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Microbiology (Reading, England) Apr 2018Azotobacter vinelandii has been studied for over 100 years since its discovery as an aerobic nitrogen-fixing organism. This species has proved useful for the study of... (Review)
Review
Azotobacter vinelandii has been studied for over 100 years since its discovery as an aerobic nitrogen-fixing organism. This species has proved useful for the study of many different biological systems, including enzyme kinetics and the genetic code. It has been especially useful in working out the structures and mechanisms of different nitrogenase enzymes, how they can function in oxic environments and the interactions of nitrogen fixation with other aspects of metabolism. Interest in studying A. vinelandii has waned in recent decades, but this bacterium still possesses great potential for new discoveries in many fields and commercial applications. The species is of interest for research because of its genetic pliability and natural competence. Its features of particular interest to industry are its ability to produce multiple valuable polymers - bioplastic and alginate in particular; its nitrogen-fixing prowess, which could reduce the need for synthetic fertilizer in agriculture and industrial fermentations, via coculture; its production of potentially useful enzymes and metabolic pathways; and even its biofuel production abilities. This review summarizes the history and potential for future research using this versatile microbe.
Topics: Azotobacter vinelandii; Biofuels; Biopolymers; Hydrogen; Metabolic Engineering; Metabolic Networks and Pathways; Nitrogen; Nitrogenase; Oxidoreductases; Oxygen
PubMed: 29533747
DOI: 10.1099/mic.0.000643 -
Critical Reviews in Biochemistry and... 2022Understanding how Nature accomplishes the reduction of inert nitrogen gas to form metabolically tractable ammonia at ambient temperature and pressure has challenged... (Review)
Review
Understanding how Nature accomplishes the reduction of inert nitrogen gas to form metabolically tractable ammonia at ambient temperature and pressure has challenged scientists for more than a century. Such an understanding is a key aspect toward accomplishing the transfer of the genetic determinants of biological nitrogen fixation to crop plants as well as for the development of improved synthetic catalysts based on the biological mechanism. Over the past 30 years, the free-living nitrogen-fixing bacterium emerged as a preferred model organism for mechanistic, structural, genetic, and physiological studies aimed at understanding biological nitrogen fixation. This review provides a contemporary overview of these studies and places them within the context of their historical development.
Topics: Nitrogen Fixation; Azotobacter vinelandii; Nitrogenase; Ammonia; Nitrogen
PubMed: 36877487
DOI: 10.1080/10409238.2023.2181309 -
Journal of Bacteriology Nov 2021Azotobacter vinelandii is a nitrogen-fixing free-living soil microbe that has been studied for decades in relation to biological nitrogen fixation (BNF). It is highly...
Azotobacter vinelandii is a nitrogen-fixing free-living soil microbe that has been studied for decades in relation to biological nitrogen fixation (BNF). It is highly amenable to genetic manipulation, helping to unravel the intricate importance of different proteins involved in the process of BNF, including the biosynthesis of cofactors that are essential to assembling the complex metal cofactors that catalyze the difficult reaction of nitrogen fixation. Additionally, A. vinelandii accomplishes this feat while growing as an obligate aerobe, differentiating it from many of the nitrogen-fixing bacteria that are associated with plant roots. The ability to function in the presence of oxygen makes A. vinelandii suitable for application in various potential biotechnological schemes. In this study, we employed transposon sequencing (Tn-seq) to measure the fitness defects associated with disruptions of various genes under nitrogen-fixing dependent growth, versus growth with extraneously provided urea as a nitrogen source. The results allowed us to probe the importance of more than 3,800 genes, revealing that many genes previously believed to be important, can be successfully disrupted without impacting cellular fitness. These results provide insights into the functional redundancy in A. vinelandii, while also providing a direct measure of fitness for specific genes associated with the process of BNF. These results will serve as a valuable reference tool in future studies to uncover the mechanisms that govern this process.
Topics: Azotobacter vinelandii; Bacterial Proteins; Base Sequence; DNA Transposable Elements; Gene Expression Regulation, Bacterial; Genetic Fitness; Molybdenum; Nitrogen; Urea
PubMed: 34570624
DOI: 10.1128/JB.00404-21 -
Critical Reviews in Biotechnology 1997Although all commercial alginates are today of algal origin, there is interest in the production of alginate-like polymers from bacteria. The species Azotobacter... (Review)
Review
Although all commercial alginates are today of algal origin, there is interest in the production of alginate-like polymers from bacteria. The species Azotobacter vinelandii seems to be the best candidate for the industrial production of alginate molecules characterized by a chemical composition, molecular mass and molecular mass distribution suited to a well defined application, especially required in the biotechnological, biomedical and pharmaceutical fields. The production of alginate by A. vinelandii has been to date widely investigated both in batch (mainly in the shaken flask scale) and in continuous cultures. This article summarizes current knowledge on the structure and properties of alginates and their applications and presents an overview of up-dated research on the physiology, genetics and kinetics of the production of alginate by Azotobacter vinelandii and its rheology, including the results of our recent studies.
Topics: Alginates; Azotobacter vinelandii; Bacterial Proteins; Biocompatible Materials; Glucuronic Acid; Hexuronic Acids
PubMed: 9397533
DOI: 10.3109/07388559709146618 -
Methods in Molecular Biology (Clifton,... 2019The biological reduction of nitrogen gas to ammonia is limited to a select group of nitrogen-fixing prokaryotes. While nitrogenase is the catalyst of nitrogen fixation...
The biological reduction of nitrogen gas to ammonia is limited to a select group of nitrogen-fixing prokaryotes. While nitrogenase is the catalyst of nitrogen fixation in these biological systems, a consortium of additional gene products is required for the synthesis, activation, and catalytic competency of this oxygen-sensitive metalloenzyme. Thus, the biochemical complexity of this process often requires functional studies and isolation of gene products from the native nitrogen-fixing organisms. The strict aerobe Azotobacter vinelandii is the best-studied model bacterium among diazotrophs. This chapter provides a description of procedures for targeted genomic manipulation and isolation of A. vinelandii strains. These methods have enabled identification and characterization of gene products with roles in nitrogen fixation and other related aspects of metabolism. The ability to modify and control expression levels of targeted sequences provides a biotechnological tool to uncover molecular details associated with nitrogen fixation, as well as to exploit this model system as a host for expression of oxygen-sensitive proteins.
Topics: Azotobacter vinelandii; Bacterial Proteins; Gene Expression Regulation, Bacterial; Genetic Engineering; Metalloproteins; Nitrogen Fixation; Nitrogenase; Phenotype
PubMed: 30317476
DOI: 10.1007/978-1-4939-8864-8_6 -
PloS One 2023In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of...
In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of Pseudomonas aeruginosa, has been a model for cellular differentiation in Gram-negative bacteria since it forms desiccation-resistant cysts. Previous work demonstrated the essential role of AlgU to withstand oxidative stress and on A. vinelandii differentiation, particularly for the positive control of alginate production. In this study, the AlgU regulon was dissected by a proteomic approach under vegetative growing conditions and upon encystment induction. Our results revealed several molecular targets that explained the requirement of this sigma factor during oxidative stress and extended its role in alginate production. Furthermore, we demonstrate that AlgU was necessary to produce alkyl resorcinols, a type of aromatic lipids that conform the cell membrane of the differentiated cell. AlgU was also found to positively regulate stress resistance proteins such as OsmC, LEA-1, or proteins involved in trehalose synthesis. A position-specific scoring-matrix (PSSM) was generated based on the consensus sequence recognized by AlgU in P. aeruginosa, which allowed the identification of direct AlgU targets in the A. vinelandii genome. This work further expands our knowledge about the function of the ECF sigma factor AlgU in A. vinelandii and contributes to explains its key regulatory role under adverse conditions.
Topics: Sigma Factor; Regulon; Azotobacter vinelandii; Proteomics; Heat-Shock Proteins; Alginates; Bacterial Proteins; Gene Expression Regulation, Bacterial; Pseudomonas aeruginosa
PubMed: 37967103
DOI: 10.1371/journal.pone.0286440 -
Current Microbiology Feb 2022Encapsulation is an immobilization method characterized by restricting microbial cells to a delimited area while preserving their metabolic viability. This technique...
Encapsulation is an immobilization method characterized by restricting microbial cells to a delimited area while preserving their metabolic viability. This technique represents an alternative to improve the adaptive capacity of bacteria in the face of interactions with native microorganisms and environmental factors that limit their inoculation. This study aimed to evaluate the effect of Azotobacter vinelandii ATCC 12837 encapsulated in alginate-Na beads as an inoculant of tomato (Solanum Lycopersicum L) seedlings. Two inoculation treatments were carried out: liquid and encapsulated, and the control without microorganisms. Physiological variables, microbial viability, and the presence of A. vinelandii were determined by qPCR. Inoculation with A. vinelandii in liquid and encapsulated form favored seedling growth. Plants with the encapsulated inoculum significantly increased germination percentage (20%), stem diameter (38%), seedling height (34%), root length (69%), NO concentration (41%), and Na (30%); compared to the control. Encapsulation of A. vinelandii in alginate-Na macrocapsules allowed its establishment in the rhizosphere and was corroborated by viable count and molecular methods. The viability of the bacteria was maintained for 28 days using both inoculation methods, and not detected in the control treatment.
Topics: Alginates; Azotobacter vinelandii; Solanum lycopersicum; Rhizosphere; Seedlings
PubMed: 35178619
DOI: 10.1007/s00284-022-02797-6 -
Current Protocols Mar 2021Azotobacter vinelandii (A. vinelandii) is a commonly used model organism for the study of aerobic respiration, the bacterial production of several industrially relevant...
Azotobacter vinelandii (A. vinelandii) is a commonly used model organism for the study of aerobic respiration, the bacterial production of several industrially relevant compounds, and, perhaps most significantly, the genetics and biochemistry of biological nitrogen fixation. Laboratory growth assessments of A. vinelandii are useful for evaluating the impact of environmental and genetic modifications on physiological properties, including diazotrophy. However, researchers typically rely on manual growth methods that are oftentimes laborious and inefficient. We present a protocol for the automated growth assessment of A. vinelandii on a microplate reader, particularly well-suited for studies of diazotrophic growth. We discuss common pitfalls and strategies for protocol optimization, and demonstrate the protocol's application toward growth evaluation of strains carrying modifications to nitrogen-fixation genes. © 2021 The Authors. Basic Protocol 1: Preparation of A. vinelandii plate cultures from frozen stock Basic Protocol 2: Preparation of A. vinelandii liquid precultures Basic Protocol 3: Automated growth rate experiment of A. vinelandii on a microplate reader.
Topics: Azotobacter vinelandii; Laboratories; Nitrogen Fixation; Nitrogenase
PubMed: 33656286
DOI: 10.1002/cpz1.57 -
Applied and Environmental Microbiology Aug 2018selectively utilizes three types of nitrogenase (molybdenum, vanadium, and iron only) to fix N, with their expression regulated by the presence or absence of different...
selectively utilizes three types of nitrogenase (molybdenum, vanadium, and iron only) to fix N, with their expression regulated by the presence or absence of different metal cofactors in its environment. Each alternative nitrogenase isoenzyme is predicted to have different electron flux requirements based on measurements, with the molybdenum nitrogenase requiring the lowest flux and the iron-only nitrogenase requiring the highest. Here, prior characterized strains, derepressed in nitrogenase synthesis and also deficient in uptake hydrogenase, were further modified to generate new mutants lacking the ability to produce poly-β-hydroxybutyrate (PHB). PHB is a storage polymer generated under oxygen-limiting conditions and can represent up to 70% of the cells' dry weight. The absence of such granules facilitated the study of relationships between catalytic biomass and product molar yields across different adaptive respiration conditions. The released hydrogen gas observed during growth, due to the inability of the mutants to recapture hydrogen, allowed for direct monitoring of nitrogenase activity for each isoenzyme. The data presented here show that increasing oxygen exposure limits equally the activities of all nitrogenase isoenzymes, while under comparative conditions, the Mo nitrogenase enzyme evolves more hydrogen per unit of biomass than the alternative isoenzymes. has been a focus of intense research for over 100 years. It has been investigated for a variety of functions, including agricultural fertilization and hydrogen production. All of these endeavors are centered around 's ability to fix nitrogen aerobically using three nitrogenase isoenzymes. The majority of research up to this point has targeted measurements of the molybdenum nitrogenase, and robust data contrasting how oxygen impacts the activity of each nitrogenase isoenzyme are lacking. This article aims to provide nitrogenase activity data using a real-time evaluation of hydrogen gas released by derepressed nitrogenase mutants lacking an uptake hydrogenase and PHB accumulation.
Topics: Azotobacter vinelandii; Bacterial Proteins; Hydrogen; Hydroxybutyrates; Iron; Molybdenum; Nitrogen; Nitrogen Fixation; Nitrogenase; Oxidation-Reduction; Oxygen; Polyesters; Vanadium
PubMed: 29915110
DOI: 10.1128/AEM.01208-18 -
Methods in Molecular Biology (Clifton,... 2021Nitrogenases are the sole enzymes known to mediate biological nitrogen fixation, an essential process for sustaining life on earth. Among the three known variants,...
Nitrogenases are the sole enzymes known to mediate biological nitrogen fixation, an essential process for sustaining life on earth. Among the three known variants, molybdenum nitrogenase is the best-studied to date. Recent work on the alternative vanadium nitrogenase provided important insights into the mechanism of nitrogen fixation since this enzyme differs from its molybdenum counterpart in some important aspects. Here, we present a protocol to obtain unmodified vanadium nitrogenase in high yield and purity from the paradigmatic diazotroph Azotobacter vinelandii, including procedures for cell cultivation, purification, and protein characterization.
Topics: Azotobacter vinelandii; Molybdenum; Nitrogen Fixation; Nitrogenase
PubMed: 34292546
DOI: 10.1007/978-1-0716-1605-5_6