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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.

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.

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.

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.

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.

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 .

The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii ( Av NifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active Av NifH protein upon co-expression of this protein with Av IscS/U and Av NifM in E. coli . Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed Av NifH protein has a high [Fe 4 S 4 ] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that Av NifM could serve as a chaperone and assist the maturation of a cluster-replete Av NifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the “clean” genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

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.

Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their in vivo cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site. IMPORTANCE Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.