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PubMed1994

In vivo modification of Azotobacter chroococcum glutamine synthetase.

A monospecific anti-(glutamine synthetase) antibody raised against glutamine synthetase of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 immunoreacted with glutamine synthetase from the N2-fixing heterotrophic bacterium Azotobacter chroococcum. In Western-blotting experiments this antibody recognized a single protein of a molecular mass of 59 kDa corresponding to glutamine synthetase subunit. This protein was in vivo-labelled in response to addition of ammonium, both [3H]adenine and H(3)32PO4 preincubation of the cells being equally effective. Nevertheless, the amount of glutamine synthetase present in A. chroococcum was independent of the available nitrogen source. Modified, inactive glutamine synthetase was re-activated by treatment with snake-venom phosphodiesterase but not by alkaline phosphatase. L-Methionine-DL-sulphoximine, an inhibitor of glutamine synthetase, prevented the enzyme from being covalently modified. We conclude that, in A. chroococcum, glutamine synthetase is adenylylated in response to ammonium and that for the modification to take place ammonium must be metabolized.

Adenine↗

PubMed2005

NifU and NifS are required for the maturation of nitrogenase and cannot replace the function of isc-gene products in Azotobacter vinelandii.

In recent years, it has become evident that [Fe-S] proteins, such as hydrogenase, nitrogenase and aconitase, require a complex machinery to assemble and insert their associated [Fe-S] clusters. So far, three different types of [Fe-S] cluster biosynthetic systems have been identified and these have been designated nif, isc and suf. In the present work, we show that the nif-specific [Fe-S] cluster biosynthetic system from Azotobacter vinelandii, which is required for nitrogenase maturation, cannot functionally replace the isc [Fe-S] cluster system used for the maturation of other [Fe-S] proteins, such as aconitase. The results indicate that, in certain cases, [Fe-S] cluster biosynthetic machineries have evolved to perform only specialized functions.

Azotobacter vinelandii↗

PubMed2000

Production of B-group vitamins by two Azotobacter strains with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions.

Azotobacter vinelandii strain ATCC 12837 and A. chroococcum strain H23 (CECT 4435) were able to grow on N-free or NH4Cl-amended chemically-defined (Burk's) media, with protocatechuic acid (1-2 mmol 1(-1)) or sodium p-hydroxybenzoate (1-10 mmol 1(-1)) as sole carbon (C) sources. At a concentration of 2 mmol 1(-1), both substrates supported nitrogen fixation (acetylene reduction assay) at similar or higher rates than bacteria grown in control media amended with 2 mmol 1(-1) sodium succinate as C source. The two strains produced the B-group vitamins niacin, pantothenic acid, thiamine, riboflavin and biotin after 72 h of growth in chemically-defined media with 2 mmol 1(-1) protocatechuic acid, sodium phydroxybenzoate or sodium succinate as sole C source, either in N-free media or in media amended with 0.1% NH4Cl. Quantitative production of all vitamins was affected by the use of the different C and N substrates.

Ammonium Chloride↗

PubMed2003

Properties of polysaccharide produced by Azotobacter vinelandii cultured on 4-hydroxybenzoic acid.

AIMS: Characterization of the exopolysaccharide produced by Azotobacter vinelandii grown on 4-hydroxybenzoic acid (EPS I), and the comparison between this exopolysaccharide and commercial alginate, constituted the main objective of this work. METHODS AND RESULTS: Total carbohydrates, uronic acids, acetyl and pyruvyl groups and proteins were determined by colorimetric methods and composition was confirmed by Nuclear Magnetic Resonance studies. Rheological properties were analysed under different physical and chemical conditions. Results showed differences between EPS I and commercial alginate, in relation to both composition and viscosity. Higher amount of guluronnosyl residues were found in EPS I, whereas commercial alginate contained the same proportion of mannuronosyl and guluronnosyl residues. In accordance with this result, EPS I gave rise to solutions of higher viscosity than commercial alginate, although solutions of this polysaccharide showed greater stability when conditions were altered. CONCLUSIONS: The exopolysaccharide produced by A. vinelandii grown on 4-hydroxybenzoic acid showed a different composition in comparison with commercial alginate, which leads to higher viscosity values for the aqueous solutions of EPS I. SIGNIFICANCE AND IMPACT OF STUDY: This work describes for the first time the characteristics of an exopolysaccharide produced by A. vinelandii from 4-hydroxybenzoic acid, a substrate rarely used as sole carbon source.

Acetates↗

PubMed1998

The redox- and fixed nitrogen-responsive regulatory protein NIFL from Azotobacter vinelandii comprises discrete flavin and nucleotide-binding domains.

Azotobacter vinelandii NIFL is a nitrogen fixation-specific regulatory flavoprotein that modulates the activity of the transcriptional activator NIFA in response to oxygen and fixed nitrogen in vivo. NIFL is also responsive to ADP in vitro. Limited proteolysis of NIFL indicates that it comprises a relatively stable N-terminal domain and a C-terminal domain that is protected from trypsin digestion in the presence of adenosine nucleotides. ATP protects the protein from cleavage in the vicinity of potential nucleotide-binding sites in the C-terminus, whereas ADP protects the entire C-terminal domain. NIFL has an apparent Kd of 130 microM for ATP and 16 microM for ADP. The purified N-terminal domain has an identical UV/visible absorption spectrum to the wild-type protein and is reduced by sodium dithionite, demonstrating that it is a flavin-binding domain. The isolated N-terminal domain does not inhibit NIFA activity. A subdomain fragment containing 160 residues of the C-terminal region, including the nucleotide-binding sites, is also not competent to inhibit NIFA. Removal of the first 146 residues of NIFL, which includes a conserved S-motif (PAS-like domain), found in a large family of sensory proteins from eubacteria, archea and eukarya eliminates the redox response. However, this truncated protein remains competent to inhibit NIFA activity in response to ADP in vitro and to the level of fixed nitrogen in vivo. The redox and nitrogen-sensing functions of A. vinelandii NIFL are therefore separable and are discrete functions of the protein.

Adenosine Diphosphate↗

PubMed2003

Azotobacter vinelandii rhodanese: selenium loading and ion interaction studies.

Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [15N]Cys, and changes in 1H and 15N NMR resonances on addition of different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions. Moreover, the 1H-NMR spectrum of a defined spectral region is indicative of the status of the enzyme and can be used to directly monitor sulfur loading even at low concentrations. Selenium loading by the addition of selenodiglutathione was monitored by fluorescence and NMR spectroscopy. It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme. These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo.

Azotobacter vinelandii↗

PubMed1998

Kinetics and specificity of reductive acylation of wild-type and mutated lipoyl domains of 2-oxo-acid dehydrogenase complexes from Azotobacter vinelandii.

The kinetics and specificity of reductive acylation of lipoyl domains derived from Azotobacter vinelandii 2-oxo-acid dehydrogenase complexes, catalysed by A. vinelandii and Escherichia coli complexes, have been investigated. With the wild-type pyruvate dehydrogenase complex from A. vinelandii the rate of reductive acetylation and deacetylation was studied by rapid mixing methods. The rate of reductive acetylation, 126 s(-1), corresponds well with the turnover rate derived from steady-state measurements. Deacetylation was rapid and specific for coenzyme A. No deacetylation was observed with reduced or oxidised lipoamide or with dithiothreitol. The rate of reductive acetylation of complex-bound lipoyl domains by pyruvate dehydrogenase (E1p) is at least 60 times higher than of free lipoyl domains under comparable conditions. This gain in catalytic rate indicates a large diffusion limitation of lipoyl domains when attached via the flexible linker segments to the complex, and illustrates the efficiency of substrate channeling in the multienzyme complex. The 2-oxo-acid dehydrogenases exhibit specificity for lipoyl domains in the reductive acylation reaction. The A. vinelandii lipoyl domain derived from the pyruvate dehydrogenase complex is a good substrate for A. vinelandii E1p, but not for A. vinelandii 2-oxoglutarate dehydrogenase (E1o), and vice versa. The A. vinelandii lipoyl domain of the pyruvate dehydrogenase complex is also, although at a lower rate, reductively acetylated by E. coli E1p and reductively succinylated by E. coli E1o. Likewise, the A. vinelandii lipoyl domain derived from the 2-oxoglutarate dehydrogenase complex is recognised by E. coli E1o, but not by E. coli E1p. This suggests that common determinants of the lipoyl domains exist that are responsible for recognition by the E1 components. On the basis of the observed specificity and lipoyl domain sequences and structures, an exposed loop of the A. vinelandii 2-oxoglutarate dehydrogenase complex lipoyl domain was subjected to mutagenesis. Although the reductive acylation experiments of mutants of the lipoyl domain indicate the importance of this loop for recognition, it is probably not the single determinant for specificity.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed2000

Kinetic properties of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii evidence for the formation of a precatalytic complex with 2-oxoglutarate.

The 2-oxoglutarate dehydrogenase complex was purified from Azotobacter vinelandii. The complex consists of three components, 2-oxoglutarate dehydrogenase/decarboxylase (E1o), lipoate succinyltransferase (E2o) and lipoamide dehydrogenase (E3). Upon purification, the E3 component dissociates partially from the complex. From reconstitution experiments, the Kd for E3 was found to be 26 nM, about 30 times higher than that for the pyruvate dehydrogenase complex. The Km values for the substrates 2-oxoglutarate, CoA and NAD+ were found to be 0.15, 0.014 and 0.17 mM, respectively. The system has a high specificity for 2-oxoglutarate, which is determined by the action of both E1o and E2o. Above 4 mM substrate inhibition is observed. From steady-state inhibition experiments with substrate analogs, two substrate-binding modes are revealed at different degrees of saturation of the enzyme with 2-oxoglutarate. At low substrate concentrations (10(-6) to 10(-5) M), the binding mainly depends on the interaction of the enzyme with the substrate carboxyl groups. At a higher degree of substrate saturation (10(-4) to 10(-3) M) the relative contribution of the 2-oxo group in the binding increases. A kinetic analysis points to a single binding site for a substrate analog under steady state conditions. Saturation of this site with an analog indicates that two kinetically different complexes are formed with 2-oxoglutarate in the course of catalysis. From competition studies with analogs it is concluded that one of these complexes is formed at the site that is sterically identical to the substrate inhibition site. The data obtained are represented by a minimal scheme that considers formation of a precatalytic complex SE between the substrate and E1o before the catalytic complex ES, in which the substrate is added to the thiamin diphosphate cofactor, is formed. The incorrect orientation of the substrate molecule in SE or the occupation of this site by analogs is supposed to cause substrate or analog inhibition, respectively.

Acyltransferases↗

PubMed2004

A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii.

NifL is an antiactivator that tightly regulates transcription of genes required for nitrogen fixation in Azotobacter vinelandii by controlling the activity of its partner protein NifA, a member of the family of sigma(54)-dependent transcriptional activators. Although the C-terminal region of A. vinelandii NifL shows homology to the transmitter domains of histidine protein kinases, signal transduction between NifL and NifA is conveyed by means of protein-protein interactions rather than by phosphorylation. Binding of the ligand 2-oxoglutarate to NifA plays a crucial role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. We have used a suppressor screen to identify a critical arginine residue (R306) in NifL that is required to release NifA from inhibition under appropriate environmental conditions. Amino acid substitutions at position 306 result in constitutive inhibition of NifA activity by NifL, thus preventing nitrogen fixation. Biochemical studies with one of the mutant proteins demonstrate that the substitution alters the conformation of NifL significantly and prevents the response of NifA to 2-oxoglutarate. We propose that arginine 306 is critical for the propagation of signals perceived by A. vinelandii NifL in response to the redox and fixed-nitrogen status and is required for a conformational switch that inactivates the inhibitory function of NifL under conditions appropriate for nitrogen fixation.

Adenosine Diphosphate↗

PubMed2005

Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii.

Rnf proteins are proposed to form membrane-protein complexes involved in the reduction of target proteins such as the transcriptional regulator SoxR or the dinitrogenase reductase component of nitrogenase. In this work, we investigate the role of rnf genes in the nitrogen-fixing bacterium Azotobacter vinelandii. We show that A. vinelandii has two clusters of rnf-like genes: rnf1, whose expression is nif-regulated, and rnf2, which is expressed independently of the nitrogen source in the medium. Deletion of each of these gene clusters produces a time delay in nitrogen-fixing capacity and, consequently, in diazotrophic growth. Deltarnf mutations cause two distinguishable effects on the nitrogenase system: (i), slower nifHDK gene expression and (ii), impairment of nitrogenase function. In these mutants, dinitrogenase reductase activity is lowered, whereas dinitrogenase activity remains essentially unaltered. Further analysis indicates that deltarnf mutants accumulate an inactive and iron-deficient form of NifH because they have lower rates of incorporation of [4Fe-4S] into NifH. Deltarnf mutations also cause a noticeable decrease in aconitase activity; however, they do not produce general oxidative stress or modification of Fe metabolism in A. vinelandii. Our results suggest the existence of a redox regulatory mechanism in A. vinelandii that controls the rate of expression and maturation of nitrogenase by the activity of the Rnf protein complexes. rnf1 plays a major and more specific role in this scheme, but the additive effects of mutations in rnf1 and rnf2 indicate the existence of functional complementation between the two homologous systems.

Azotobacter vinelandii↗

PubMed2006

Phenolic lipid synthesis by type III polyketide synthases is essential for cyst formation in Azotobacter vinelandii.

Cysts of Azotobacter vinelandii are resting cells that are surrounded by a protective coat, conferring resistance to various chemical and physical agents. The major chemical components of the cyst coat are alkylresorcinols, which are amphiphilic molecules possessing an aromatic ring with a long aliphatic carbon chain. Although alkylresorcinols are widely distributed in bacteria, fungi, plants, and animals, no enzyme systems for their biosynthesis are known. We report here an ars operon in A. vinelandii that is responsible for the biosynthesis of the alkylresorcinols in the cysts. The ars operon consisted of four genes, two of which encoded a type III polyketide synthase, ArsB and ArsC. In vitro experiments revealed that ArsB and ArsC, sharing 71% amino acid sequence identity, were an alkylresorcinol synthase and an alkylpyrone synthase, respectively, indicating that ArsB and ArsC are not isozymes but enzymatically distinct polyketide synthases. In addition, ArsB and ArsC accepted several acyl-CoAs with various lengths of the side chain as a starter substrate and gave corresponding alkylresorcinols and alkylpyrones, respectively, which suggests that the mode of the ring folding is uninfluenced by the structure of the starter substrates. The importance of the alkylresorcinols for encystment was confirmed by gene inactivation experiments; the lack of alkylresorcinols synthesis caused by ars mutations resulted in the formation of severely impaired cysts, as observed by electron microscopy.

Azotobacter vinelandii↗

PubMed2006

Flavodoxin hydroquinone reduces Azotobacter vinelandii Fe protein to the all-ferrous redox state with a S = 0 spin state.

Azotobacter vinelandii flavodoxin hydroquinone (FldHQ) is a physiological reductant to nitrogenase supporting catalysis that is twice as energy efficient (ATP/2e- = 2) as dithionite (ATP/2e- = 4). This catalytic efficiency results from reduction of Fe protein from A. vinelandii (Av2) to the all-ferrous oxidation state ([Fe4S4]0), in contrast to dithionite, which only reduces Av2 to the [Fe4S4]1+ state. Like FldHQ, Ti(III) citrate yields ATP/2e- = 2, and Ti(III)-reduced [Fe4S4]0 Av2 has a S = 4 spin state and characteristic Mossbauer spectrum, a parallel mode g = 16.4 EPR signal, and a shoulder at 520 nm in its UV-vis spectrum, each of which distinguish the S = 4 [Fe4S4]0 Av2 from other states. In this study, we demonstrate that FldHQ makes [Fe4S4]0 Av2, which is sufficiently characterized to demonstrate unique physical properties that distinguish it from the previously characterized Ti(III)-reduced [Fe4S4]0 Av2. In particular, Evans NMR magnetic susceptibility and EPR measurements indicate that FldHQ-reduced [Fe4S4]0 Av2 has an S = 0 spin state (like [Fe4S4]2+ Av2). There is no g = 16.4 EPR signal and no shoulder at 520 nm in its absorbance spectrum, which resembles that of [Fe4S4]1+ Av2. That the physiological reductant to Av2 is capable of forming [Fe4S4]0 Av2 has important implications for in vivo nitrogenase activity.

Azotobacter vinelandii↗

PubMed1970

Nitrogenaseless mutants of Azotobacter vinelandii.

Mutants of Azotobacter which grow normally on excess ammonia under a variety of conditions and which grow slowly or not at all on atmospheric nitrogen have been isolated. Extracts of these strains have low or no detectable nitrogenase activity. There are three classes of mutants. Cell-free preparations of members of the first class possess an enhancement factor (EF+) which stimulates wild-type nitrogenase in vitro. Homogenates of members of the second class possess an enhanceable factor (EF-) which complements in vitro with extracts of the first class of mutants to give substantial nitrogenase activity. Preparations of members of the third class contain neither EF+ nor EF- activity. EF+ and EF- are repressed by the same conditions that repress nitrogenase. Molybdenum-deficient cells of the second class of mutants do not appear to contain EF- activity, but molybdenum deficient cells of the first class of mutants contain EF+. Because of these observations, EF+ is tentatively equated to azoferredoxin and EF- to molybdoferredoxin.

Azotobacter↗

PubMed1972

Nature of oxygen inhibition of nitrogenase from Azotobacter vinelandii.

The reduction of nitrogen, acetylene, azide, and cyanide at various oxygen concentrations by nitrogenase from Azotobacter vinelandii was measured with a well-defined system. Oxygen inhibited the reduction of each substrate uncompetitively. The inhibition constants (K(i)) were 0.014, 0.023, 0.008, and 0.003 atm of oxygen for reduction of nitrogen, acetylene, azide, and cyanide, respectively. The system used included ATP-generating components, subcellular particles from A. vinelandii with high nitrogenase specific activity, and illuminated spinach chloroplasts plus carriers to supply electrons. Oxygen did not affect the photochemical electron donating system, but it did inhibit nitrogenase-dependent ATP hydrolysis.

Acetylene↗

PubMed1980

Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii.

Two Azotobacter vinelandii strains capable of growing on N2(Nif+) were isolated from two different mutant strains that lacked dinitrogenase activity (Nif-). Extracts of N2-grown cells of the two Nif+ strains lacked significant amounts of the "conventional" dinitrogenase protein subunits, as determined by two-dimensional gel electrophoresis. Instead, the extracts contained at least four new proteins that appeared to be ammonia-repressible (i.e., they were not detected in extracts of ammonia-grown cells). Based on the results of genetic backcrosses, the two Nif+ strains were shown to be pseudorevertants. Both Nif+ pseudorevertant strains were able to grow in N-free media lacking molybdenum but containing tungsten (conditions that prevented growth of the wild-type strain). The four new proteins were observed in extracts of N2-fixing cells of the Nif+ pseudorevertants regardless of whether the cells were grown in the presence of molybdenum-starved wild-type A. vinelandii cells grown under N2-fixing conditions. Under conditions of molybdenum deprivation, Nif- mutant strains of several different phenotypic classes underwent phenotypic reversal to Nif+, as shown by their ability to incorporate 15N2 and to grow in N-free media. These results provide evidence that A. vinelandii possesses an alternative N2-fixation system that is expressed during conditions of molybdenum deficiency.

Azotobacter↗

PubMed1980

Comparison of the iron proteins from the nitrogen fixation complexes of Azotobacter vinelandii, Clostridium pasteurianum, and Klebsiella pneumoniae.

The molecular weights, amino acid compositions, amino- and carboxyl-terminal sequences, and ion-exchange peptide maps of the cysteine-containing tryptic peptides were determined for the iron proteins from the nitrogen fixation complexes of Azotobacter vinelandii (Av2) and Klebsiella pneumoniae (Kp2). Our results are compared to the known amino acid sequence of the iron protein from Clostridium pasteurianum (Cp2) [Tanaka, M., Haniu, M., Yasunobu, K. & Mortenson, L. E. (1977) J. Biol. Chem. 252, 7093-7100]. Previous studies have shown the iron proteins to have similar enzymatic functions and spectroscopic properties. Furthermore, the DNAs coding for the iron protein from many different species cross-hybridize [Ruvkun, G. B. & Ausubel, F. M. (1980) Proc. Natl. Acad. Sci. USA 77, 191-195]. Our results indicate that the protein structures are similar yet have significant differences. The amino-terminal sequences of Av2 and Kp2 are extended compared to the amino-terminal methionine of Cp2 and may indicate a different initiation site in these proteins. The aminoterminal sequences for Av2 and Kp2 are more homologous with each other than either of these are with Cp2. The carboxyl-terminal sequences are extended in Av2(14 residues) and Kp2 ( approximately 30 residues) compared to Cp2. The amino- and carboxyl-terminal sequences establish that either the structural gene sizes are different in the three organisms or extensive posttranslational modification must occur in some species. Because cysteinyl residues are involved at the active site of the iron protein, a sensitive peptide mapping technique was used to compare cysteinyl peptides of the iron protein from the three species. Av2 and Kp2 have a redistribution of cysteinyl residues when compared to Cp2. Three important differences in the cysteine distributions were found, namely, residue 4 is valine and residue 148 is alanine in Cp2, but cysteinyl residues occupy these positions in Av2, whereas residue 231 is cysteine in Cp2 but alanine in Av2. The peptide mapping technique provides a method for the investigation of selective chemical modification of cysteinyl residues.

Amino Acid Sequence↗

PubMed1982

Crystallographic properties of the MoFe proteins of nitrogenase from Clostridium pasteurianum and Azotobacter vinelandii.

Preliminary x-ray diffraction data from single crystals of the MoFe proteins of nitrogenase from Clostridium pasteurianum and Azotobacter vinelandii have been obtained. Both protein crystals belong to the P2(1) space group. The MoFe protein crystals from C. pasteurianum and A. vinelandii diffract to angles corresponding to resolutions as great as 2.4 A and 3.0 A, respectively. The cell dimensions of the MoFe protein crystals from the two species have been determined from precession photographs.

Azotobacter↗

PubMed1984

H2-uptake activity of the MoFe protein component of Azotobacter vinelandii nitrogenase.

The MoFe protein from Azotobacter vinelandii catalyzes the reduction of methylene blue and other oxidants by H2 under anaerobic conditions. H2 uptake followed manometrically or by 3H2 transfer from the gas to aqueous phase occurs concomitantly with methylene blue disappearance monitored optically or coulometrically. The stoichiometry was found to be 1:1 methylene blue/H2. MoFe protein oxidized by transfer of approximately 4 e- seems to be the redox state of the protein most active in the catalytic step, although both the S2O4(2-)-reduced and 6-e- oxidized state have been shown to react, but at a much lower rate. The presence of H2 in the atmosphere above the MoFe protein offers increased protection against O2 inactivation.

Azotobacter↗