Azospirillum Brasilense Sp7 Research Articles

Identification and functional characterization of a fructose-inducible phosphotransferase system in Azospirillum brasilense Sp7.

Plant growth-promoting rhizobacterium Azospirillum brasilense Sp7 utilizes fructose efficiently via a fructose phosphotransferase system (Fru-PTS). Its genome encodes two putative Fru-PTS, each consisting of FruB (EIIA), FruK (Pfk), and FruA (EIIBC) proteins. We compared the proteomes of A. brasilense Sp7 grown with malate or fructose as sole carbon source, and noticed upregulation of the constituent proteins of Fru-PTS1 only on fructose. Inactivation of fruA gene of both the Fru-PTS showed that Fru-PTS1 is the main PTS involved in fructose utilization. Overexpression of fruA1 in A. brasilense Sp7 enhanced its growth on fructose showing improved consumption of fructose. This suggested that fructose utilization in A. brasilense Sp7 is limited due to the limitation of EIIBC component. A FruR-type regulator, encoded divergently to the Fru-PTS1 operon, was required for chemotaxis toward fructose. Although not an absolute necessity for the growth of fructose, FruR was required for the optimal growth of fructose. The fruB1 promoter was activated by fructose and repressed by malate, but FruR does not seem to regulate its expression. A 27-nucleotide stem-loop structure located between the -125 and -99 promoter proximal region of fruB1 was involved in fructose inducibility and malate repression. Fructose also upregulated several proteins involved in the biogenesis of a Type 6 secretion system. Here, we have shown that A. brasilense Sp7 was able to inhibit the growth of Escherichia coli and Agrobacterium tumefaciens in the presence of fructose, and that an intact T6SS was required for contact-dependent growth inhibition of the two Gram-negative bacteria.IMPORTANCEAzospirillum brasilense, a plant growth-promoting rhizobacterium, has limited ability to utilize carbohydrates and sugars. Although it is known to utilize fructose via a fructose phosphotransferase system (fructose-PTS), the genes involved in fructose utilization and the role of fructose in its biology were not well characterized. This study has shown that out of the two units of fructose-PTS encoded in its genome, fructose-PTS1 plays the major role in fructose utilization. Overexpression of the membrane component (EIIBC) improved the growth of A. brasilense on fructose. The ability of fructose to induce proteins of the Type 6 Secretion System (T6SS) enables A. brasilense to cause contact-dependent inhibition of the growth of Escherichia coli as well as A. tumefaciens. This is the first report on the fructose inducibility of T6SS in A. brasilense, which may provide a handle to control the growth of undesirable bacteria using T6SS of A. brasilense in a mixed culture.

Building blocks toward sustainable biofertilizers: variation in arbuscular mycorrhizal spore germination when immobilized with diazotrophic bacteria in biodegradable hydrogel beads.

We investigated whether there was interspecies and intraspecies variation in spore germination of 12 strains of arbuscular mycorrhizal fungi when co-entrapped with the diazotrophic plant growth-promoting bacteria, Azospirillum brasilense Sp7 in alginate hydrogel beads. Twelve Rhizophagus irregularis, Rhizophagus intraradices, and Funneliformis mosseae strains were separately combined with a live culture of Azospirillum brasilense Sp7. Each fungal-bacterial consortia was supplemented with sodium alginate to a 2% concentration (v/v) and cross-linked in calcium chloride (2%w/v) to form biodegradable hydrogel beads. One hundred beads from each combination (total of 1200) were fixed in solidified modified Strullu and Romand media. Beads were observed for successful spore germination and bacterial growth over 14 days. In all cases, successful growth of A. brasilense was observed. For arbuscular mycorrhizal fungi, interspecies variation in spore germination was observed, with R. intraradices having the highest germination rate (64.3%), followed by R. irregularis (45.5%) and F. mosseae (40.3%). However, a difference in intraspecies germination was only observed among strains of R. irregularis and F. mosseae. Despite having varying levels of germination, even the strains with the lowest potential were still able to establish with the plant host Brachypodium distachyon in a model system. Arbuscular mycorrhizal spore germination varied across strains when co-entrapped with a diazotrophic plant growth-promoting bacteria. This demonstrates that hydrogel beads containing a mixed consortium hold potential as a sustainable biofertilizer and that compatibility tests remain an important building block when aiming to create a hydrogel biofertilizer that encases a diversity of bacteria and fungi. Moving forward, further studies should be conducted to test the efficacy of these hydrogel biofertilizers on different crops across varying climatic conditions in order to optimize their potential.

Spatial distribution of Azospirillum brasilense in 2D flow cell: Effect of irrigation and inoculation regimes

AbstractRecent advances in the use of plant growth‐promoting bacteria (PGPB) have highlighted their potential to significantly enhance crop yield and plant health. In desert areas with sandy soil, employing drip irrigation systems to inoculate PGPB serves as an efficient method that saves both time and labour. This study examined the absorption, transport and spatial distribution of two strains of Azospirillum brasilense (Sp7 and Cd) under two‐dimension (2D) unsaturated transient water flow. We used sand as a substitute for sandy soil and evaluated bacterial surface characteristics, adsorption isotherms and transport under different irrigation and inoculation regimes. The research determined that, owing to its smaller size and lower adsorption, A. brasilense Cd exhibited enhanced mobility and occupied an inoculated area 33% larger than that of A. brasilense Sp7. Moreover, subsurface drip irrigation (SSDI) exhibited a 29% higher inoculation area than surface drip irrigation (SDI). The sequence of introducing PGPB suspension and irrigation water impacted the distribution, particularly for A. brasilense Sp7. The attachment/detachment numerical model adequately described the 2D bacterial distribution (R2 ranged from 0.75 to 0.99), providing a useful tool for predicting bacterial distribution in soils and optimizing agricultural practices to enhance crop productivity. Overall, smaller bacteria, SSDI inoculation and inoculation before irrigation could enhance the extent of inoculation. This study provides novel insights into optimizing PGPB inoculation strategies in agricultural settings, highlighting the importance of considering bacterial physical properties, irrigation techniques and inoculation sequences to improve PGPB distribution within the rhizosphere.

Plant seedlings of peas, tomatoes, and cucumbers exude compounds that are needed for growth and chemoattraction of Rhizobium leguminosarum bv. viciae 3841 and Azospirillum brasilense Sp7.

This study characterizes seedling exudates of peas, tomatoes, and cucumbers at the level of chemical composition and functionality. A plant experiment confirmed that Rhizobium leguminosarum bv. viciae 3841 enhanced growth of pea shoots, while Azospirillum brasilense Sp7 supported growth of pea, tomato, and cucumber roots. Chemical analysis of exudates after 1 day of seedling incubation in water yielded differences between the exudates of the three plants. Most remarkably, cucumber seedling exudate did not contain detectable sugars. All exudates contained amino acids, nucleobases/nucleosides, and organic acids, among other compounds. Cucumber seedling exudate contained reduced glutathione. Migration on semi solid agar plates containing individual exudate compounds as putative chemoattractants revealed that R. leguminosarum bv. viciae was more selective than A. brasilense, which migrated towards any of the compounds tested. Migration on semi solid agar plates containing 1:1 dilutions of seedling exudate was observed for each of the combinations of bacteria and exudates tested. Likewise, R. leguminosarum bv. viciae and A. brasilense grew on each of the three seedling exudates, though at varying growth rates. We conclude that the seedling exudates of peas, tomatoes, and cucumbers contain everything that is needed for their symbiotic bacteria to migrate and grow on.

Computational Analysis of the Tripartite Interaction of Phasins (PhaP4 and 5)-Sigma Factor (σ24)-DNA of Azospirillum brasilense Sp7.

Azospirillum brasilense Sp7 produces PHB, which is covered by granule-associated proteins (GAPs). Phasins are the main GAPs. Previous studies have shown phasins can regulate PHB synthesis. When A. brasilense grows under stress conditions, it uses sigma factors to transcribe genes for survival. One of these factors is the σ24 factor. This study determined the possible interaction between phasins and the σ24 factor or phasin-σ24 factor complex and DNA. Three-dimensional structures of phasins and σ24 factor structures were predicted using the I-TASSER and SWISS-Model servers, respectively. Subsequently, a molecular docking between phasins and the σ24 factor was performed using the ClusPro 2.0 server, followed by molecular docking between protein complexes and DNA using the HDOCK server. Evaluation of the types of ligand-receptor interactions was performed using the BIOVIA Discovery Visualizer for three-dimensional diagrams, as well as the LigPlot server to obtain bi-dimensional diagrams. The results showed the phasins (Pha4Abs7 or Pha5Abs7)-σ24 factor complex was bound near the -35 box of the promoter region of the phaC gene. However, in the individual interaction of PhaP5Abs7 and the σ24 factor, with DNA, both proteins were bound to the -35 box. This did not occur with PhaP4Abs7, which was bound to the -10 box. This change could affect the transcription level of the phaC gene and possibly affect PHB synthesis.

Improving the Sustainability of Rice Cultivation in Central Thailand with Biofertilizers and Laser Land Leveling

Rice production in the Central Plains of Thailand plays a key role in the country’s food security. However, the overuse of inputs coupled with the rising production costs are making it increasingly difficult for smallholder rice farming to remain economically and environmentally sustainable. Replicated production-scale field trials of Cost Reduction Operating Principles (CROP)—Thailand’s national package of best management practices for rice production—were established in tandem with laser land leveling (LLL), mechanical drum seeder, and the application of two biofertilizer products (i.e., PGPR II, that contains Azospirillum brasilense Sp. TS29 and Burkholderia vietnamiensis S45; and LDD #12, that contains Azotobacter tropicalis, Burkholderia unamae and Bacillus subtilis) and compared with farmer’s practices (FP). Performance indicators (PI) promoted by the Sustainable Rice Platform (SRP) were used to assess economic and environmental indicators. CROP + PGPR had significantly higher net income (79%) and nitrogen-use efficiency (57%) compared with FP. Pesticide use (28%), seed (60%), inorganic fertilizer N (41%) and total production costs (19%) were reduced in all CROP treatments compared with FP. These results demonstrate that the application of CROP, LLL, mechanical drum seeder, and biofertilizers can substantially improve the economic and environmental sustainability of rice production in the Central Plains of Thailand.

The role of rhizospheric bacteria in the formation of photosynthetic apparatus and productiv-ity of winter wheat in the conditions of the Left Bank of the Saratov region

The article discusses the ability of strains of plant-stimulating rhizobacteria (plant-growth-promoting rhizobacteria, PGPR) Azospirillum baldaniorum Sp245, Azospirillum brasilense Sp7, Azospirillum brasilense SR80, Azospirillum brasilense SR88, Azospirillum brasilense Cd, Ochrobactrum cytisi IPA7.2 and Enterobacter ludwigii K7 to stimulate the growth of the assimilation apparatus and productivity of winter soft wheat of the Novoershovskaya variety in the conditions of the Left Bank of the Saratov region. The bacteria stimulated the growth of the assimilation apparatus of plants, in addition, the bacteria of the genus Azospirillum as a whole more significantly increased the net productivity of photosynthesis. As a result, bacterization significantly increased the yield of winter soft wheat by 11-27,7%.

Plant-growth promotion by proteobacterial strains depends on the availability of phosphorus and iron in Arabidopsis thaliana plants.

Phosphorus (as phosphate, Pi) and iron (Fe) are critical nutrients in plants that are often poorly available in the soil and can be microbially affected. This work aimed to evaluate how plant-rhizobacteria interaction changes due to different Pi or Fe nutritional scenarios and to study the underlying molecular mechanisms of the microbial modulation of these nutrients in plants. Thus, three proteobacteria (Paraburkholderia phytofirmans PsJN, Azospirillum brasilense Sp7, and Pseudomonas putida KT2440) were used to inoculate Arabidopsis seeds. Additionally, the seeds were exposed to a nutritional factor with the following levels for each nutrient: sufficient (control) or low concentrations of a highly soluble source or sufficient concentrations of a low solubility source. Then, the effects of the combinatorial factors were assessed in plant growth, nutrition, and genetic regulation. Interestingly, some bacterial effects in plants depended on the nutrient source (e.g., increased aerial zones induced by the strains), and others (e.g., decreased primary roots induced by Sp7 or KT2440) occurred regardless of the nutritional treatment. In the short-term, PsJN had detrimental effects on plant growth in the presence of the low-solubility Fe compound, but this was not observed in later stages of plant development. A thorough regulation of the phosphorus content was detected in plants independent of the nutritional treatment. Nevertheless, inoculation with KT2440 increased P content by 29% Pi-deficiency exposed plants. Conversely, the inoculation tended to decrease the Fe content in plants, suggesting a competition for this nutrient in the rhizosphere. The P-source also affected the effects of the PsJN strain in a double mutant of the phosphate starvation response (PSR). Furthermore, depending on the nutrient source, PsJN and Sp7 strains differentially regulated PSR and IAA- associated genes, indicating a role of these pathways in the observed differential phenotypical responses. In the case of iron, PsJN and SP7 regulated iron uptake-related genes regardless of the iron source, which may explain the lower Fe content in inoculated plants. Overall, the plant responses to these proteobacteria were not only influenced by the nutrient concentrations but also by their availabilities, the elapsed time of the interaction, and the specific identities of the beneficial bacteria. Graphical AbstractThe effects of the different nutritional and inoculation treatments are indicated for plant growth parameters (A), gene regulation (B) and phosphorus and iron content (C). Figures created with BioRender.com with an academic license.

Engineering D-glucose utilization in Azospirillum brasilense Sp7 promotes rice root colonization.

Bacteria of the genus Azospirillum include several plant associated bacteria which often promote the growth of their host plants. Although the host range of Azospirillum brasilense Sp7 is much wider than its close relative Azospirillum lipoferum 4B, it lacks the ability to efficiently utilize D-glucose for its growth. By comparing the genomes of both the species, the genes of A. lipoferum 4B responsible for conferring D-glucose utilization ability in A. brasilese Sp7 were identified by cloning individual or a combination of genes in a broad host range expression vector, mobilizing them in A. brasilense Sp7 and examining the ability of exconjugants to use D-glucose as sole carbon source for growth. These genes also included the homologs of genes involved in N-acetyl glucosamine utilization in Pseudomonas aeruginosa PAO1. A transcriptional fusion of the 5 genes encoding glucose-6-phosphate dehydrogenase and 4 components of glucose phosphotransferase system were able to improve D-glucose utilization ability in A. brasilense Sp7. The A. brasilense Sp7 strain engineered with D-glucose utilization ability showed significantly improved root colonization of rice seedling. The improvement in the ability of A. brasilense Sp7 to colonize rice roots is expected to bring benefits to rice by promoting its growth. KEY POINTS: • Genes required for glucose utilization in Azospirillum lipoferum were identified. • A gene cassette encoding glucose utilization was constructed. • Transfer of gene cassette in A. brasilense improves glucose utilization and rice root colonization..

Assessment of Shelf-life and Efficacy of the Seed-Coating Delivery System of Biofertilizers in Maize

Film-coating of seeds is a widely accepted technology in agriculture for effectively protecting germinating seeds and seedlings against biotic and abiotic stresses and successfully delivering agro-chemicals, biomolecules, and bio-inoculants to the rhizosphere region. The present experiments explored the possibility of using hydroxypropyl methylcellulose (HPMC) and dextrin as film-coating agents for impregnating maize seed with four different biofertilizer strains. Azospirillum brasilense (Sp7), Bacillus megaterium var phosphaticum (Pb1), Paenibacillus mucilaginosus (KRB9), and Pseudomonas chlororaphis (ZSB15) were prepared as consortium containing an equimolar ratio of each strain. The in vitro growth experiment revealed that HPMC, dextrin, or a combination of both did not affect the growth of all four biofertilizers. When biofertilizer consortium was film-coated on maize seed using HPMC and dextrin as film-coating /binding and protecting agents, no apparent changes were observed in the size, shape, weight, and volume of maize seeds. The shelf-life study on film-coated maize seeds revealed that the microbial inoculants accounted for a one-log reduction (from 104 cells at initial to 103 cells after 3 months) when stored at low temperature. The pre-coated seeds with microbial-inoculants showed significant improvement in seedling growth. Hence, the film-coating of maize seed with microbial inoculants using HPMC and dextrin could be an effective delivery system for ensuring rhizosphere colonization of biofertilizers for sustainable maize cultivation.

Deciphering the role of the two conserved motifs of the ECF41 family σ factor in the autoregulation of its own promoter in Azospirillum brasilense Sp245.

In Azospirillum brasilense, an extra-cytoplasmic function σ factor (RpoE10) shows the characteristic 119 amino acid long C-terminal extension found in ECF41-type σ factors, which possesses three conserved motifs (WLPEP, DGGGR, and NPDKV), one in the linker region between the σ2 and σ4 , and the other two in the SnoaL_2 domain of the C-terminal extension. Here, we have described the role of the two conserved motifs in the SnoaL_2 domain of RpoE10 in the inhibition and activation of its activity, respectively. Truncation of the distal part of the C-terminal sequence of the RpoE10 (including NPDKV but excluding the DGGGR motif) results in its promoter's activation suggesting autoregulation. Further truncation of the C-terminal sequence up to its proximal part, including NPDKV and DGGGR motif, abolished promoter activation. Replacement of NPDKV motif with NAAAV in RpoE10 increased its ability to activate its promoter, whereas replacement of DGGGR motif led to reduced promoter activation. We have explored the dynamic modulation of σ2 -σ4 domains and the relevant molecular interactions mediated by the two conserved motifs of the SnoaL2 domain using molecular dynamics simulation. The analysis enabled us to explain that the NPDKV motif located distally in the C-terminus negatively impacts transcriptional activation. In contrast, the DGGGR motif found proximally of the C-terminal extension is required to activate RpoE10.

TOR participation on the root system changes of Arabidopsis during its interaction with Azospirillum

The root system of the plant is essential for taking up water and nutrients, serves as an anchor and is the organ where plant-microorganism interaction takes place. When the Plant Growth Promoting Rhizobacteria (PGPR) Azospirillum brasilense Sp245 colonizes the root of the plants, it halts the growth of the primary root and stimulates the development of the lateral roots and root hairs which support vegetative, green biomass. Target of Rapamycin (TOR) is a highly conserved protein in all eukaryotes, and it controls anabolic processes, such as cell cycle, ribosome biogenesis, protein synthesis, cell wall changes and photosynthesis among others. TOR in plants forms part of the TORC1 complex, which when is activated by auxins and light, activates anabolic processes and represses autophagy. TOR regulates the growth of the primary root of Arabidopsis through cell proliferation and elongation. In the present investigation, the participation of TOR during the Arabidopsis-Azospirillum interaction was determined using two approaches, a pharmacology and other genetic. The results showed that TOR is involved in the development of the lateral roots of A. thaliana seedlings inoculated with A. brasilense.

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.

Cometabolism of Ethanol in Azospirillum brasilense Sp7 Is Mediated by Fructose and Glycerol and Regulated Negatively by an Alternative Sigma Factor RpoH2.

Azospirillum brasilense is a plant growth-promoting rhizobacterium that is not known to utilize ethanol as a sole source of carbon for growth. This study shows that A. brasilense can cometabolize ethanol in medium containing fructose or glycerol as a carbon source and contribute to its growth. In minimal medium containing fructose or glycerol as a carbon source, supplementation of ethanol caused enhanced production of an alcohol dehydrogenase (ExaA) and an aldehyde dehydrogenase (AldA) in A. brasilense. However, this was not the case when malate was used as a carbon source. Inactivation of aldA in A. brasilense resulted in the loss of the AldA protein and its ethanol utilizing ability in fructose- or glycerol-supplemented medium. Furthermore, ethanol inhibited the growth of the aldA::Km mutant. The exaA::Km mutant also lost its ability to utilize ethanol in fructose-supplemented medium. However, in glycerol-supplemented medium, A. brasilense utilized ethanol due to the synthesis of a new paralog of alcohol dehydrogenase (ExaA1). The expression of exaA1 was induced by glycerol but not by fructose. Unlike exaA, expression of aldA and exaA1 were not dependent on σ54. Instead, they were negatively regulated by the RpoH2 sigma factor. Inactivation of rpoH2 in A. brasilense conferred the ability to use ethanol as a carbon source without or with malate, overcoming catabolite repression caused by malate. This is the first study showing the role of glycerol and fructose in facilitating cometabolism of ethanol by inducing the expression of ethanol-oxidizing enzymes and the role of RpoH2 in repressing them. IMPORTANCE This study unraveled a hidden ability of Azospirillum brasilense to utilize ethanol as a secondary source of carbon when fructose or glycerol were used as a primary growth substrate. It opens the possibility of studying the regulation of expression of the ethanol oxidation pathway for generating high yielding strains that can efficiently utilize ethanol. Such strains would be useful for economical production of secondary metabolites by A. brasilense in fermenters. The ability of A. brasilense to utilize ethanol might be beneficial to the host plant under the submerged growth conditions.

Role of a fasciclin domain protein in photooxidative stress and flocculation in Azospirillum brasilense Sp7

Fasciclin domain proteins (FDP) are found in all domains of life, but their biological role and regulation are not clearly understood. While studying the proteome of a mutant (Car1) of Azospirillum brasilense Sp7 with a Tn5 insertion in the gene encoding an anti-sigma factor (ChrR1), we found that FDP was maximally expressed. To study the biological role of this FDP, we inactivated fdp in A. brasilense Sp7 and in its Car1 mutant, which rendered them sensitive to methylene blue (MB) and toluidine blue (TB) in the presence of light. The transcription of fdp was also strongly upregulated by an ECF sigma factor (RpoE1) and photooxidative stress. The fdp null mutants of A. brasilense Sp7 and its Car1 mutant produced relatively fewer carotenoids and showed reduced flocculation. The reduced ability of fdp null mutants to flocculate was partly due to their reduced ability to produce carotenoids as inhibition of carotenoid synthesis by diphenylamine reduced their flocculation ability by 15–20%. Hence, FDP plays an important role in protecting A. brasilense Sp7 against photo-oxidative stress by supporting carotenoid accumulation and cell aggregation.

Azospirillum brasilense Sp245 triggers cytokinin signaling in root tips and improves biomass accumulation in Arabidopsis through canonical cytokinin receptors.

The plant growth promoting rhizobacterium Azospirillum brasilense Sp245 enhances biomass production in cereals and horticultural species and is an interesting model to study the physiology of the phytostimulation program. Although auxin production by Azospirillum appears to be critical for root architectural readjustments, the role of cytokinins in the growth promoting effects of Azospirillum remains unclear. Here, Arabidopsis thaliana seedlings were co-cultivated in vitro with A. brasilense Sp245 to assess whether direct contact of roots with bacterial colonies or exposure to the bacterial volatiles using divided Petri plates would affect biomass production and root organogenesis. Both interaction types increased root and shoot fresh weight but had contrasting effects on primary root length, lateral root formation and root hair development. Cell proliferation in root meristems analyzed with the CYCB1;1::GUS reporter decreased over time with direct contact, but was augmented by plant exposure to volatiles. Noteworthy, the expression of the cytokinin-inducible reporters TCS::GFP and ARR5::GUS increased in root tips in response to bacterial contact, without being affected by the volatiles. In A. thaliana having single (cre1-12, ahk2-2, ahk3-3), double (cre1-12/ahk2-2, cre1-12/ahk3-3, ahk2-2/ahk3-3) or triple (cre1-12/ahk2-2/ahk3-3) mutations in canonical cytokinin receptors, only the triple mutant had a marked effect on plant growth in response to A. brasilense. These results show that different mechanisms are elicited by A. brasilense, which influence the cytokinin-signaling pathway.

Fourier Transform Infrared (FTIR) Spectroscopic Analyses of Microbiological Samples and Biogenic Selenium Nanoparticles of Microbial Origin: Sample Preparation Effects.

To demonstrate the importance of sample preparation used in Fourier transform infrared (FTIR) spectroscopy of microbiological materials, bacterial biomass samples with and without grinding and after different drying periods (1.5–23 h at 45 °C), as well as biogenic selenium nanoparticles (SeNPs; without washing and after one to three washing steps) were comparatively studied by transmission FTIR spectroscopy. For preparing bacterial biomass samples, Azospirillum brasilense Sp7 and A. baldaniorum Sp245 (earlier known as A. brasilense Sp245) were used. The SeNPs were obtained using A. brasilense Sp7 incubated with selenite. Grinding of the biomass samples was shown to result in slight downshifting of the bands related to cellular poly-3-hydroxybutyrate (PHB) present in the samples in small amounts (under ~10%), reflecting its partial crystallisation. Drying for 23 h was shown to give more reproducible FTIR spectra of bacterial samples. SeNPs were shown to contain capping layers of proteins, polysaccharides and lipids. The as-prepared SeNPs contained significant amounts of carboxylated components in their bioorganic capping, which appeared to be weakly bound and were largely removed after washing. Spectroscopic characteristics and changes induced by various sample preparation steps are discussed with regard to optimising sample treatment procedures for FTIR spectroscopic analyses of microbiological specimens.

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.

Electroduction on Azospirillum brasilense Sp7 and Chromobacterium violaceum

Electroduction is a fast, simple and very efficient transformation method with a wider range of bacteria strains which involved physical process by exposing to high electricity. A successful transfer of a 21.6 kb cosmid vector pLAFR1 into Azospirillum brasilense Sp7 and Chromobacterium violaceum was achieved with the maximum efficiency of about 35 X 102 and 24 X 102 transformants, respectively, at the electrical field strength of 5kV/cm with longer pulse length range between 8.3 and 8.5 milliseconds. Doubling the pulse length and a decrease or increase in the electrical field strength of 5kV/cm resulted in a reduction in the total number of transformants. The electroduction parameters were very important to make this transformation method works efficiently.

Genome-wide identification of Azospirillum brasilense Sp245 small RNAs responsive to nitrogen starvation and likely involvement in plant-microbe interactions

BackgroundSmall RNAs (sRNAs) are non-coding RNAs known to regulate various biological functions such as stress adaptation, metabolism, virulence as well as pathogenicity across a wide range of bacteria, mainly by controlling mRNA stabilization or regulating translation. Identification and functional characterization of sRNAs has been carried out in various plant growth-promoting bacteria and they have been shown to help the cells cope up with environmental stress. No study has been carried out to uncover these regulatory molecules in the diazotrophic alpha-proteobacterium Azospirillum brasilense Sp245 to date.ResultsExpression-based sRNA identification (RNA-seq) revealed the first list of ~ 468 sRNA candidate genes in A. brasilense Sp245 that were differentially expressed in nitrogen starvation versus non-starved conditions. In parallel, in silico tools also identified 2 of the above as candidate sRNAs. Altogether, putative candidates were stringently curated from RNA-seq data based on known sRNA parameters (size, location, secondary structure, and abundance). In total, ~ 59 significantly expressed sRNAs were identified in this study of which 53 are potentially novel sRNAs as they have no Rfam and BSRD homologs. Sixteen sRNAs were randomly selected and validated for differential expression, which largely was found to be in congruence with the RNA-seq data.ConclusionsDifferential expression of 468 A. brasilense sRNAs was indicated by RNA-seq data, a subset of which was confirmed by expression analysis. Four of the significantly expressed sRNAs were not observed in nitrogen starvation while 16 sRNAs were found to be exclusively expressed in nitrogen depletion. Putative candidate sRNAs identified have potential mRNA targets primarily involved in stress (abiotic and biotic) adaptability; regulation of bacterial cellular, biological and molecular pathways such as nitrogen fixation, polyhydroxybutyrate synthesis, chemotaxis, biofilm formation and transcriptional regulation. In addition to directly influencing bacteria, some of these sRNAs also have targets influencing plant-microbe interactions through adhesion of bacteria to plant roots directly, suppressing host response, inducing plant defence and signalling.