Reduction of Selenite by Azospirillum brasilense with the Formation of Selenium Nanoparticles

The ability to reduce selenite (SeO₃ ^2?) ions with the formation of selenium nanoparticles was demonstrated in Azospirillum brasilense for the first time. The influence of selenite ions on the growth of A. brasilense Sp7 and Sp245, two widely studied wild-type strains, was investigated. Growth of cultures on both liquid and solid (2 % agar) media in the presence of SeO₃ ^2? was found to be accompanied by the appearance of the typical red colouration. By means of transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS) and X-ray fluorescence analysis (XFA), intracellular accumulation of elementary selenium in the form of nanoparticles (50 to 400 nm in diameter) was demonstrated for both strains. The proposed mechanism of selenite-to-selenium (0) reduction could involve SeO₃ ^2? in the denitrification process, which has been well studied in azospirilla, rather than a selenite detoxification strategy. The results obtained point to the possibility of using Azospirillum strains as endophytic or rhizospheric bacteria to assist phytoremediation of, and cereal cultivation on, selenium-contaminated soils. The ability of A. brasilense to synthesise selenium nanoparticles may be of interest to nanobiotechnology for “green synthesis” of bioavailable amorphous red selenium nanostructures.     

Effect of genomic rearrangement on heavy metal tolerance in the plant-growth-promoting rhizobacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245

A derivative of Azospirillum brasilense Sp245, Sp245.5, which spontaneously lost 85 and 120 MDa replicons upon the formation of a new megaplasmid, has been shown to produce a novel lipopolysaccharide and to lose Calcofluor-binding polysaccharides. As compared to Sp245, the derivative displays notably increased heavy metal tolerance. The phenotypes of Sp245 and Sp245.5 are characterized by the following minimal inhibitory concentrations (MICs) of heavy metals: 0.5 and 0.9 μmol l⁻¹ of Ag⁺, 0.4 and 0.7 mmol l⁻¹ of Co²⁺, 0.9 and 4.7 mmol l⁻¹ of Cu²⁺, and 3.1 and 11.5 mmol l⁻¹ of Zn²⁺, respectively. In Sp245, in the presence of a nonlethal concentration (0.625 μmol l⁻¹) of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), the MIC of cobalt, copper, and zinc drop 1.3- to 1.6-fold, but the low tolerance to silver is unaffected. In Sp245.5, CCCP does not affect cobalt tolerance, suppresses tolerance to copper and silver to the wild-type levels, and causes a 1.4-fold decrease in resistance to zinc. Therefore, significant elevation of heavy metal tolerance in Sp245.5 seems caused by the induction/overexpression of the proton-dependent efflux of certain metal ions. The novel cell surface and other unknown factors could also be responsible for the increased tolerance of A. brasilense Sp245.5 to heavy metals.     

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.     

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.     

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.     

Arsenic Transformation by Azospirillum Brasilense Sp245 in Association with Wheat (Triticum Aestivum L.) Roots

The transformation of sodium arsenite and sodium arsenate by the rhizospheric nitrogen-fixing bacterium Azospirillum brasilense Sp245 in association with wheat (Triticum aestivum L. ‘Saratovskaya 29’) was studied. The effect produced by the A. brasilense strain on the morphological parameters of wheat in an As-polluted environment was examined. The plants were cultivated in a hydroponic system, with glass beads serving as a support for root growth. The plant-growth medium (an artificial soil solution) was deficient in P and Fe. The total initial As concentrations used were 75, 750, and 7500 μg l⁻¹. The As compounds used contained sodium arsenate and sodium arsenite at an As(V):As(III) ratio of 1:3.6 (in terms of As) in all experiments. Inoculation of A. brasilense Sp245 led to a decrease in the overall root length and to the formation of lateral roots; both effects are possibly related to the bacteria’s ability to synthesize auxins. Inoculation also changed the As(V): As(III) ratio of the plant-growth medium. In all experiments, the concentration of As(V) in the nutrient medium increased relative to the initial one and was approximately 1.5-fold higher than that in the medium of uninoculated plants. This value slightly decreased (1.6 > 1.5 > 1.4) with increasing concentration of As in the medium. Azospirillum-inoculated plants accumulated less As than did the surface-sterilized uninoculated plants. This study shows that A. brasilense Sp245 in association with wheat changes the speciation, bioavailability, and plant uptake of As.     

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.     

Biosynthesis of gold nanoparticles by Azospirillum brasilense

Plant-associated nitrogen-fixing soil bacteria Azospirillum brasilense were shown to reduce the gold of chloroauric acid to elemental gold, resulting in formation of gold nanoparticles. Extracellular phenoloxidizing enzymes (laccases and Mn peroxidases) were shown to participate in reduction of Au⁺³ (HAuCl₄) to Au⁰. Transmission electron microscopy revealed accumulation of colloidal gold nanoparticles of diverse shape in the culture liquid of A. brasilense strains Sp245 and Sp7. The size of the electron-dense nanospheres was 5 to 50 nm, and the size of nanoprisms varied from 5 to 300 nm. The tentative mechanism responsible for formation of gold nanoparticles is discussed.     

Detection of Vascular Endothelial Growth Factor Based on Gold Nanoparticles and Immunoreaction Using Resonance Light Scattering

In the study, a new assay of vascular endothelial growth factor (VEGF) has been developed by the use of gold nanoparticles (GNPs)–anti-VEGF conjugates. The immunoreaction between GNPs–anti-VEGF conjugates and VEGF took place in pH?7.5 PBS buffer solution after the addition of VEGF. The formation of GNPs modified VEGF immunocomplex resulted in the enhanced resonance light scattering (RLS) intensity at 388.0 nm. Under the optimal conditions, the magnitude of enhanced RLS intensity (ΔI _RLS) was proportional to the VEGF concentration in the range from 100 to 1,500 pg?mL^?1, with a detection limit of 60 pg?mL^?1. The surface plasma resonance absorption spectrum, the characteristics of RLS, the VEGF immunocomplex, and the optimum conditions of the immunoreaction have all been investigated. The VEGF concentrations of 20 serum specimens detected by the developed assay showed consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay kit.     

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:

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.     

Spontaneous Super-Swarming Derivatives of Azospirillum brasilense Sp245 have Different DNA Profiles and Behavior in the Presence of Various Nitrogen Sources

Azospirillum brasilense swims in liquid environments and swarms in semisolid media. Five variants of A. brasilense Sp245, Sp245.P1–Sp245.P5, which swarmed faster than Sp245 in a semisolid malate–salt medium, have been isolated. In Sp245.P1–Sp245.P4, a new megaplasmid was revealed instead of an indigenous 85-MDa plasmid (p85). By polymerase chain reactions (PCR) with primers to the segments of p85 important for proper bacterial motility/flagellation and for dissimilatory nitrite and NO reduction, that DNA of p85 was found retained by all the variants. In ERIC- and RAPD-PCR, microdiversity between the total DNAs of Sp245 and its variants was detected. Interstrain differences in growth characteristics in liquid peptone–succinate–salt medium with KNO₃ or KNO₂ and in KNO₂ production/consumption were revealed. Although all the variants swam and swarmed faster than Sp245 in the medium supplemented with NH₄Cl or KNO₃, not all of them could do so in MPSS with KNO₂.     

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.     

The ability of the rhizobacterium Azospirillum brasilense to reduce selenium(IV) to selenium(0)

Effect of selenium(+4) as selenite (Se ₃ ^2? ) on two Azospirillum brasilense strains, which occupy different ecological niches (an epiphyte Sp7 and a facultative endophyte Sp245), was studied. The cultures grown in the medium with sodium selenite exhibited intense red coloration. Transmission electron microscopy and X-ray fluorescence analysis revealed accumulation of elementary selenium within the cells of both strains as nanoparticles 50–400 nm in diameter. The ability to reduce inorganic selenium(+4) to elementary selenium (as nanoparticles) has not been previously reported for azospirilla. Our results indicate the possibility to apply Azospirillum strains as microsymbionts for phytoremediation of, and cereal cultivation on, selenium-contaminated soils. The ability of azospirilla to synthesize selenium nanoparticles may be of interest for nanobiotechnology.     

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.     

Effect of Azospirillum brasilense Sp245 lipopolysaccharide on the functional activity of wheat root meristematic cells

We studied changes in the physiological and biochemical parameters of wheat (Triticum aestivum L. ??Saratovskaya 29??) seedlings treated with lipopolysaccharide isolated from the outer membrane of the associative bacterium Azospirillum brasilense Sp245. The obtained data were compared with (i) the results of plant inoculation with whole Sp245 cells and (ii) the effects exerted by the lipopolysaccharide and whole cells of the enterobacterium Escherichia coli K12 and the specific legume symbiont Rhizobium leguminosarum 249. The functional activity of meristematic cells was judged by their mitotic index and by the results of immunochemical determination of the proliferative antigen of initials, a molecular marker for wheat meristem cells. Treatment of the seedling root system with 10 ??g mL^?1 of Sp245 lipopolysaccharide increased the mitotic index (1.8-fold) and the antigen content (approximately 1.4-fold). These increases were comparable to the effects produced by whole cell inoculation (2- and 1.4-fold, respectively). Our findings give grounds to consider lipopolysaccharide as an active component of the Azospirillum cell surface that not only determines bacterial contact interactions with wheat roots but also participates in the induction of plant responses to these interactions. We finally discuss the linkage between the proliferative antigen of initials and the transduction of a hormonal signal to the cell, as well as the informational value of this antigen as an indicator of effectiveness of plant?Cbacterial interactions.     

Obtaining phage mini-antibodies and using them for detection of microbial cells with an electroacoustic sensor

Phage mini-antibodies to bacterial cells of strain Azospirillum brasilense Sp245 were obtained, and the possibility of using them for detection of microbial cells with a lateral field excited piezoelectric resonator was studied. It has been found that the frequency dependences of the real and imaginary parts of electrical impedance of such a resonator loaded with a suspension of A. brasilense Sp245 cells with the mini-antibodies differ significantly from the dependences of the resonator with a control cell suspension without mini-anti-bodies. The limit of possible determination of the concentration of microbial cells is found to be 10³ cells/mL upon interaction with mini-antibodies. It has been ascertained that detection of A. brasilense Sp245 cells with the aid of mini-antibodies is possible even in the presence of other cultures, for example, E. coli BL-Ril and A. brasilense Sp7. Therefore, it has been shown for the first time that detection of microbial cells with an electroacoustic sensor is feasible.     

Short hydrogen bond: Spectral studies and X-ray structure of bis(alpha-picoline N-oxide)-hydronium tetrachloroaurate(III)

Spectral and the X-ray diffraction studies of the title compound [H(2-picNO)₂] [AuCl₄] (2-picNO = alpha-picoline N-oxide) were done to investigate the short hydrogen bond in the dimeric cation [H(2-picNO)₂]⁺. The compound exhibits several narrow transmission bands overlaid on a broad continuous absorption characteristic of a short hydrogen bond; it crystallizes with four molecules in a monoclinic unit cell of space group P2₁/n with lattice parameters a = 22.376(9), b = 9.874(6), c = 7.957(5) Å and β = 94.98(6)°. The structure was solved using 2623 data points collected on a CAD4 to the final value for R(F_o) = 0.039. The structure consists of discrete [AuCl₄]^? and [H(2-picNO)₂]⁺ ions. Contrary to all known examples, the dimeric cation in this compound has a short hydrogen bond having no symmetry constraints making the present structural analysis unique in this series of compounds. The O—O distance is 2.393(6) Å and the dihedral angle between the two planar 2-picNO moieties in the cation is 44.1°. The [AuCl₄]^? anion is planar with an average AuCl distance of 2.268(2) Å. The infrared spectrum and the structure of the compound is compared with the related structures.     

Impact of plant growth-promoting bacteria on grain yield,protein content,and urea-<Superscript>15</Superscript> N recovery by maize in a Cerrado Oxisol

Background and aims

The inoculation of cereal crops with plant growth-promoting bacteria (PGPB) is a potential strategy to improve fertilizer-N acquisition by crops in soils with low capacity to supply N. A study was conducted to assess the impact of three inoculants on grain yield, protein content, and urea-¹⁵ N recovery in maize (Zea mays L.) under Cerrado soil and climate conditions.

Methods

The main treatments included inoculants containing (i) Azospirillum brasilense strain Sp245, (ii) A. brasilense strains AbV5 + AbV6, (iii) Herbaspirillum seropedicae strain ZAE94, and (iv) a non-inoculated control. The subtreatments were (i) urea-N fertilization (100 kg N ha^?1) at 30 days after sowing and (ii) no N addition at the stage. To determine fertilizer-N recovery, ¹⁵N–labelled urea was applied in microplots.

Results

Inoculants carrying A. brasilense improved urea-¹⁵ N acquisition efficiency in maize and also improved grain yield compared to the non-inoculated control, while urea-N fertilization enhanced grain quality by providing higher protein content.

Conclusion

Our results suggest that the inoculation of maize grains with PGPB represents a strategy to improve fertilizer-N recovery and maize yield in Cerrado soil with a low capacity to supply N.

     

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.