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The influence of nanomaterials on the ecological environment is becoming an increasingly hot research field, and many researchers are exploring the mechanisms of nanomaterial toxicity on microorganisms. Herein, we studied the effect of two different sizes of nanosilver (10 nm and 50 nm) on the soil nitrogen fixation by the model bacteria Azotobacter vinelandii. Smaller size AgNPs correlated with higher toxicity, which was evident from reduced cell numbers. Flow cytometry analysis further confirmed this finding, which was carried out with the same concentration of 10 mg/L for 12 h, the apoptotic rates were20.23% and 3.14% for 10 nm and 50 nm AgNPs, respectively. Structural damage to cells were obvious under scanning electron microscopy. Nitrogenase activity and gene expression assays revealed that AgNPs could inhibit the nitrogen fixation of A. vinelandii. The presence of AgNPs caused intracellular reactive oxygen species (ROS) production and electron spin resonance further demonstrated that AgNPs generated hydroxyl radicals, and that AgNPs could cause oxidative damage to bacteria. A combination of Ag content distribution assays and transmission electron microscopy indicated that AgNPs were internalized in A. vinelandii cells. Overall, this study suggested that the toxicity of AgNPs was size and concentration dependent, and the mechanism of antibacterial effects was determined to involve damage to cell membranes and production of reactive oxygen species leading to enzyme inactivation, gene down-regulation and death by apoptosis.

Bacterial alginate initially consists of 1–4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α- L -guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum . The three A. chroococcum genes, here designated AcalgE1 , AcalgE2 and AcalgE3 , were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions − 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.

Alginate is a linear polysaccharide that can be used for different applications in the food and pharmaceutical industries. These polysaccharides have a chemical structure composed of subunits of (1–4)-β- d -mannuronic acid (M) and its C-5 epimer α- l -guluronic acid (G). The monomer composition and molecular weight of alginates are known to have effects on their properties. Currently, these polysaccharides are commercially extracted from seaweed but can also be produced by Azotobacter vinelandii and Pseudomonas spp. as an extracellular polymer. One strategy to produce alginates with different molecular weights and with reproducible physicochemical characteristics is through the manipulation of the culture conditions during fermentation. This mini-review provides a comparative analysis of the metabolic pathways and molecular mechanisms involved in alginate polymerization from A. vinelandii and Pseudomonas spp. Different fermentation strategies used to produce alginates at a bioreactor laboratory scale are described.

The two-component system GacS/A and the posttranscriptional control system Rsm constitute a genetic regulation pathway in Gammaproteobacteria; in some species of Pseudomonas, this pathway is part of a multikinase network (MKN) that regulates the activity of the Rsm system. In this network, the activity of GacS is controlled by other kinases. One of the most studied MKNs is the MKN-GacS of Pseudomonas aeruginosa, where GacS is controlled by the kinases RetS and LadS; RetS decreases the kinase activity of GacS, whereas LadS stimulates the activity of the central kinase GacS. Outside of the Pseudomonas genus, the network has been studied only in Azotobacter vinelandii. In this work, we report the study of the RetS kinase of A. vinelandii; as expected, the phenotypes affected in gacS mutants, such as production of alginates, polyhydroxybutyrate, and alkylresorcinols and swimming motility, were also affected in retS mutants. Interestingly, our data indicated that RetS in A. vinelandii acts as a positive regulator of GacA activity. Consistent with this finding, mutation in retS also negatively affected the expression of small regulatory RNAs belonging to the Rsm family. We also confirmed the interaction of RetS with GacS, as well as with the phosphotransfer protein HptB.

Nitrogen fixation is advantageous in microbial competition when bioavailable nitrogen is scarce, but has substantial costs for growth rate and growth efficiency. To quantify these costs, we have developed a model of a nitrogen-fixing bacterium that constrains mass, electron and energy flow at the scale of the individual. When tested and calibrated with laboratory data for the soil bacterium Azotobacter vinelandii , the model reveals that the direct energetic cost of nitrogen fixation is small relative to the cost of managing intracellular oxygen. It quantifies the costs and benefits of several potential oxygen protection mechanisms present in nature including enhanced respiration (respiratory protection) as well as the production of extracellular polymers as a barrier to O 2 diffusion, and increasing cell size. The latter mechanisms lead to higher growth efficiencies relative to respiratory protection alone. This simple, yet mechanistic framework provides a quantitative model of nitrogen fixation, which can be applied in ecological simulations.

The integration of microbial nitrogen (N 2 ) fixation with photochemical processes using inorganic light-absorbing nanomaterials is a burgeoning field in sustainable energy production. Here, we explore the synergistic combination of inorganic semiconductor nanowires (NWs) with whole-cell microorganisms to create an inorganic-bacterial biohybrid system. Specifically, we employ Cu 2 O@TiO 2 NWs with a core/shell structure to harness sunlight and generate photoexcited electrons. Azotobacter vinelandii , serving as a biocatalyst, adsorbs onto these NWs and facilitates the reception of photoexcited electrons, thereby enhancing the efficiency of the photoelectrochemical N 2 fixation reaction (PEC-NRR). The biohybrid system achieves an impressive ammonia (NH 3 ) yield of (1.49 ± 0.05) × 10 -9   mol s -1  cm -2 (5.36 ± 0.18 μmol h -1  cm -2 ). The enhancement in NH 3 synthesis within the Cu 2 O@TiO 2 NWs/ A. vinelandii biohybrid is attributed to the increased concentrations of nicotinamide adenine dinucleotide-hydrogen (NADH) and adenosine 5’-triphosphate (ATP), as well as the overexpression of N 2 -fixing genes like nif H and nif D within the nitrogenase enzyme complex. This study underscores the potential of inorganic-bacterial biohybrid systems in solar-chemical conversion, paving the way for more diverse and functional approaches to harnessing solar energy for sustainable chemical production. Converting atmospheric nitrogen (N2) to ammonia (NH3) is challenging. Here, the authors develop a photoelectrochemical biohybrid system for fixing N2 into NH3, which consists of Cu2O/TiO2 nanowires and the N2-fixing diazotroph Azotobacter vinelandii .

Biological nitrogen fixation requires substantial metabolic energy in form of ATP as well as low-potential electrons that must derive from central metabolism. During aerobic growth, the free-living soil diazotroph Azotobacter vinelandii transfers electrons from the key metabolite NADH to the low-potential ferredoxin FdxA that serves as a direct electron donor to the dinitrogenase reductases. This process is mediated by the RNF complex that exploits the proton motive force over the cytoplasmic membrane to lower the midpoint potential of the transferred electron. Here we report the cryogenic electron microscopy structure of the nitrogenase-associated RNF complex of A. vinelandii , a seven-subunit membrane protein assembly that contains four flavin cofactors and six iron–sulfur centers. Its function requires the strict coupling of electron and proton transfer but also involves major conformational changes within the assembly that can be traced with a combination of electron microscopy and modeling. Biological nitrogen fixation requires low-potential electrons from ferredoxin or flavodoxin. Here the authors show how the soil diazotroph Azotobacter vinelandii employs the NADH:ferredoxin oxidoreductase RNF1 complex to lower the midpoint potential of the electron from NADH to reduce ferredoxin.

Background The emerging roles of rhizobacteria in improving plant nutrition and stress protection have great potential for sustainable use in saline soils. We evaluated the function of the salt-tolerant strain Azotobacter chroococcum 76A as stress protectant in an important horticultural crop, tomato. Specifically we hypothesized that treatment of tomato plants with A. chroococcum 76A could improve plant performance under salinity stress and sub-optimal nutrient regimen. Results Inoculation of Micro Tom tomato plants with A. chroococcum 76A increased numerous growth parameters and also conferred protective effects under both moderate (50 mM NaCl) and severe (100 mM NaCl) salt stresses. These benefits were mostly observed under reduced nutrient regimen and were less appreciable in optimal nitrogen conditions. Therefore, the efficiency of A. chroococcum 76A was found to be dependent on the nutrient status of the rhizosphere. The expression profiles of LEA genes indicated that A. chroococcum 76A treated plants were more responsive to stress stimuli when compared to untreated controls. However, transcript levels of key nitrogen assimilation genes revealed that the optimal nitrogen regimen, in combination with the strain A. chroococcum 76A, may have saturated plant’s ability to assimilate nitrogen. Conclusions Roots inoculation with A. chroococcum 76A tomato promoted tomato plant growth, stress tolerance and nutrient assimilation efficiency under moderate and severe salinity. Inoculation with beneficial bacteria such as A. chroococcum 76A may be an ideal solution for low-input systems, where environmental constraints and limited chemical fertilization may affect the potential yield.

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2 heterohexamer.

The presence of Azotobacter bacteria in the soil plays an important role in increasing its fertility and enhancing plant health. Azotobacter diversity depends on several environmental factors, particularly soil texture, pH, and nutrient content. The current study investigated the diversity of Azotobacter in various soil samples collected from 10 different governorates along the river Nile valley and its delta, Northern Mediterranean shore, Sinai, and Upper Egypt regions. The sampling sites spanned different environmental and ecological conditions of the Egyptian land either cultivated (agricultural land) or uncultivated (desert land). Fifty Azotobacter isolates were isolated and characterized based on cell morphology, culture properties, physiological, biochemical, and molecular characteristics. In addition, the alginate production capacity of the isolates was investigated. The results indicated that Egyptian soils are rich in Azotobacter diversity. The isolates were Gram-negative short rods, appearing either as single cells or in diploid structures. The isolates showed high variability in alginate production where two isolates (BH3 and AST4) were the highest alginate producers (3.12 and 4.22 g alginate L − 1 ), respectively. 16S-rDNA sequencing and 16S-rDNA RFLP analyses indicated that despite the presence of Azotobacter salinestris and Azotobacter vinelandii in the Egyptian soil, Azotobacter chroococcum was the predominant species. In addition, sequence analysis of the gene coding for the transcription factor AlgU confirmed the results of 16S-rRNA gene sequence analysis. RAPD-REP and BOX-PCR were used to study the polymorphism among the isolates. High levels of microbial diversity were found using these DNA primers as 6–9 fingerprinting profiles were retrieved.