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Applied and Environmental Microbiology Jan 2016In this study, we performed a detailed characterization of the siderophore metabolome, or "chelome," of the agriculturally important and widely studied model organism...
In this study, we performed a detailed characterization of the siderophore metabolome, or "chelome," of the agriculturally important and widely studied model organism Azotobacter vinelandii. Using a new high-resolution liquid chromatography-mass spectrometry (LC-MS) approach, we found over 35 metal-binding secondary metabolites, indicative of a vast chelome in A. vinelandii. These include vibrioferrin, a siderophore previously observed only in marine bacteria. Quantitative analyses of siderophore production during diazotrophic growth with different sources and availabilities of Fe showed that, under all tested conditions, vibrioferrin was present at the highest concentration of all siderophores and suggested new roles for vibrioferrin in the soil environment. Bioinformatic searches confirmed the capacity for vibrioferrin production in Azotobacter spp. and other bacteria spanning multiple phyla, habitats, and lifestyles. Moreover, our studies revealed a large number of previously unreported derivatives of all known A. vinelandii siderophores and rationalized their origins based on genomic analyses, with implications for siderophore diversity and evolution. Together, these insights provide clues as to why A. vinelandii harbors multiple siderophore biosynthesis gene clusters. Coupled with the growing evidence for alternative functions of siderophores, the vast chelome in A. vinelandii may be explained by multiple, disparate evolutionary pressures that act on siderophore production.
Topics: Azotobacter vinelandii; Bacterial Proteins; Biosynthetic Pathways; Chromatography, Liquid; Mass Spectrometry; Metabolome; Siderophores
PubMed: 26452553
DOI: 10.1128/AEM.03160-15 -
Applied and Environmental Microbiology Mar 2022The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A....
The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an antiactivator that tightly controls the activity of the nitrogen fixation-specific transcriptional activator NifA in response to redox, nitrogen, and carbon status. While several studies reported that mutations in A. vinelandii resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonium, knowledge about the specific determinants for this ammonium-excreting phenotype is lacking. In this work, we report that only specific disruptions of lead to large quantities of ammonium accumulated in liquid culture (∼12 mM). The ammonium excretion phenotype is associated solely with deletions of NifL domains combined with the insertion of a promoter sequence in the orientation opposite that of transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonium excreted by affecting gene expression as an additional requirement for ammonium excretion. These ammonium-excreting mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops. There is considerable interest in the engineering of ammonium-excreting bacteria for use in agriculture to promote the growth of plants under fixed-nitrogen-limiting conditions. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.
Topics: Ammonium Compounds; Azotobacter vinelandii; Bacterial Proteins; Gene Expression Regulation, Bacterial; Nitrogen Fixation; Nitrogenase
PubMed: 35138932
DOI: 10.1128/AEM.01876-21 -
ELife Feb 2023The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always...
The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always followed the same set of strategies for performing these critical tasks. Today, microbes access atmospheric sources of bioessential nitrogen through the activities of just one family of enzymes, nitrogenases. Here, we show that the only dinitrogen reduction mechanism known to date is an ancient feature conserved from nitrogenase ancestors. We designed a paleomolecular engineering approach wherein ancestral nitrogenase genes were phylogenetically reconstructed and inserted into the genome of the diazotrophic bacterial model, enabling an integrated assessment of both in vivo functionality and purified nitrogenase biochemistry. Nitrogenase ancestors are active and robust to variable incorporation of one or more ancestral protein subunits. Further, we find that all ancestors exhibit the reversible enzymatic mechanism for dinitrogen reduction, specifically evidenced by hydrogen inhibition, which is also exhibited by extant nitrogenase isozymes. Our results suggest that life may have been constrained in its sampling of protein sequence space to catalyze one of the most energetically challenging biochemical reactions in nature. The experimental framework established here is essential for probing how nitrogenase functionality has been shaped within a dynamic, cellular context to sustain a globally consequential metabolism.
Topics: Nitrogenase; Nitrogen Fixation; Azotobacter vinelandii; Amino Acid Sequence; Nitrogen
PubMed: 36799917
DOI: 10.7554/eLife.85003 -
The Journal of Biological Chemistry Jan 1996We have identified the molecular basis for the nitrogenase negative phenotype exhibited by Azotobacter vinelandii UW97. This strain was initially isolated following... (Comparative Study)
Comparative Study
We have identified the molecular basis for the nitrogenase negative phenotype exhibited by Azotobacter vinelandii UW97. This strain was initially isolated following nitrosoguanidine mutagenesis. Recently, it was shown that this strain lacks the Fe protein activity, which results in the synthesis of a FeMo cofactor-deficient apodinitrogenase. Activation of this apodinitrogenase requires the addition of both MgATP and wild-type Fe protein to the crude extracts made by A. vinelandii UW97 (Allen, R.M., Homer, M.J., Chatterjee R., Ludden, P.W., Roberts, G.P., and Shah, V.K. (1993) J. Biol. Chem. 268 23670-23674). Earlier, we proposed the sequence of events in the MoFe protein assembly based on the biochemical and spectroscopic analysis of the purified apodinitrogenase from A. vinelandii DJ54 (Gavini, N., Ma, L., Watt, G., and Burgess, B.K. (1994) Biochemistry 33, 11842-11849). Taken together, these results imply that the assembly process of apodinitrogenase is arrested at the same step in both of these strains. Since A. vinelandii DJ54 is a delta nifH strain, this strain is not useful in identifying the features of the Fe protein involved in the MoFe protein assembly. Here, we report a systematic analysis of an A. vinelandii UW97 mutant and show that, unlike A. vinelandii DJ54, the nifH gene of A. vinelandii UW97 has no deletion in either coding sequence or the surrounding sequences. The specific mutation responsible for the Nif- phenotype of A. vinelandii UW97 is the substitution of a non-conserved serine at position 44 of the Fe protein by a phenylalanine as shown by DNA sequencing. Furthermore, oligonucleotide site-directed mutagenesis was employed to confirm that the Nif- phenotype in A. vinelandii UW97 is exclusively due to the substitution of the Fe protein residue serine 44 by phenylalanine. By contrast, replacing Ser-44 with alanine did not affect the Nif phenotype of A. vinelandii. Therefore, it seems that the Nif- phenotype of A. vinelandii UW97 is caused by a general structural disturbance of the Fe protein due to the presence of the bulky phenylalanine at position 44.
Topics: Amino Acid Sequence; Azotobacter vinelandii; Consensus Sequence; Genes, Bacterial; Molecular Sequence Data; Molybdoferredoxin; Mutagenesis, Site-Directed; Nitrogen Fixation; Nitrogenase; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship
PubMed: 8567634
DOI: 10.1074/jbc.271.4.1884 -
Journal of Applied Microbiology Jul 2018The sigma E (AlgU) in Azotobacter vinelandii has been shown to control the expression of cydR gene, a repressor of genes of the alternative respiratory chain, and...
AIMS
The sigma E (AlgU) in Azotobacter vinelandii has been shown to control the expression of cydR gene, a repressor of genes of the alternative respiratory chain, and alginate has been considered a barrier for oxygen diffusion. Therefore, the aim of the present study was to compare the respiratory activity of an alginate nonproducing strain, lacking the sigma factor E (algU-), and polymer-producing strains (algU+) of A. vinelandii under diazotrophic conditions at different aeration conditions.
METHODS AND RESULTS
Our results reveal that under diazotrophic and high aeration conditions, A. vinelandii strain OP (algU-) had a specific oxygen consumption rate higher (30 and 54%) than those observed in the OP algU+-complemented strain, named OPAlgU+, and the ATCC 9046 respectively. However, the specific growth rate and biomass yields (based on oxygen and sucrose) were lower for OP cultivations as compared to the algU+ strains. These differences were partially explained by an increase in 1·5-fold of cydA relative expression in the OP strain, as compared to that obtained in the isogenic OPAlgU+ strain.
CONCLUSIONS
Overall, our results confirm the important role of algU gene on the regulation of respiratory metabolism under diazotrophic growth when A. vinelandii is exposed to high aeration.
SIGNIFICANCE AND IMPACT OF THE STUDY
This study highlights the role of AlgU to control respiration of A. vinelandii when exposed to diazotrophy.
Topics: Alginates; Azotobacter vinelandii; Glucuronic Acid; Hexuronic Acids; Nitrogen Fixation; Oxygen
PubMed: 29573518
DOI: 10.1111/jam.13760 -
Microbiology (Reading, England) Sep 2019is a metabolically versatile bacterium and also an important opportunistic pathogen. It has a remarkable genomic structure since the genetic information encoding its...
is a metabolically versatile bacterium and also an important opportunistic pathogen. It has a remarkable genomic structure since the genetic information encoding its pathogenicity-related traits belongs to its core-genome while both environmental and clinical isolates are part of the same population with a highly conserved genomic sequence. Unexpectedly, considering the high level of sequence identity and homologue gene number shared between different isolates, the presence of specific essential genes of the two type strains PAO1 and PA14 has been reported to be highly variable. Here we report the detailed bioinformatics analysis of the essential genes of PAO1 and PA14 that have been previously experimentally identified and show that the reported gene variability was owed to sequencing and annotation inconsistencies, but that in fact they are highly conserved. This bioinformatics analysis led us to the definition of 348 . general essential genes. In addition we show that 342 of these 348 essential genes are conserved in a nitrogen-fixing, cyst-forming, soil bacterium. These results support the hypothesis of having a polyphyletic origin with a Pseudomonads genomic backbone, and are a challenge to the accepted theory of bacterial evolution.
Topics: Azotobacter vinelandii; Bacteria; Biological Evolution; Computational Biology; Conserved Sequence; Evolution, Molecular; Genes, Bacterial; Genes, Essential; Genome, Bacterial; Pseudomonas aeruginosa
PubMed: 31274400
DOI: 10.1099/mic.0.000833 -
Microbial Cell Factories Jan 2018Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P) ratios on the metabolic fluxes through...
BACKGROUND
Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P) ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P) ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions.
RESULTS
The results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP ratio, showing that at the lowest OTR (2.4 mmol L h), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L h). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L h. At the highest OTR condition, a decrease in the NADPH/NADP ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH).
CONCLUSIONS
We have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes.
Topics: Alginates; Azotobacter vinelandii; Biomass; Biosynthetic Pathways; Carbon; Citric Acid Cycle; Culture Media; Metabolic Flux Analysis; NAD; NADP; Oxidation-Reduction; Oxygen; Pentose Phosphate Pathway
PubMed: 29357933
DOI: 10.1186/s12934-018-0860-8 -
Journal of Bacteriology Jul 2009Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from...
Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.
Topics: Azotobacter vinelandii; Bacterial Proteins; Base Sequence; DNA, Bacterial; Genome, Bacterial; Metabolism; Molecular Sequence Data; Phylogeny; Sequence Analysis, DNA
PubMed: 19429624
DOI: 10.1128/JB.00504-09 -
Scientific Reports Jun 2022While biodiesel is drawing attention as an eco-friendly fuel, the use of crude glycerol, a byproduct of the fuel production process, has increasingly become a concern to...
While biodiesel is drawing attention as an eco-friendly fuel, the use of crude glycerol, a byproduct of the fuel production process, has increasingly become a concern to be addressed. Here we show the development of a low-cost fermentation technology using an atmospheric nitrogen-fixing bacterium to recycle crude glycerol into functional biopolymers. Azotobacter vinelandii showed substantial growth on tap water-diluted crude glycerol without any pretreatment. The number of viable A. vinelandii cells increased over 1000-fold under optimal growth conditions. Most of the glycerol content (~ 0.2%) in the crude glycerol medium was completely depleted within 48 h of culture. Useful polymers, such as polyhydroxybutyrate and alginate, were also produced. Polyhydroxybutyrate productivity was increased ten-fold by blocking the alginate synthesis pathway. Although there are few examples of using crude glycerol directly as a carbon source for microbial fermentation, there are no reports on the use of crude glycerol without the addition of a nitrogen source. This study demonstrated that it is possible to develop a technology to produce industrially useful polymers from crude glycerol through energy-saving and energy-efficient fermentation using the atmospheric nitrogen-fixing microorganism A. vinelandii.
Topics: Alginates; Azotobacter vinelandii; Fermentation; Glycerol; Nitrogen; Polymers
PubMed: 35672418
DOI: 10.1038/s41598-022-11728-1 -
PloS One 2018The influence of nanomaterials on the ecological environment is becoming an increasingly hot research field, and many researchers are exploring the mechanisms of...
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.
Topics: Apoptosis; Azotobacter vinelandii; Bacterial Proteins; Environmental Pollutants; Gene Expression; Hydroxyl Radical; Metal Nanoparticles; Nitrogen Fixation; Oxidative Stress; Particle Size; Reactive Oxygen Species; Silver Compounds
PubMed: 30566461
DOI: 10.1371/journal.pone.0209020