells with plants is discussed.     

Structure of the O-specific polysaccharide of the lipopolysaccharide of Azospirillum brasilense Sp245

An O-specific polysaccharide was isolated from the lipopolysaccharide of a plant-growth-promoting bacterium Azospirillum brasilense Sp245 and studied by sugar analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY. The polysaccharide was found to be a new rhamnan with a pentasaccharide repeating unit having the following structure:-->2)-beta-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->     

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 Isolation and Partial Characterization of the Lipopolysaccharides from Several Rhizobium trifolii Mutants Affected in Root Hair Infection

The lipopolysaccharides (LPSs) from Rhizobium trifolii ANU843 and several transposon (Tn5) symbiotic mutants derived from ANU843 were isolated and partially characterized. The mutant strains are unable to induce normal root hair curling (Hac- phenotype) or nodulation (Nod-phenotype) in clover plants. The LPSs from the parent and mutants are very similar in composition. Analysis by PAGE shows that the LPSs consist of higher and lower molecular weight forms. The higher molecular weight form of the LPSs exists in several aggregation states when PAGE is done in 0.1% SDS but collapses into a single band when PAGE is done in 0.5% SDS. Mild acid hydrolysis of all the LPSs releases two polysaccharides, PS1 and PS2. Immunoblots of the PAGE gels and enzyme linked immunosorbant assay inhibition assays show that the PS1 fractions contain the immunodominant sites of the LPSs and that these sites are present in the higher molecular weight form of the LPSs. All the PS1 fractions contain methylated sugars, 2-amino-2,6-dideoxyhexose, heptose, glucuronic acid, and 2-keto-3-deoxyoctonic acid (KDO). All the PS2 fractions contain galacturonic acid, mannose, galactose, and KDO. The PS2 fractions have a molecular weight of about 700. The KDO is present at the reducing end of both the PS1 and the PS2 fractions. The PS1 and PS2 fractions from the mutants contain more glucose than these fractions from the parent. The LPS from a deletion mutant contains less acyl groups than the other LPSs. Immunoblots of the LPSs show that the parent and nod A mutant LPSs contain an additional antigenic band which is not observed in the other LPSs.     

Nitrogen-fixing bacterium Burkholderia brasiliensis produces a novel yersiniose A-containing O-polysaccharide

Burkholderia brasiliensis, a Gram-negative diazotrophic endophytic bacterium, was first isolated from roots, stems, and leaves of rice plant in Brazil. The polysaccharide moiety was released by ammonolysis from the B. brasiliensis lipopolysaccharide (LPS), allowing the unambiguous characterization of a 3,6-dideoxy-4-C-(1-hydroxyethyl)-D-xylo-hexose (yersiniose A), an uncommon feature for Burkholderia LPS. The complete structure of the yersiniose A-containing O-antigen was identified by sugar and methylation analyses and NMR spectroscopy. Our results show that the repeating oligosaccharide motif of LPS O-chain consists of a branched tetrasaccharide with the following structure:-->2-alpha-d-Rhap-(1-->3)-[alpha-YerAp-(1-->2)]-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->.     

The O-chain structure from the LPS of the bacterium Naxibacter alkalitolerans YIM 31775T

The O-chain polysaccharide of the lipopolysaccharide from the bacterium Naxibacter alkalitolerans strain YIM 31775(T) was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy and shown to be built up by the following tetrasaccharide repeating unit: -->3)-alpha-D-FucpNAc-(1-->2)-beta-D-Quip3NHBu-(1-->2)-alpha-D-Rhap-(1-->)-beta-D-Galp-(1--> where HBu is hydroxy-butanoyl.     

Structure of the O-polysaccharide of Pseudomonas putida FERM P-18867

The O-polysaccharide of the lipopolysaccharide of Pseudomonas putida FERM P-18867 was found to contain D-mannose and D-rhamnose and have the following structure of the trisaccharide repeating unit:-->2)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-beta-D-Manp-(1-->     

Isolation and characterization of lectin from the surface of Grifola frondosa (FR.) S.F. Gray mycelium

For the first time, from the surface of the dikaryotic mycelium of the xylotrophic basidiomycete Grifola frondosa 0917 a lectin has been isolated with a molecular mass of 68 +/- 1 kDa, consisting of two subunits of 33-34 kDa each. The lectin is a hydrophilic glycoprotein with the protein : glycan ratio of 3 : 1. It exhibits high affinity to native rabbit erythrocytes and to human erythrocytes of the 0 blood group, but not to trypsin-treated ones. The hemagglutination (HA) caused by lectin was not blocked by any of the 25 tested mono-, di-, and amino sugars; it was also not blocked by some of glyco derivatives. Only 13.9 microg/ml of the homogeneous preparation of a polysaccharide, a linear D-rhamnan with the structure of the repetitive component --> 2)-beta-D-Rhap-(1 --> 3)-alpha-D-Rhap-(1 --> 3)-alpha-D-Rhap-(1 --> 2)-alpha-D-Rhap-(1 --> 2)-alpha-D-Rhap-(1 --> blocked hemagglutination completely. The analysis of the amino acid composition of the lectin showed the greatest percentage of amino acids with positively charged R groups, arginine, lysine, and histidine, as well as the complete absence of sulfur-containing amino acids, cysteine, and methionine. D-glucose and D-glucosamine were detected in the carbohydrate part.     

Hemagglutinating activity and motility of the bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> in the presence of various nitrogen sources

Hemagglutinating activity of the Azospirillum brasilense strain Sp245 grown in liquid media and the swarming motility of those bacteria grown in semisolid media vary significantly depending on the nitrogen source. In media with nitrate or nitrite, an increase in the hemagglutinating activity and a decrease in the swarming circles’ diameter of Sp245 were observed, compared to bacteria grown in the presence of ammonium or N₂. A ∼67-kDa hemagglutinin exhibiting affinity to the O-specific polysaccharide, an acidic D-rhamnan (OPS-I), was isolated from the surface of Sp245 cells. Introduction of the hemagglutinin into the media resulted in a decrease in the Sp245 cell motility while not affecting its mutants lacking the acidic D-rhamnan or the Sp245.5 mutant with a different OPS structure. Cells of strain Sp245.5 demonstrated hemagglutinating activity two times higher than that of the parent Sp245 strain and formed “diffuse” colonies, rather than distinct swarming circles Sp245 formed when grown in a semisolid medium. The data obtained demonstrate that intercellular contacts mediated by the interaction between the surface hemagglutinin and OPS-I, which is sensitive to environmental factors, affect the collective motility of cells.     

Lipopolysaccharides possessing two L-glycero-D-manno-heptopyranosyl-alpha -(1-->5)-3-deoxy-D-manno-oct-2-ulopyranosonic acid moieties in the core region. The structure of the core region of the lipopolysaccharides from Burkholderia caryophylli.

The carbohydrate backbone of the core-lipid A region was characterized from the lipopolysaccharides (LPSs) of the plant-pathogenic bacterium Burkholderia caryophylli. For the first time, the presence of two moieties of l-glycero-d-manno-heptopyranosyl-alpha-(1-->5)-3-deoxy-d-manno-oct-2-ulopyranosonic acid was identified in a core region, which is of particular interest with regard to the biosynthesis of this and of LPSs in general. The LPSs of B. caryophylli were degraded by mild hydrazinolysis (de-O-acylation), treatment with 48% aqueous HF at 4 degrees C (cleavage of phosphate groups and destruction of the O-specific polysaccharides), reduction with NaBH4, and de-N-acylation utilizing hot KOH. The major oligosaccharide representing the carbohydrate backbone of the core region and lipid A was isolated by high-performance anion-exchange chromatography. Its analysis employing compositional and methylation analyses, matrix-assisted laser desorption/ionization mass spectrometry, and (1)H and (13)C NMR spectroscopy applying various one-dimensional and two-dimensional experiments identified the following structure. Structure I All sugars are pyranoses and alpha-linked, if not stated otherwise. Hep is l-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid.     

Structural and serological studies on Hafnia alvei O-specific polysaccharide of alpha-D-mannan type isolated from the lipopolysaccharide of strain PCM 1223

On the basis of chemical and methylation analyses, one- and two-dimensional (1)H- and (13)C-NMR spectroscopy, including COSY, TOCSY, NOESY and (1)H, (13)C HSQC experiments, a neutral O-specific polysaccharide isolated from Hafnia alvei strain PCM 1223 lipopolysaccharide (LPS) was found to be an alpha-mannan composed of pentasaccharide repeating units having the following structure:-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->. Immunoblotting showed a strong cross-reactivity between anti-H. alvei PCM 1223 serum and LPSs of Escherichia coli O9 and Klebsiella pneumoniae O3. The serological relationship of the LPSs of these bacteria is due to the structural identity of their O-specific polysaccharides, though the LPSs differ in their core regions.     

Changes in Azospirillum brasilense motility and the effect of wheat seedling exudates

The rhizobacterium Azospirillum brasilense Sp245 swims, swarms (Swa⁺ phenotype) or, very rarely, migrates with the formation of granular macrocolonies (Gri⁺ phenotype). Our aims were (i) to identify Sp245 mutants that swarm faster than the parent strain or differ from it in the mode of spreading and (ii) to compare the mutants’ responses to wheat seedling exudates. In isotropic liquid media, the swimming speeds of all motile A. brasilense strains were not influenced by the exudates. However, the exudates significantly stimulated the swarming of Sp245. In several Sp245 mutants, the superswarming phenotype was insensitive to local colonial density and to the presence of wheat seedling exudates. An A. brasilense polar-flagellum-defective Gri⁺ mutant BK759.G gave rise to stable Swa⁺⁺ derivatives with restored flagellum production. This transition was concurrent with plasmid rearrangements and was stimulated in the presence of wheat seedling exudates. The swarming rate of the Swa⁺⁺ derivatives of BK759.G was affected by the local density of their colonies but not by the presence of the exudates.     

Complementation analysis of mutants of the associative bacteria Azospirillum brasilense Sp245 and S27, defective in mobility and flagellation

Three mutants of Azospirillum brasilense Sp245 incapable of both formation of the polar flagellum (Fla-phenotype) and swarming in semisolid media (Swa-phenotype) were characterized. These mutants were shown to have lost the 85-MDa plasmid and to carry the Tn5-Mob transposon and pSUP5011 vector in different regions of their genomes. With the use of A. brasilense Sp245 gene bank, the capacity for both polar flagellum formation and swarming was restored in the above mutants and in the previously generated transposon mutants A. brasilense Sp245 and S27. The transconjugants obtained were only slightly motile in the liquid culture. In the gene bank of Sp245, the recombinant plasmids carrying wild-type fla/swa loci were identified.     

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.     

Complementation Analysis of Associative Bacteria Azospirillum brasilense Sp245 and S27 Defective for Motility and Flagellation

Three mutants of Azospirillum brasilense Sp245 incapable of both formation of the polar flagellum (Fla^–-phenotype) and swarming in semisolid media (Swa^–-phenotype) were characterized. These mutants were shown to have lost the 85-MDa plasmid and to carry the Tn5-Mob transposon and pSUP5011 vector in different regions of their genomes. With the use of A. brasilense Sp245 gene bank, the capacity for both polar flagellum formation and swarming was restored in the above mutants and in the previously generated transposon mutants A. brasilense Sp245 and S27. The transconjugants obtained were only slightly motile in the liquid culture. In the gene bank of Sp245, the recombinant plasmids carrying wild-type fla/swa loci were identified.     

High temperature-induced changes in exopolysaccharides, lipopolysaccharides and protein profile of heat-resistant mutants of Rhizobium sp. (Cajanus)

A thermosensitive wild-type strain (PP201) of Rhizobium sp. (Cajanus) and its 14 heat-resistant mutants were characterized biochemically with regard to their cell surface (exopolysaccharides (EPSs) and lipopolysaccharides (LPSs)) properties and protein profile. Differences were observed between the parent strain and the mutants in all these parameters under high temperature conditions. At normal temperature (30 °C), only half of the mutant strains produced higher amounts of EPSs than the parent strain, but at 43 °C, all the mutants produced higher quantities of EPS. The LPS electrophoretic pattern of the parent strain PP201 and the heat-resistant mutants was almost identical at 30 °C. At 43 °C, the parent strain did not produce LPS but the mutants produced both kinds of LPSs. The protein electrophoretic pattern showed that the parent strain PP201 formed very few proteins at high temperature, whereas the mutants formed additional new proteins. A heat shock protein (Hsp) of 63–74 kDa was overproduced in all mutant strains.     

The isolation and characterization of lipopolysaccharide-defective mutants of Pseudomonas aeruginosa PAC1.

Mutants with defective lipopolysaccharides (LPSs) were isolated from Pseudomonas aeruginosa PACIR (Habs serogroup 3) by selection for resistance to aeruginocin from P. aeruginosa PI6 Carbenicillin-sensitive mutants were isolated from P. aeruginosa PACI but not all had defective LPSs. Rough colonial morphology and resistance to bacteriophage II9X appeared to be independent of LPS composition. The LPSs from five mutants were analysed and compared with that of the parent strain. Separation of partially-degraded polysaccharides from LPS from PACI on Sephadex G75 yielded two different high molecular weight fractions and a phosphorylated low molecular weight fraction (L). The mutant LPSs lacked most or all of the high molecular weight fractions but retained some low molecular weight material. That from PACI and two of the mutants was separated by elution from Biogel P6 into two fractions. One, L2, was the core polysaccharide while the other, LI, contained short antigenic side-chains attached to the core like the semi-rough (SR) LPSs of the Enterobacteriaceae. The two mutants which gave the LI fraction with Habs 3 and PACI antisera as did the parent strain. The other three mutants were unreactive and their LPSs contained core components only. One appeared to have a complete core while the other two lacked rhamnose and rhammose plus glucose respectively. Thus there may be four types of LPS in PACI: one contains unsubstituted core polysaccharide and yields L2 on acid hydrolysis, another has short antigenic side-chains of the SR type and yields the LI fraction, while the two high molecular weight fractions are derived from core polysaccharides with different side-chains.     

Mutants of bacterium Azospirillum brasilense Sp245 with Omegon insertion in mmsB or fabG genes of lipid metabolism are defective in motility and flagellation

Bacteria Azospirillum brasilense have mixed flagellation: in addition to the polar flagellum, numerous lateral flagella are formed on their cells on medium with increased density. Flagella determine the active swimming and swarming capacities of azospirilla. Using A. brasilense Sp245 as an example, it was shown that the Omegon-Km artificial transposon insertion into the chromosomal gene for 3-hydroxyisobutyrate dehydrogenase (mmsB) was concurrent with the appearance of significant defects in the formation of polar flagella and with the paralysis of lateral flagella. The Sp245 mutant with the Omegon insertion into the plasmid AZOBR-p1-borne gene for 3-oxoacyl-[acyl-carrier protein]-reductase (fabG) showed the complete loss of flagella and the swarming capacity, as well as significant defects in polar flagellar assembly (though some cells are still motile in liquid medium). The viability of the A. brasilense Sp245 mutants with the Omegon insertion into the mmsB or fabG gene was not reduced. No considerable differences in the fatty acid composition of whole cell lipid extracts were found for the A. brasilense Sp245 strain and its mmsB and fabG mutants.