Antigenic identity of the capsule lipopolysaccharides,exopolysaccharides, and O-specific polysaccharides in Azospirillum brasilense

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.     

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.     

Determination of the Structure of the Repeated Unit of the Azospirillum brasilense SR75 O-Specific Polysaccharide and Homology of the lps Loci in the Plasmids of Azospirillum brasilense strains SR75 and Sp245

The structural identity of the repeated unit in O-specific polysaccharides (OPSs) present in the outer membrane of strain SR75 of the bacterium Azospirillum brasilense, isolated from wheat rhizosphere in Saratov oblast, and the previously studied OPSs of A. brasilense strain Sp245, isolated from surfacesterilized wheat roots in Brazil, has been demonstrated. Plasmid profiles, DNA restriction, and hybridization assays suggested that A. brasilense strains SR75 and Sp245 have different genomic structures. It was shown that homologous lps loci of both strains were localized in their plasmid DNA. This fact allows us to state that, despite their different origin, the development of the strains studied was convergent. Presumably, the habitation of these bacteria in similar ecological niches influenced this process in many respects. __________ Translated from Mikrobiologiya, Vol. 74, No. 5, 2005, pp. 626–632. Original Russian Text Copyright ? 2005 by Fedonenko, Borisov, O. Konnova, Zdorovenko, Katsy, S. Konnova, Ignatov.     

The Use of Fragments of the 85- and 120-MDa Plasmids of Azospirillum brasilense Sp245 to Study the Plasmid Rearrangement in This Bacterium and to Search for Homologous Sequences in Plasmids of Azospirillum brasilense Sp7

A spontaneous loss of the 85- (p85) and 120-MDa (p120) replicons and simultaneous generation of a plasmid of more than 300 MDa were associated with defects in synthesis of O-specific and Calcofluor-binding polysaccharides and had no effect on flagellation and motility of theAzospirillum brasilenseSp245.5 mutant. The plasmid rearrangement was studied by hybridization of DNAs from the wild-type Sp245 strain and the Sp245.5 mutant with p85 and p120 fragments that contained loci involved in formation of the polar (fla) and lateral (laf) flagella, synthesis of O-specific and Calcofluor-binding polysaccharides (lps/cal), swimming (mot), and swarming (swa) of bacteria. Hybridization with the p120 fragments revealed incorporation of the intact fla/swa loci and the altered lps/cal loci into a new megaplasmid. Two EcoRI fragments homologous to the fla/laf/mot/swa loci of p85 were found in A. brasilense Sp245 DNA, whereas only one copy was preserved in the Sp245.5 mutant. Hybridization of the p120 and p85 fragments of Sp245 to the A. brasilenseSp7 DNA for the first time revealed regions of substantial homology to these fragments in the 90- and 115-MDa Sp7 plasmids, respectively.     

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 .     

Azospirillum brasilense colonisation of wheat roots and the role of lectin–carbohydrate interactions in bacterial adsorption and root-hair deformation

The dynamics of adsorption of the nitrogen-fixing soil bacteria Azospirillum brasilense 75 and 80 (isolated from soil samples collected in Saratov Oblast, southern Russia) and A. brasilense Sp245 to the roots of seedlings of common spring wheat was studied in relation to inoculum size, period of incubation with the roots and bacterial-growth phase. The number of root-attached cells increased with increasing size of inoculum and time of contact. The saturation of root-surface adsorption was observed by 24 h of co-incubation for A. brasilense 75, by 6 h for A. brasilense 80, and by 3 h for A. brasilense Sp245. The firmness of bacterial–root attachment increased after extended co-incubation. Differences in the adsorption kinetics of the azospirilla were found that were associated with bacterial-growth phases. Azospirilla attached to the roots of their host cultivar more actively than they did to the roots of a non-host cultivar. Adsorption was partially inhibited when the roots were treated with N-acetyl-D-glucosamine. Maximal inhibition occurred after a 3-h exposure of the roots to the bacteria. Root-hair deformation induced with polysaccharide-containing complexes from the Azospirillum capsular material was inhibited by N-acetyl-D-glucosamine and chitotriose, specific haptens of wheat germ agglutinin. A possible mechanism of the mutual influence of bacteria and plants may involve key roles of wheat germ agglutinin, present on the roots, and the polysaccharide-containing components of the Azospirillum capsule.     

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.     

Effect of inoculation ofAzospirillum spp. on nitrogen accumulation by field-grown wheat

Summary Two experiments were performed to examine the effects of inoculation of field grown wheat with various Azospirillum strains. In the first experiment the soil was sterilized with methyl bromide to reduce the Azospirillum population and¹⁵N labelled fertilizer was added to all treatments. Two strains ofAzospirillum brasilense isolated from surface sterilized wheat roots and theA. brasilense type strain Sp7 all produced similar increases in grain yield and N content. From the¹⁵N and acetylene reduction data it was apparent that these increases were not due to N₂ fixation. In the second experiment performed in the same (unsterilized) soil, twoA. brasilense strains (Sp245, Sp246) and oneA. amazonense strain (Am YTr), all isolated from wheat roots, produced responses of dry matter and N content while the response to the strain Sp7 was much smaller. These data confirm earlier results which indicate that if natural Azospirillum populations in the soil are high (the normal situation under Brazilian conditions), strains which are isolated from wheat roots are better able to produce inoculation responses than strains isolated from other sources. The inoculation of a nitrate reductase negative mutant of the strain Sp245 produced only a very small inoculation response in wheat. This suggests that the much greater inoculation response of the original strain was not due to N₂ fixation but to an increased nitrate assimilation due to the nitrate reductase activity of the bacteria in the roots. Consultant Inter-American Institute for Cooperation in Agriculture IICA/EMBRAPA World Bank Project.     

Improved Potential for Nitrogen Fixation in Azospirillum brasilense Sp7‐S Associated with Wheat nifH Expression as a Function of Oxygen Pressure

The use of a nifH‐lacZ fusion as an indicator of nitrogen fixing conditions is investigated in relation to two strains of Azospirillum brasilense with contrasting patterns of colonization on wheat roots. The degree of expression of nifH‐lacZ of Azospirillum brasilense could be manipulated by controlling the oxygen pressure. A strong correlation between nitrogenase activity and nifH expression was found in pure cultures. nifH expression was maximal at 0.5% oxygen in pure cultures of both the wild type Sp7 and spontaneous mutant Sp7‐S. Differentiation of the maximal expression was observed when the two strains were in association with para‐nodulated wheat, resulting in greater expression by Sp7‐S over a broader range of external oxygen concentrations than by Sp7. This result was observed when expressed as activity per mg of plant protein as well as per bacterium. An increase in nifH expression was also noted with para‐nodulated (2,4‐D treated) wheat inoculated with Sp7‐S when compared with untreated wheat. No significant difference was found between treated and untreated wheat inoculated with Sp7. The results indicate that the majority of the Azospirillum brasilense Sp7‐S cells occupy a more protected niche when in association with wheat roots, resulting in conditions that support a greater potential for nitrogen fixation as judged by nifH expression.     

The Role of Cell-Surface Lectins in the Aggregation of Azospirilla

The mutant strain Azospirillum brasilenseSp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilenseSp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilenseSp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense75, A. brasilenseSp7, and A. lipoferum59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilenseSp7 cells with Bacillus polymyxa1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.     

Capsular polysaccharide of the bacterium <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis> Sp59b: Structure and antigenic specificity

Antigenic differences were revealed between the cell wall outer membrane lipopolysaccharides and the capsular high molecular weight bioglycans for a typical strain of the nitrogen-fixing rhizobacterium Azospirillum lipoferum Sp59b using antibodies prepared against the homologous lipopolysaccharide and lipopolysaccharide-protein complex. From the capsular lipopolysaccharide-protein and polysaccharide-lipid complexes of A. lipoferum Sp59b, polysaccharides were isolated and their structure was for the first time established in Azospirillum by monosaccharide analysis which included determination of the absolute configurations, methylation, O-deacetylation, and one- and two-dimensional NMR spectroscopy. The polysaccharides of the capsular complexes were shown to have identical structure of the branched tetrasaccharide repeating unit, which differs from the structure of the O-specific polysaccharide within the outer membrane lipopolysaccharide of this strain.     

Effect of lectins from <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> to peroxidase and oxalate oxidase activity regulation in wheat roots

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability of lectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect of lectin in under 10 min in a concentration of 10 μg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.     

Identification of salt stress inducible genes that control cell envelope related functions in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7

Plant growth promoting rhizobacteria such as Azospirillum brasilense are agronomically important as they are frequently used for crop inoculation. But adverse factors such as increasing soil salinity limit their survival, multiplication and phytostimulatory effect. In order to understand the role of the genes involved in the adaptation of A. brasilense Sp7 to salt stress, a mutant library (6,800 mutants) was constructed after random integration of a mini-Transposon Tn5 derivative containing a promoterless gusA and oriV. The library was screened for salt stress inducible Gus activity on minimal malate agar medium containing NaCl and 5-bromo-4-chloro-3-indolyl-β-d-glucuronide. Salt stress responsiveness of the promoters was estimated by quantifying GusA activity in the presence and absence of NaCl stress using p-nitrophenyl-β-d-glucuronide as a substrate. In 11 mutants showing high levels of gusA expression in the presence of salt-stress, the partial nucleotide sequence of the DNA region flanking the site of Tn5 insertion was determined and analysed using the NCBI-BLAST programs. Similarity searches revealed that 10 out of the 11 genes sequenced showed notable similarity with genes involved in functions related to modulation in the composition of exopolysaccharides, capsular polysaccharides, lipopolysaccharides, peptidoglycan and lipid bilayer of the cell envelope. Induction of cell envelope related genes in response to salt stress and salt sensitive phenotype of several mutants in A. brasilense indicate a prominent role of cell envelope in salt-stress adaptation.     

The structure of the O-specific polysaccharide from a mutant of nitrogen-fixing rhizobacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 with an altered plasmid content

The rhizobacteria Azospirillum brasilense Sp245 produce immunochemically different lipopolysaccharides LPSI and LPSII, both containing identical pentasaccharides built from D-rhamnose residues as the repeating units of O-specific polysaccharides (OPS). In this study, we report the structure of the OPS from A. brasilense LPSI⁻LPSII⁻ mutant Sp245.5, which spontaneously lost the p85 and p120 plasmids upon the formation of a new 300-MDa megaplasmid after the long-term storage of the bacteria in a rich medium. The repeating unit of the OPS of A. brasilense Sp245.5 appeared to be a disaccharide consisting of residues of N-acetyl-D-galactosamine and N-acetyl-D-mannosaminuronic acid:

Azospirillum brasilense Sp 245 produces ABA in chemically-defined culture medium and increases ABA content in arabidopsis plants

Azospirillum sp. are plant growth promoting bacteria (PGPB) that increase grain yield in cereals and other species via growth promotion and/or stress alleviation. The PGPB beneficial effects have been partially attributed to bacterial production of plant hormones, especially growth promoters like auxins, gibberellins and cytokinins. This paper reports the characterization of the stress-like plant hormone abscisic acid (ABA) by GC-EIMS in cultures of A. brasilense Sp 245 after 120 h of incubation in chemically-defined media, and chemically-defined media with moderate stress (100 mM NaCl). Chemical characterization of ABA was done by gas chromatography-electron impact mass spectrometry (GC-EIMS) and quantification by selected ion monitoring (SIM) with a stable isotope of the hormone as internal standard in the media. A. brasilense cultures produced higher amounts of ABA per ml of culture when NaCl was incorporated in the culture medium. Inoculation of Arabidopsis thaliana with A. brasilense Sp 245 enhanced two-fold the plant’s ABA content. These results contribute to explain, at least to some extent, the beneficial effects of Azospirillum sp. previously found in inoculated plants placed under adverse environmental conditions.     

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.     

Wheat root colonization by Azospirillum brasilense strains with different motility

Migration of associative bacteria Azospirillum brasilense in semisolid media is performed mainly by swarming (Swa⁺ phenotype), which depends on the flagellar functioning and intercellular contacts. Non-swarming mutants of A. brasilense Sp245 lacking a polar flagellum migrate in semisolid media with microcolony formation using a unrevealed mechanism (Gri⁺ phenotype). The study of wheat root colonization dynamics demonstrated that A. brasilense Sp245 Gri⁺ mutants exhibited lower capacity for wheat root adsorption. However, after “anchoring” has occurred, both A. brasilense Sp245 and its Swa-Gri⁺ mutants colonized the growing roots with virtually the same efficiency. All strains under study formed microcolonies on the surface of roots, stimulated root branching, and exhibited changes in the composition of protein antigens exposed on the bacterial cell surface. Indirect evidence was obtained for enhanced production of genus-specific protein antigens in the process of A. brasilense Sp245 adaptation to growth on plant roots.