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PubMed1985

Design of a system for the control of low dissolved oxygen concentrations: critical oxygen concentrations for Azotobacter vinelandii and Escherichia coli.

The physiological activity of microorganisms in environments with low dissolved oxygen concentrations often differs from the metabolic activity of the same cells growing under fully aerobic or anaerobic conditions. This article describes a laboratory-scale system for the control of dissolved oxygen at low levels while maintaining other parameters, such as agitator speed, gas flowrate, position of sparger outlet, and temperature at fixed values. Thus, it is possible to attribute in dilute nonviscous fermentations all physiologic changes solely to changes in dissolved oxygen. Experiments were conducted with Azotobacter vinelandii and Escherichia coli. Critical oxygen concentrations for growth (that value of oxygen allowing growth at 97% of mu max) were measured as 0.35 +/- 0.03 mg/L for A. vinelandii and 0.12 +/- 0.03 mg/L for E. coli. These values are significantly different from the commonly quoted values for critical oxygen concentrations based on respiration rates. Because of the superior dissolved oxygen control system and an improved experimental protocol preventing CO2 limitation, we believe that the values reported in this work more closely represent reality.

Azotobacter vinelandii↗

PubMed2005

A new type of metalloprotein: The Mo storage protein from azotobacter vinelandii contains a polynuclear molybdenum-oxide cluster.

Azotobacter vinelandii is a diazotrophic bacterium characterized by the outstanding capability of storing Mo in a special storage protein, which guarantees Mo-dependent nitrogen fixation even under growth conditions of extreme Mo starvation. The Mo storage protein is constitutively synthesized with respect to the nitrogen source and is regulated by molybdenum at an extremely low concentration level (0-50 nM). This protein was isolated as an alpha4beta4 octamer with a total molecular mass of about 240 kg mol(-1) and its shape was determined by small-angle X-ray scattering. The genes of the alpha and beta subunits were unequivocally identified; the amino acid sequences thereby determined reveal that the Mo storage protein is not related to any other known molybdoprotein. Each protein molecule can store at least 90 Mo atoms. Extended X-ray absorption fine-structure spectroscopy identified a metal-oxygen cluster bound to the Mo storage protein. The binding of Mo (biosynthesis and incorporation of the cluster) is dependent on adenosine triphosphate (ATP); Mo release is ATP-independent but pH-regulated, occurring only above pH 7.1. This Mo storage protein is the only known noniron metal storage system in the biosphere containing a metal-oxygen cluster.

Amino Acid Sequence↗

PubMed1990

Growth of Azotobacter vinelandii with correlation of Coulter cell size, flow cytometric parameters, and ultrastructure.

When Azotobacter vinelandii is grown under nitrogen-fixing conditions, the mean cell volume fluctuates from 2.7 to 6.6 microns 3 as determined using a Coulter counter. When NH4Cl is supplied as nitrogen source, the mean cell volume fluctuates from 4.6 to 7.4 microns3. Parallel experiments using flow cytometric measurements show similar characteristic fluctuations in the narrow forward angle light scattering signal and also in cellular protein content as determined using fluorescein isothiocyanate (FITC) fluorescence. Fluctuations in the perpendicular light scatter signal during batch growth are similar for both sets of growth conditions. Changes in cell morphology and ultrastructure are also similar for both sets of growth conditions, as demonstrated by electron microscopic examination. We conclude that narrow forward angle light scatter is a close correlate of cell size, whereas right angle scatter is an indicator of morphological variations other than size.

Ammonium Chloride↗

PubMed1986

Second gene (nifH*) coding for a nitrogenase iron protein in Azotobacter chroococcum is adjacent to a gene coding for a ferredoxin-like protein.

Azotobacter chroococcum MCD1 contains a cluster of nitrogen fixation (nif) genes coding for the structural polypeptides for nitrogenase (nifH for the Fe-protein and nifD and nifK for the MoFe protein) and a second sequence in the genome homologous to nifH. DNA fragments bearing this second nifH-like sequence were cloned and the DNA sequence around the homologous region determined. Two open reading frames were identified in this region. One codes for a protein of 289 amino acid residues and is highly homologous to other Fe-proteins but is different from the gene adjacent to the nifDK genes in A. chroococcum. This putative gene we call nifH*. The following open reading frame codes for a protein of 63 amino acids, nine of which are cysteine residues. The protein is homologous to the small low-potential ferredoxins found in anaerobic bacteria, and in particular those from Chlorobium limicola. Linkage between a structural gene for nitrogenase and a small ferredoxin has not previously been observed. Sequence analysis suggests that the two genes form an operon. Transcription of the ferredoxin gene on a 1320-bp transcript was only detectable under conditions in which A. chroococcum MCD1155, which carries a chromosomal deletion of 6.3 kb removing the entire nifHDK cluster, is capable of fixing N2, i.e. in media containing no added molybdenum or high levels of NH3. The size of the observed transcript agrees well with the predicted size for a transcript encoding nifH* and the ferredoxin genes. Expression of the nifH* promoter was not significantly activated in Escherichia coli even when nifA, the positive activator of nif genes in Klebsiella pneumoniae, was supplied in multiple copies. The results are discussed in relation to an alternative pathway for N2 fixation in A. chroococcum.

Amino Acid Sequence↗

PubMed1989

Structural genes for the vanadium nitrogenase from Azotobacter chroococcum.

Structural genes for the VFe-protein (Ac1V) of the vanadium nitrogenase from Azotobacter chroococcum were cloned and sequenced. The VFe-protein contains three subunit types with Mr of 53,793 (alpha), 52,724 (beta) and 13,274 (delta). alpha and beta subunits show 18 and 15% sequence identity respectively, with alpha and beta subunits of the MoFe-protein of A.chroococcum molybdenum nitrogenase. The genes for the three subunits vnfD (alpha), vnfG (delta) and vnfK (beta) are contiguous and form an operon whose transcription is repressed in response to ammonia. The Fe-protein component of the V-nitrogenase (Ac2V) is the product of nifH* that we have previously cloned and sequenced. This gene was located 2.5 kb upstream of vnfD. A deletion in the vnfD, G and K gene cluster prevents V-dependent nitrogen fixation. A strain defective in both V-nitrogenase and Mo-nitrogenase structural genes showed no residual nitrogen fixing capacity arguing against the presence of a third nitrogen fixation system in this organism.

Amino Acid Sequence↗

PubMed1996

Investigations on the cell volumes of Azotobacter vinelandii by scanning electron microscopy.

Previous experiments by other investigators on the DNA content of Azotobacter vinelandii have demonstrated that the DNA content in these cells is several folds higher than that of E. coli. On the basis of this observation, it was hypothesized that A. vinelandii has at least 40 to 80 identical chromosomes per cell. However, the gene dosage analysis in A. vinelandii cells suggested that many genetic operations can be performed in these cells without the constraints expected in a polyploid bacterium. In an attempt to explain this apparent discrepancy, we have done systematic analysis of the relationship between the DNA content and the cell volume of this bacterium. Since a linear correlation is observed between the DNA content and the cell size in many other cell types, we hypothesized that if A. vinelandii is polyploid in nature, it should have a much larger cell volume to accommodate such a large amount of DNA. Our scanning electron microscopic analysis revealed that the cell volume of the vegetative cells of A. vinelandii is about 16 times larger than the cell volume of E. coli. This result is apparently consistent with the concept that the A. vinelandii is a polyploid bacterium. It was also reported that the encysted cells of A. vinelandii contain about 25% of the DNA content of the vegetative cells. This would mean that an encysted cell of A. vinelandii could contain about 10 copies of its chromosome. Since the estimated molecular weight of A. vinelandii chromosome is very similar to that of E. coli chromosome, the DNA content of the encysted cells also should be about 10 times higher than that of E. coli cells. If we assume that the relationship between the DNA content and the cell size is linear, then the encysted cells should have a cell volume larger than that of E. coli and smaller than that of the vegetative cells of A. vinelandii. However our scanning electron microscopic analysis showed that the cell volume of the encysted cells of A. vinelandii is in fact very similar to the cell volume of E. coli.

Azotobacter vinelandii↗

PubMed1977

Isolation of membrane vesicles with inverted topology by osmotic lysis of Azotobacter vinelandii spheroplasts.

Membrane vesicles were prepared from Azotobacter vinelandii spheroplasts by lysis in either potassium phosphate (pH 7.0) or Tris1-acetate (pH 7.8) buffers. These 2 types of preparations differ considerably in their properties: 1) Examination by scanning electron microscopy reveals that the Pi vesicles consist primarily of closed structures 0.6-0.8 micrometer in diameter with a rough or particulate surface similar to that of spheroplasts. The Tris vesicles are significantly smaller, 0.1-0.3 micrometer in diameter, and have a much smoother surface structure. 2) Antisera from rabbits immunized with A. vinelandii lipopolysaccharide antigen will agglutinate Pi vesicles but not Tris vesicles. 3) Tris vesicles have a fourfold higher specific activity of latent H+-ATPase than Pi vesicles. After exposure to Triton X-100 similar ATPase activities are observed for both types of vesicles. 4) Pi vesicles transport calcium in the presence of ATP or lactate at less than 30% of the rats observed for Tris vesicles. 5) Tris vesicles have less than 22% of the transport capacity of Pi vesicles for accumulation of labeled sucrose and less than 3% of the capacity for valinomycin-induced uptake of rubidium observed during respiration. 6) Quinacrine fluorescence intensity is reduced by 30% during lactate oxidation and 20% during ATP hydrolysis by Tris vesicles. Under similar conditions, fluorescence in Pi vesicles is quenched by only 7% and less than 2%, respectively. These findings suggest that Pi vesicles have the normal orientation of the intact cell whereas Tris vesicles have an inverted topology.

Adenosine Triphosphatases↗

PubMed1994

Docking of nitrogenase iron- and molybdenum-iron proteins for electron transfer and MgATP hydrolysis: the role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein.

Docking of the nitrogenase component proteins, the iron protein (FeP) and the molybdenum-iron protein (MoFeP), is required for MgATP hydrolysis, electron transfer between the component proteins, and substrate reductions catalyzed by nitrogenase. The present work examines the function of 3 charged amino acids, Arg 140, Glu 141, and Lys 143, of the Azotobacter vinelandii FeP in nitrogenase component protein docking. The function of these amino acids was probed by changing each to the neutral amino acid glutamine using site-directed mutagenesis. The altered FePs were expressed in A. vinelandii in place of the wild-type FeP. Changing Glu 141 to Gln (E141Q) had no adverse effects on the function of nitrogenase in whole cells, indicating that this charged residue is not essential to nitrogenase function. In contrast, changing Arg 140 or Lys 143 to Gln (R140Q and K143Q) resulted in a significant decrease in nitrogenase activity, suggesting that these charged amino acid residues play an important role in some function of the FeP. The function of each amino acid was deduced by analysis of the properties of the purified R140Q and K143Q FePs. Both altered proteins were found to support reduced substrate reduction rates when coupled to wild-type MoFeP. Detailed analysis revealed that changing these residues to Gln resulted in a dramatic reduction in the affinity of the altered FeP for binding to the MoFeP. This was deduced in FeP titration, NaCl inhibition, and MoFeP protection from Fe2+ chelation experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1998

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.

Amino Acid Sequence↗

PubMed1998

The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate.

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of alpha/beta doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.

Apoproteins↗

PubMed1998

The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii.

NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN phosphate with Arg51 in a conserved FAD binding motif. The fourth feature, common to homologous oxidoreductases, is a concentration of 10 basic residues on the face of the protein surrounding the active site, in addition to Arg51 and Lys258.

Azotobacter vinelandii↗

PubMed1995

New open reading frames, one of which is similar to the nifV gene of Azotobacter vinelandii, found on a 12.5 kbp fragment of chromosome IV of Saccharomyces cerevisiae.

The nucleotide sequence of a 12.5 kbp segment of the left arm of chromosome IV is described. Five open reading frames (ORFs) longer than 100 amino acids were detected, all of which are completely confined to the 12.5 kbp region. Two ORFs (D1271 and D1286) correspond to previously sequenced genes (PPH22 and VMA1 or TFP1, respectively). ORF D1298 shows the characteristics of alpha-isopropylmalate and homocitrate synthase genes and is similar to the nifV gene of Azotobacter vinelandii. Two more ORFs have no apparent homologue in the data libraries. Conversely, two smaller ORFs of 25 and 85 amino acids encoding the ribosomal protein YL41A and an ATPase inhibitor, respectively, were detected. Although a substantial part of the 12.5 kbp fragment apparently lacks protein-coding characteristics, no other elements, such as tRNA genes or transposons, were found.

Amino Acid Sequence↗

PubMed1995

The concentration of cellular nitrogenase proteins in Azotobacter vinelandii whole cells as determined by activity measurements and electron paramagnetic resonance spectroscopy.

The concentration of MoFe protein (Av1) in Azotobacter vinelandii whole-cell crude extract was measured by electron paramagnetic resonance spectroscopy at g = 3.7 resonance. The Av1 concentration was also measured from the activity of crude extract to which increasing amounts of purified Av1 and Av2 were added. The Av2 concentration was determined by fitting activity measurements of crude extract and crude extract to which purified Av2 was added. The Av1 concentration was found to be 26-28 microM and that for Av2 was 42-45 microM in whole cells, with a Av2/Av1 ratio of 1.6. In vitro activity measurements carried out as a function of Av1 concentration at Av2/Av1 ratios of 1 and 4 showed a dilution effect below 0.08 microM, a factor of 2 below that observed for nitrogenase reactivity for Klebsiella pneumoniae. No deviations from linearity were observed up to 26 microM for the Av1-Av2 interaction. The flavoprotein (AvFlp) was shown to enhance nitrogenase reactivity at low Av2/Av1 ratios, a result attributed to decreasing the Km for Av2-Av1 interaction. Direct reduction of bound Av2 is possibly the source of this kinetic enhancement. The kinetic results are considered in terms of the Thorneley and Lowe scheme.

Azotobacter vinelandii↗

PubMed1995

Purification and characterization of Azotobacter vinelandii glucose-6-phosphate dehydrogenase: dual coenzyme specificity.

Azotobacter vinelandii glucose-6-phosphate dehydrogenase isolated from cell sonicates was purified 81-fold to electrophoretic homogeneity and a specific activity of 73 units/mg protein using ion-exchange and Matrex Dye chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular exclusion chromatography indicated the enzyme to be a tetramer composed of 52,000 M(r) subunits. The enzyme utilized both NAD and NADP as coenzymes with Km values of 220 and 50 microM, respectively. In addition, the purified enzyme functioned well with the thionicotinamide analogs of NAD and NADP. A sigmoidal response was observed in studies of the effect of glucose 6-phosphate concentration on initial velocities. Evidence in support of one enzyme with dual coenzyme specificity was obtained in purification, thermodenaturation, and inhibitor studies. The enzyme exhibited a pH optimum of 8.5. Coenzyme-competitive inhibition was observed with nine adenosine derivatives with no significant selectivity shown for 2'-phosphoryl derivatives. Ki values for product inhibition by NADH and NADPH were higher than the Km values for the respective oxidized forms of the coenzymes.

Adenine Nucleotides↗

PubMed1995

Reductant-independent ATP hydrolysis catalyzed by homologous nitrogenase proteins from Azotobacter vinelandii and heterologous crosses with Clostridium pasteuranium.

Reductant-independent ATPase activity was initiated and studied for Azotobacter vinelandii and Clostridium pasteuranium nitrogenase proteins (Av1, Cp1 and Av2, Cp2, 1 designating the iron molybdenum protein and 2 the iron protein) and their heterologous crosses by two methods: (1) allowing dithionite to be depleted from a normal assay in the presence of substrate levels of MgATP and (2) using reduced but reductant-free nitrogenase proteins in the presence of substrate levels of MgATP. In both cases, at a 1:1 protein ratio, MgATP is converted initially to MgADP with a specific activity of 400-500 nmol MgATP hydrolyzed/min.mg Av1, but in slower steps the MgADP is converted to AMP and, after 12 h, AMP is ultimately converted to adenosine. This reactivity requires the presence of both proteins, increases with increasing Av2/Av1 ratio, and is not a result of unique redox states of either protein. For Av1-Av2, ATP hydrolysis in the absence of Mg2+ occurred at nearly the same rate as reductant-dependent MgATP hydrolysis. Reductant-independent ATPase activity also occurred for the Av1-Cp2 and Cp1-Av2 heterologous crosses and was 2-fold and 18-fold slower than the Av1-Av2 or Cp1-Cp2 combinations. In both cases further hydrolysis of MgADP to AMP and AMP to adenosine occurred. A unique nucleotide hydrolysis system is apparently operating in the complex formed between the two nitrogenase proteins in the absence of reductant. The relationship between the reductant-independent and reductant-dependent activities of nitrogenase catalysis is explored.

Adenosine Triphosphate↗

PubMed1994

Purification and characterization of 2-keto-3-deoxy-6-phosphogluconate aldolase from Azotobacter vinelandii: evidence that the enzyme is bifunctional towards 2-keto-4-hydroxy glutarate cleavage.

2-keto-3-deoxy-6-phosphogluconate aldolase (E.C. 4.1.2.14) has been purified in two chromatographic steps to 99% purity in 73% overall yield from Azotobacter vinelandii. The pure enzyme is a 70 kD trimeric Class I aldolase, inhibitable by bromopyruvate or pyruvate plus sodium borohydride, with a specific activity of 625 mumol per min per mg protein and a Km of 38 microM for 2-keto-3-deoxy-6-phosphogluconate. The enzyme also has 2-keto-4-hydroxy glutarate aldolase (E.C. 4.1.3.16) activity, with a specific activity of 4.8 mumol per min per mg protein and a Km of 39 microM. 2-keto-4-hydroxy glutarate inhibits the 2-keto-3-deoxy-6-phosphogluconate aldolase activity of the enzyme with an apparent Ki of 0.17 mM. Slow steps following formation of the Schiff base intermediate between KHG and the enzyme are responsible for both the slower turnover of this substrate and for its inhibitory effect.

Aldehyde-Lyases↗

PubMed1996

Improved catalytic performance of a 2-haloacid dehalogenase from Azotobacter sp. by ion-exchange immobilisation.

The stability and catalytic efficacy of the L-2-haloacid dehalogenase isolated from Azotobacter sp. RC26 were studied after immobilisation on a DEAE Sephacel solid matrix. While the optimum temperature for the soluble dehalogenase falls in the range of 30-40 degrees C, the activity of the immobilised enzyme shows a four-fold increase at 60 degree C. Immobilisation on a plug-flow bioreactor extends the range of usable substrate concentration. The improved catalytic characteristics after immobilisation of the haloacid dehalogenase may be relevant for its possible utilization in biotechnological applications ranging from waste treatment to synthesis of stereoisomers.

Azotobacter↗

PubMed1997

Nucleotide sequence and genetic complementation analysis of lep from Azotobacter vinelandii.

The lep of Azotobacter vinelandii is an 852-base-pair open reading frame (ORF) which encodes a protein of 284 amino acid residues. The translated protein shares 75% homology with leader peptidase I isolated from Pseudomonas fluorescens and 37% homology with leader peptidase I isolated from Escherichia coli. Five highly conserved regions found in the family of leader peptidase I proteins are conserved in A. vinelandii Lep. The putative membrane topology of the protein seems similar to that of E. coli leader peptidase I based on the hydrophobicity analysis of the predicted amino acid sequence. Southern blotting analysis of the A. vinelandii chromosome by probing with lep specific DNA revealed that lep is present as a single copy per the chromosome. A multicopy plasmid carrying A. vinelandii lep could complement a temperature sensitive lep mutant of E. coli strain IT41, suggesting that we have identified the functional copy of lep present on A. vinelandii genome.

Amino Acid Sequence↗