Bacterium Azospirillum Brasilense Sp245 Research Articles

Lipopolysaccharide and flagellin of Azospirillum brasilense Sp7 influence callus morphogenesis and plant regeneration in wheat.

In vitro somatic callus culturing is used widely in plant biotechnology, but its effectiveness depends largely on the donor plant genotype. Bacteria or components of their cells are rarely used to activate morphogenesis. In this work, inoculation of explants from immature wheat (Triticum aestivum L.) embryos with a suspension of living cells of the bacterium Azospirillum brasilense Sp7 resulted in callus death after 7days of growth, in contrast to explant treatment with a suspension of heat-killed whole cells of Sp7. The experiments used two wheat lines, LRht-B1a and LRht-B1c, which differ in morphogenic activity. Growing calluses with the lipopolysaccharide of A. brasilense Sp7 increased the yield of regenerated plants 2- to 3.5-fold in both lines. This increase was through the activation of regenerant formation from morphogenic calluses. We have demonstrated for the first time the effects of bacterial flagellin on plant tissue culture. The polar-flagellum flagellin of A. brasilense Sp7 leveled the genotypic differences in the morphogenic ability of callus tissue. Specifically, it increased the yield of morphogenic calluses in the weakly morphogenic line LRht-B1a to the yield value in the highly morphogenic line LRht-B1c but lowered the yield of regenerants in the highly morphogenic line LRht-B1c to the yield value in the weakly morphogenic line LRht-B1a. Thus, bacterial lipopolysaccharides and flagellins can be used to regulate the formation of morphogenic calluses and regenerants in plant tissue culturing in vitro.

Poly-3-hydroxybutyrate synthesis by different Azospirillum brasilense strains under varying nitrogen deficiency: A comparative in-situ FTIR spectroscopic analysis

Monitoring of poly-3-hydroxybutyrate accumulation and changes in its relative contents in biomass of the plant-growth-promoting bacteria Azospirillum brasilense (strains Sp7, Cd and Sp245) was performed during aerobic cultivation for 1 to 8 days at various initial concentrations of bound nitrogen (0.1 to 0.5 g∙L–1 NH4Cl) in the culture medium using in-situ transmission FTIR spectroscopy. A methodology has been proposed based on calculating band areas in FTIR spectra (instead of band intensities commonly used earlier) for determining relative contents of PHB in dry bacterial biomass, using the ester ν(C=O) band as a PHB marker (in the region 1750–1720 cm−1) and amide II band of cellular proteins (at ca. 1540 cm−1). Differences in PHB accumulation levels and their changes in the course of cultivation under various trophic stress for the three strains are discussed in relation to their different ecological niches which they occupy in the rhizosphere.

Auxin-dependent alleviation of oxidative stress and growth promotion of Scenedesmus obliquus C1S by Azospirillum brasilense

There is currently an increasing interest in the use of microalgae for wastewater treatment and the use of its biomass as a feedstock for biofuels. Both of these applications are often performed more efficiently by microalgal-bacteria consortia. However, the mechanisms that account for the stability and robustness of this kind of interactions are poorly understood. In this study, we confirmed the growth promotion activity of the plant growth-promoting bacterium Azospirillum brasilense Sp245 on the microalgae Scenedesmus obliquus C1S. We show that this activity is critically dependent on bacterial indole-3 acetic acid (IAA) production, which results in a decrease in algal reactive oxygen species levels, higher cell densities and ameliorates algal cells bleaching after nitrogen deprivation. We also show a close inter-species interaction between both partners and an active expression of the bacterial Indole-3-pyruvate decarboxylase (ipdC) gene involved in production of IAA when co-cultivated.This study extends the current knowledge of the mechanisms underlying bacteria-microalgae consortia to improve their technological applications and to better understand ecological relationships in the environment.

Functioning of plant-bacterial associations under osmotic stress in vitro.

The search for effective plant-growth-promoting strains of rhizospheric bacteria that would ensure the resistance of plant-microbial associations to environmental stressors is essential for the design of environmentally friendly agrobiotechnologies. We investigated the interaction of potato (cv. Nevsky) microplants with the plant-growth-promoting bacteria Azospirillum brasilense Sp245 and Ochrobactrum cytisi IPA7.2 under osmotic stress in vitro. The bacteria improved the physiological and biochemical variables of the microplants, significantly increasing shoot length and root number (1.3-fold, on average). Inoculation also led a more effective recovery of the plants after stress. During repair, inoculation contributed to a decreased leaf content of malonic dialdehyde. With A. brasilense Sp245, the decrease was 1.75-fold; with O. cytisi IPA7.2, it was 1.4-fold. During repair, the shoot length, node number, and root number of the inoculated plants were greater than the control values by an average of 1.3-fold with A. brasilense Sp245 and by an average of 1.6-fold with O. cytisi IPA7.2. O. cytisi IPA7.2, previously isolated from the potato rhizosphere, protected the physiological and biochemical processes in the plants under stress and repair better than did A. brasilense Sp245. Specifically, root weight increased fivefold during repair, as compared to the noninoculated plants, while chlorophyll a content remained at the level found in the nonstressed controls. The results indicate that these bacteria can be used as components of biofertilizers. A. brasilense Sp245 has favorable prospects for use in temperate latitudes, whereas O. cytisi IPA7.2 can be successfully used in saline and drought-stressed environments.

Phasin PhaP1 is involved in polyhydroxybutyrate granules morphology and in controlling early biopolymer accumulation in Azospirillum brasilense Sp7

Phasins are amphiphilic proteins involved in the regulation of the number and size of polyhydroxybutyrate (PHB) granules. The plant growth promoting bacterium Azospirillum brasilense Sp7 accumulates high quantities of bioplastic PHB as carbon and energy source. By analyzing the genome, we identified six genes that code for proteins with a Phasin_2 domain. To understand the role of A. brasilense Sp7 PhaP1 (PhaP1Abs) on PHB synthesis, the phaP1 gene (AMK58_RS17065) was deleted. The morphology of the PHB granules was analyzed by transmission electron microscopy (TEM) and the PHB produced was quantified under three different C:N ratios in cultures subjected to null or low-oxygen transfer. The results showed that PhaP1Abs is involved in PHB granules morphology and in controlling early biopolymer accumulation. Using RT-PCR it was found that phasin genes, except phaP4, are transcribed in accordance with the C:N ratio used for the growth of A. brasilense. phaP1, phaP2 and phaP3 genes were able to respond to the growth conditions tested. This study reports the first analysis of a phasin protein in A. brasilense Sp7.

Antimony Causes Mortality and Induces Mutagenesis in the Soil Functional Bacterium Azospirillum brasilense Sp7

Antimony (Sb) is increasing in the environment but effects of exposure in ecosystems are not well understood. The aim of this work was to examine effects of antimony exposure on the multifunctional, plant growth promoting, ubiquitous soil bacterium Azospirillum brasilense Sp7. Contaminated mine water with high Sb concentrations (0.13 ± 0.09 mg L−1) was lethal to A. brasilense Sp7 in laboratory experiments. Exposure-dose- and time-dependent incubation toxicity assays on A. brasilense Sp7 with Sb(III) and Sb(V) at different concentrations (0.05–5 mg L−1) also resulted in cell mortality which was dose and time dependent. Median effect concentrations of 0.004–0.049 and 0.019–0.467 mg L−1 were estimated for Sb(III) and Sb(V), respectively. Exposure to Sb(III) resulted in greater cell mortality than Sb(V) at all concentrations tested. Exposure also resulted in the emergence of phenotypic variants that were more frequent with exposure to Sb(III). The toxicity assays demonstrated that Sb alone could have been responsible for the mortality observed with exposure to the contaminated mine water even without any other contaminants present. A. brasilense Sp7 was highly sensitive to Sb exposure and the observed effects suggest possible consequences for microbial function, plant-bacterial symbioses and ecosystem health with Sb contamination.

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.

Structural properties of capsular and O-specific polysaccharides of Azospirillum brasilense 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.

Mössbauer spectroscopic study of iron and cobalt metabolic transformations in cells of the bacterium Azospirillum brasilense Sp7

The transmission (57Fe) and emission (57Co) variants of Mossbauer spectroscopy are used to monitor metabolic transformations of 57FeIII/57FeII and 57CoII in cells of the bacterium Azospirillum brasilense Sp7. (The spectra are measured at 80 K.) For 57CoII, metabolic changes in the living cells are observed after 1 h. Two components of high-spin 57FeII are observed for the bacteria grown with 57FeIII as a source of iron; the parameters for 57FeIII correspond to ferritin-like components.

Insertional mutation in the AZOBR_p60120 gene is accompanied by defects in the synthesis of lipopolysaccharide and calcofluor-binding polysaccharides in the bacterium Azospirillum brasilense Sp245

In the bacterium Azospirillum brasilense Sp245, extracellular calcofluor-binding polysaccharides (Cal+ phenotype) and two types of lipopolysaccharides, LPSI and LPSII, were previously identified. These lipopolysaccharides share the same repeating O-polysaccharide unit but have different antigenic structures and different charges of their O-polysaccharides and/or core oligosaccharides. Several dozens of predicted genes involved in the biosynthesis of polysaccharides have been localized in the AZOBR_p6 plasmid of strain Sp245 (GenBank accession no. HE577333). In the present work, it was demonstrated that an artificial transposon Omegon-Km had inserted into the central region of the AZOBR_p60120 gene in the A. brasilense Sp245 LPSI- Cal- KM252 mutant. In A. brasilense strain Sp245, this plasmid gene encodes a putative glycosyltransferase containing conserved domains characteristic of the enzymes participating in the synthesis of O-polysaccharides and capsular polysaccharides (accession no. YP004987664). In mutant KM252, a respective predicted protein is expected to be completely inactivated. As a result of the analysis of the EcoRI fragment of the AZOBR_p6 plasmid, encompassing the AZOBR_p60120 gene and a number of other loci, novel data on the structure of AZOBR_p6 were obtained: an approximately 5-kb gap (GenBank accession no. KM189439) was closed in the nucleotide sequence of this plasmid.

Signal effects of the lectin from the associative nitrogen-fixing bacterium Azospirillum brasilense Sp7 in bacterial–plant root interactions

Azospirillum brasilense, which has the potential to stimulate plant growth, belongs to the group of plant growth-promoting bacteria. The lectin found on the surface of A. brasilense strain Sp7 has the ability to bind specific carbohydrates and ensures adhesion of the bacteria to the root surface. The aim of this work was to investigate possible inductive effects of the Sp7 lectin on the plant cell signal systems. Enzyme-linked immunosorbent assay, spectrophotometry, and thin-layer and gas–liquid chromatography were used to determine the content of signal intermediates in the cells of wheat root seedlings. Laser scanning confocal microscopy was used to examine the localization of fluorescently labeled lectin on the plant cell. The Sp7 lectin acted on the signal system components in wheat seedling roots by regulating the contents of cAMP, nitric oxide, diacylglycerol, and salicylic acid, as well as by modifying the activities of superoxide dismutase and lipoxygenase. The revealed cell membrane localization of the lectin is of deciding importance for its signal function. The results of the study suggest that the A. brasilense Sp7 lectin acts as a signal molecule involved in the interaction of growth-promoting rhizobacteria with plant roots.

Phenotypical and molecular responses of Arabidopsis thaliana roots as a result of inoculation with the auxin‐producing bacterium Azospirillum brasilense

The auxin-producing bacterium Azospirillum brasilense Sp245 can promote the growth of several plant species. The model plant Arabidopsis thaliana was chosen as host plant to gain an insight into the molecular mechanisms that govern this interaction. The determination of differential gene expression in Arabidopsis roots after inoculation with either A.brasilense wild-type or an auxin biosynthesis mutant was achieved by microarray analysis. Arabidopsis thaliana inoculation with A.brasilense wild-type increases the number of lateral roots and root hairs, and elevates the internal auxin concentration in the plant. The A.thaliana root transcriptome undergoes extensive changes on A.brasilense inoculation, and the effects are more pronounced at later time points. The wild-type bacterial strain induces changes in hormone- and defense-related genes, as well as in plant cell wall-related genes. The A.brasilense mutant, however, does not elicit these transcriptional changes to the same extent. There are qualitative and quantitative differences between A.thaliana responses to the wild-type A.brasilense strain and the auxin biosynthesis mutant strain, based on both phenotypic and transcriptomic data. This illustrates the major role played by auxin in the Azospirillum-Arabidopsis interaction, and possibly also in other bacterium-plant interactions.

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 in their cells on medium with increased density. Flagella determine the active swimming and swarming capacities of azospirilla. Using A. brasilense Sp245 as an example, we showed 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.

Transposon insertion into a chromosomal copy of flhB gene is concurrent with defects in the formation of polar and lateral flagella in the bacterium Azospirillum brasilense Sp245

Based on an example of Azospirillum brasilense Sp245, it was shown that, in bacteria with mixed flagellation, insertional mutagenesis of one of the copies of the flhB gene, which encodes a component of the flagellar protein export apparatus, may be concurrent with defects in the formation of both a constitutive polar flagellum and inducible lateral flagella. Despite the presence of a second copy of flhB in the plasmid-located gene cluster, which seems necessary for the formation of lateral flagella, the flhB1::Omegon-Km Sp245 mutant completely lost the ability to produce them. The described effect of the inactivation of flhB1 might be explained by the use of FlhBl for the assembly of both types of flagella. Since the open reading frame AZOBR_150176, which is transcribed in the same direction and codes for a hypothetical multisensor hybrid histidine kinase/response regulator, adjoins to the 3’-end of flhB1, the participation of the latter protein in the induction of the lateral flagellar synthesis in response to the increase in the density of the environment was not excluded.

Insertional Mutagenesis of a Chromosomal Copy offlhBGene is Concurrent with Defects in the Formation of Polar and Lateral Flagella in BacteriumAzospirillum brasilenseSp245

Insertional Mutagenesis of a Chromosomal Copy of<i>flhB</i>Gene is Concurrent with Defects in the Formation of Polar and Lateral Flagella in Bacterium<i>Azospirillum brasilense</i>Sp245

Co2 + interaction with Azospirillum brasilense Sp7 cells: a 57Co emission Mössbauer spectroscopic study

Preliminary 57Co emission Mossbauer spectroscopic data were obtained for the soil bacterium Azospirillum brasilense Sp7 (T = 80 K) in frozen 57Co2 + -containing suspensions and in their dried residues. The Mossbauer parameters were compared with those for A. brasilense strain Sp245 differing from strain Sp7 by ecological behaviour. Live cells of both strains showed metabolic transformations of 57Co2 + within an hour. Differences in the parameters observed for the two strains under similar conditions suggest dissimilarities in their metabolic response to Co2 + .

Alterations in the primary structure of an 85-MDa plasmid affecting flagellation and motility in the bacterium Azospirillum brasilense Sp245

Earlier such Azospirillum brasilense Sp245 mutants as flagellation-defective SK051, SK248 with immobilized flagella, and BK570 swimming and swarming faster than Sp245 were obtained. In SK051 and SK248 the self-killer vector pJFF350 integrated into the 18.3-kb XhoI fragment ofplasmid 85MDa (p85) while in BK570, it integrated into the 9.1-kb XhoI-fragment of p85. In the present work, analysis of the nucleotide sequence of fusion products of p85 and pJFF350 was performed. In p85, in addition to three IS elements (two of which caused cointegrate formation) and phage integrase gene, 22 open reading frames with coding sequence properties were identified. Possible participation of predicted translation products of several p85 genes in bacterial motility detection is discussed. Since differences in the primary structure of p85::pJFF350 cointegrates from SK051 and SK248 cells are localized within pJFF350 DNA, different effects of DNA-folding changes on expression of corresponding p85 genes are suggested.

Изучение влияния лектинов азоспирилл на образование перекиси водорода в корнях проростков пшеницы

We show that the lectins isolated from the surface of the nitrogenfixing soil bacterium Azospirillum brasilense Sp7 and its mutant defective in lectin activity, A. brasilense Sp7.2.3., can regulate the production of hydrogen peroxide in wheat seedling roots, which is associated with the activation of superoxide dismutase, peroxidase and oxalate oxidase, as well as with the inhibition of catalase activity. We show that activation of oxalate oxidase is the most rapidly inducible pathway for the formation of hydrogen peroxide in wheat seedling roots under the effect of lectins. The obtained data indicate that the Azospirillum lectins can act as inducers of adaptation processes in wheat seedling roots.

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 μg 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–bacterial interactions.

Localization of denitrification genes in plasmid DNA of bacteria Azospirillum brasilense

In 85-Mda plasmid (p85) of plant-associated bacteria Azospirillum brasilense Sp245 model strain, the genes encoding copper-containing nitrite reductase (nirK); heterodimeric NO-reductase (norCB); NorQ and NorD proteins affecting synthesis and (or) activation of NirK and (or) NO-reductase (norQD); catalytic subunit I ofcytochrom c oxidase (CccoN); presumable NO sensor carrying two hemeerythrine domains (orf181); and an enzyme required for synthesis of presumable NO antagonist, homocystein (metC) were identified. In the same region of p85, orf293 encoding transcriptional regulator of LysR type, orf208 whose protein product carries a formylmethanofuran dehydrogenase subunit E domain, and an orf164-encoding conservative secretory protein with unknown function were also found. Localization of a set of denitrification genes in the plasmid DNA A. brasilense Sp245 adjacent to IS elements ISAzba1 and ISAzba2 indicates potential mobility of these genes and high probability of their horizontal transfer among populations of rhizospheric bacteria. A site homologous to p85 nirK-orf208-orf181 genes was detected in the 115 kb plasmid of A. brasilense Sp7 type strain.