Characterization of genes involved in molybdenum transport in Azotobacter vinelandii

Expression of alternative nitrogenases in Azotobacter vinelandii is repressed by molybdenum. Two strains with Tn5 insertion mutations showed alternative nitrogenase-dependent diazotrophic growth in the presence of Mo. The mutations were in a region which contained four open reading frames (ORFs 1-4). The genetic structure and predicted products of ORFs 2, 3 and 4 are typical of the membrane-associated elements of the ATP-binding cassette (ABC) superfamily of transport systems. The products of ORF3 and ORF4 are homologous with the products of the Escherichia coli genes chlD and the partially sequenced chlJ, respectively, both of which are implicated in molybdenum transport. ORF1, which is in the relative position of bacterial permease genes commonly specifying periplasmic binding proteins, encodes a 29 kDa protein with a novel primary structure. It lacks a potential signal sequence, and its C-terminal half consists of a tandem repeat of a segment which is homologous with the M(r) 7 kDa molybdenum-pterin binding protein Mop from Clostridium pasteurianum. This suggests that a substituted pterin may be involved in the initial capture or early metabolism of molybdenum.     

Alginate production by Azotobacter vinelandii

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.     

Characterization of a spontaneous mutant of Azotobacter vinelandii in which vanadium-dependent nitrogen fixation is not inhibited by molybdenum

Neopterin is a sensitive indicator for cellular immune activation. Its concentrations were determined in cerebrospinal fluid (CSF) and serum specimens from 91 children with no evidence of central nervous system (CNS) or peripheral inflammations, 43 with definite neuroborreliosis, 51 with other CNS infections, and 33 with peripheral infections. The aim of our study was (a) to establish a range of normal CSF neopterin concentrations in control children, and (b) to inquire into the diagnostic potential of neopterin measurements in both body compartments for aiding in differential diagnosis of inflammatory vs noninflammatory diseases, and CNS vs peripheral inflammations. CSF neopterin concentrations in controls were invariably low (up to 9.3 nmol/L), but in children with neuroborreliosis and, even more so, with other CNS infections neopterin concentrations were significantly (P <0.0001) increased. Children with peripheral infections, however, rarely showed raised CSF neopterin concentrations. Serum concentrations of neopterin, on the other hand, were not significantly different between controls and children with neuroborreliosis. Although serum concentrations were significantly different between controls and children with other CNS infections, diagnostic efficiency was poor for this comparison. Peripheral infections, in contrast, were associated with significantly higher (P <0.0001) serum neopterin concentrations when compared with controls. A classification tree was constructed on the basis of CSF and serum neopterin concentrations, allowing with high accuracy the discrimination between controls, children with CNS infections, and children with peripheral infections. Thus, on the basis of a comparatively large control group, our data underline the diagnostic validity of neopterin as an aid in differential diagnosis of inflammatory vs noninflammatory diseases, and confirm that CSF neopterin concentrations are not correlated with serum neopterin concentrations, and, therefore, CSF neopterin appears to be produced intrathecally.     

Characterization and mutagenesis of the leucine biosynthetic genes of Azotobacter vinelandii: an analysis of the rarity of amino acid auxotrophs

The Wechsler Intelligence Scale for Children-Revised (WISC-R) was applied (in a Swedish version) in 120 children with Asperger syndrome, autistic disorder, and attention disorders. Using stepwise logistic regression analysis, the WISC's discriminating ability was investigated. The overall rate of correct diagnostic classification was 63%. Further, WISC profiles were analysed within each group. The group with autistic disorder was characterised by a peak on Block Design. The Asperger syndrome group had good verbal ability and troughs on Object Assembly and Coding. The group with attention disorders had troughs on Coding and Arithmetic. The results suggest that Kaufman's Verbal Comprehension, Perceptual Organisation and Freedom from Distractibility factors rather than verbal IQ and performance IQ account for the variance on the WISC. Furthermore, the Asperger syndrome and autistic disorder groups differed in respect of "fluid" and "crystallised" cognitive ability.     

Inhibition of Azotobacter vinelandii RNA polymerase by cibacron blue F3GA

Cibacron blue F3GA is a potent inhibitor of the Azotobacter vinelandii DNA-directed RNA polymerase. Addition of 8 micrometer Cibacron blue F3GA prior to initiation results in a greater than 90% inhibition of the poly[d(A-T]-directed synthesis of poly[r(A-U)] while addition of the dye during the course of the reaction is without effect on chain elongation. Binding of RNA polymerase to [3H]poly[d(A-T)] is inhibited by only 15% in the presence of 8 micrometer Cibacron blue F3GA. Inhibition by Cibacron blue F3GA is noncompetitive with regard to ATP, UTP, or template. The poly[d(A-T)]-directed pyrophosphate exchange reaction is relatively resistant to inhibition by Cibacron blue F3GA. Rifampicin added to a similar reaction (in the presence of absence of Cibacron blue F3GA) results in 95% inhibition of the exchange reaction. The interaction of the RNA polymerase core enzyme with Cibacron blue F3GA is shown by the formation of a difference spectrum with a positive maximum at 675 nm which is not affected by the presence of a high concentration (4 micrometer) of rafampicin. The data indicate that Cibacron blue F3GA acts by binding to RNA polymerase and inhibits a step between the synthesis of the initial phosphodiester bond and formation of a stable ternary elongation complex.     

Characterization of VNFG, the delta subunit of the vnf-encoded apodinitrogenase from Azotobacter vinelandii. Implications for its role in the formation of functional dinitrogenase 2

The vnf-encoded apodinitrogenase (apodinitrogenase 2) from Azotobacter vinelandii is an alpha2beta2delta2 hexamer. The delta subunit (the VNFG protein) has been characterized in order to further delineate its function in the nitrogenase 2 enzyme system. Two species of VNFG were observed in cell-free extracts resolved on anoxic native gels; one is composed of VNFG associated with the VNFDK polypeptides, and the other is a homodimer of the VNFG protein. Both species of VNFG are observed in extracts of A. vinelandii strains that accumulate dinitrogenase 2, whereas extracts of strains impaired in the biosynthetic pathway of the iron-vanadium cofactor (FeV-co) that accumulate apodinitrogenase 2 (a catalytically inactive form of dinitrogenase 2 that lacks FeV-co) exhibit only the VNFG dimer on native gels. FeV-co and nucleotide are required for the stable association of VNFG with the VNFDK polypeptides; this stable association can be correlated with the formation of active dinitrogenase 2. The iron-molybdenum cofactor was unable to replace FeV-co in promoting the stable association of VNFG with VNFDK. FeV-co specifically associates with the VNFG dimer in vitro to form a complex of unknown stoichiometry; combination of this VNFG-FeV-co species with apodinitrogenase 2 results in its reconstitution to dinitrogenase 2. The results presented here suggest that VNFG is required for processing apodinitrogenase 2 to functional dinitrogenase 2.     

Complex formation between Azotobacter vinelandii ferredoxin I and its physiological electron donor NADPH-ferredoxin reductase

In Azotobacter vinelandii, deletion of the fdxA gene, which encodes ferredoxin I (FdI), leads to activation of the expression of the fpr gene, which encodes NADPH-ferredoxin reductase (FPR). In order to investigate the relationship of these two proteins further, the interactions of the two purified proteins have been examined. AvFdI forms a specific 1:1 cross-linked complex with AvFPR through ionic interactions formed between the Lys residues of FPR and Asp/Glu residues of FdI. The Lys in FPR has been identified as Lys258, a residue that forms a salt bridge with one of the phosphate oxygens of FAD in the absence of FdI. UV-Vis and circular dichroism data show that on binding FdI, the spectrum of the FPR flavin is hyperchromatic and red-shifted, confirming the interaction region close to the FAD. Cytochrome c reductase assays and electron paramagnetic resonance data show that electron transfer between the two proteins is pH-dependent and that the [3Fe-4S]+ cluster of FdI is specifically reduced by NADPH via FPR, suggesting that the [3Fe-4S] cluster is near FAD in the complex. To further investigate the FPR:FdI interaction, the electrostatic potentials for each protein were calculated. Strongly negative regions around the [3Fe-4S] cluster of FdI are electrostatically complementary with a strongly positive region overlaying the FAD of FPR, centered on Lys258. These proposed interactions of FdI with FPR are consistent with cross-linking, peptide mapping, spectroscopic, and electron transfer data and strongly support the suggestion that the two proteins are physiological redox partners.     

The Azotobacter vinelandii mannuronan C-5-epimerase AlgE1 consists of two separate catalytic domains

The Azotobacter vinelandii enzyme AlgE1 is a member of a family of secreted mannuronan C-5-epimerases. These enzymes convert beta-D-mannuronic acid residues (M) to alpha-L-guluronic acid residues (G) at the polymer level in the industrially important polysaccharide alginate, leading to altered physical and immunological properties of the polymer. The reaction product of AlgE1 was found to be a mixture of blocks of continuous G residues (G-blocks) and blocks containing alternating M and G residues (MG-blocks). The enzyme is dependent on Ca2+ for activity, and only Sr2+ of those tested was able to replace Ca2+. Zn2+ blocked the activity even at low concentrations. algE1 has been divided into two parts based on the modular type of structure previously reported to be a characteristic of the secreted epimerases, and each part has been expressed in Escherichia coli. These experiments showed that AlgE1 contains two catalytic domains, AlgE1-1, which introduces both G-blocks and MG-blocks, and AlgE1-2, which only introduces MG-blocks. AlgE1-1 has a much lower specific activity than both AlgE1-2 and AlgE1. However, the two halves of AlgE1 seem to cooperate in such a way that they contribute approximately equally to the overall epimerization reaction.     

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.     

The Azotobacter vinelandii NifEN complex contains two identical [4Fe-4S] clusters

The nifE and nifN gene products from Azotobacter vinelandii form an alpha2beta2 tetramer (NifEN complex) that is required for the biosynthesis of the nitrogenase FeMo cofactor. In the current model for NifEN complex organization and function, the complex is structurally analogous to the nitrogenase MoFe protein and provides an assembly site for a portion of FeMo cofactor biosynthesis. In this work, gene fusion and immobilized metal-affinity chromatography strategies were used to elevate the in vivo production of the NifEN complex and to facilitate its rapid and efficient purification. The NifEN complex produced and purified in this way exhibits an FeMo cofactor biosynthetic activity similar to that previously described for the NifEN complex purified by traditional chromatography methods. UV-visible, EPR, variable-temperature magnetic circular dichroism, and resonance Raman spectroscopies were used to show that the NifEN complex contains two identical [4Fe-4S]2+ clusters. These clusters have a predominantly S = 1/2 ground state in the reduced form, exhibit a reduction potential of -350 mV, and are likely to be coordinated entirely by cysteinyl residues on the basis of spectroscopic properties and sequence comparisons. A model is proposed where each NifEN complex [4Fe-4S] cluster is bridged between a NifE-NifN subunit interface at a position analogous to that occupied by the P clusters in the nitrogenase MoFe protein. In contrast to the MoFe protein P clusters, the NifEN complex [4Fe-4S] clusters are proposed to be asymmetrically coordinated to the NifEN complex where NifE cysteines-37, -62, and -124 and NifN cysteine-44 are the coordinating ligands. On the basis of a homology model of the three-dimensional structure of the NifEN complex, the [4Fe-4S] cluster sites are likely to be remote from the proposed FeMo cofactor assembly site and are unlikely to become incorporated into the FeMo cofactor during its assembly.     

Catalytic and biophysical properties of a nitrogenase Apo-MoFe protein produced by a nifB-deletion mutant of Azotobacter vinelandii

A Zn-immobilized metal-affinity chromatography technique was used to purify a poly-histidine-tagged, FeMo-cofactorless MoFe protein (apo-MoFe protein) from a nifB-deletion mutant of Azotobacter vinelandii. Apo-MoFe protein prepared in this way was obtained in sufficient concentrations for detailed catalytic, kinetic, and spectroscopic analyses. Metal analysis and electron paramagnetic resonance spectroscopy (EPR) were used to show that the apo-MoFe protein does not contain FeMo-cofactor. The EPR of the as-isolated apo-MoFe protein is featureless except for a minor S = 1/2 signal probably arising from the presence of either a damaged P cluster or a P cluster precursor. The apo-MoFe protein has an alpha2beta2 subunit composition and can be activated to 80% of the theoretical MoFe protein value by the addition of isolated FeMo-cofactor. Oxidation of the as-isolated apo-MoFe protein by indigodisulfonate was used to elicit the parallel mode EPR signal indicative of the two-electron oxidized form of the P cluster (P2+). The midpoint potential of the PN/P2+ redox couple for the apo-MoFe protein was shown to be shifted by -63 mV when compared to the same redox couple for the intact MoFe protein. Although the apo-MoFe protein is not able to catalyze the reduction of substrates under turnover conditions, it does support the hydrolysis of MgATP at 60% of the rate supported by the MoFe protein when incubated in the presence of Fe protein. The ability of the apo-MoFe protein to specifically interact with the Fe protein was also shown by stopped-flow techniques and by formation of an apo-MoFe protein-Fe protein complex. Finally, the two-electron oxidized form of the apo-MoFe protein could be reduced to the one-electron oxidized form (P1+) in a reaction that required Fe protein and MgATP. These results are interpreted to indicate that the apo-MoFe protein produced in a nifB-deficient genetic background [corrected] contains intact P clusters and P cluster polypeptide environments. Small changes in the electronic properties of P clusters contained within the apo-MoFe protein are most likely caused by slight perturbations in their polypeptide environments.     

Hydrodynamic properties of nitrogenase--the MoFe protein from Azotobacter vinelandii studied by dynamic light scattering and hydrodynamic modelling

Trichloroethylene (TRI) is an industrial solvent with a history of use in anesthesia, and is a common groundwater contaminant. Cytochrome P450 (CYP)-dependent metabolism of TRI produces chloral hydrate (CH) and is rate limiting in the ultimate production of trichloro- and/or dichloroacetic acid from TRI. Exposure of rodents to TRI results in lung and liver tumors (mice) and nephrotoxicity (rats). The toxicity is exacerbated by pretreatment of mice with CYP inducers. We report significant variability in TRI metabolism in a sample of 23 human hepatic microsomal samples and demonstrate the dependence of TRI metabolism on CYP2E1. K(m) values in this limited sample population are not normally distributed. We have correlated microsomal CH formation with the activity toward routine CYP2E1 substrates and with immunologically detectable CYP2E1 protein. Further, TRI metabolism in microsomes from lymphoblastoid cell lines expressing CYP2E1, CYP1A1, CYP1A2, or CYP3A4 indicated minimal involvement of the latter forms, with CYP2E1 catalyzing more than 60% of total microsomal TRI metabolism. These results indicate that humans are not uniform in their capacity for CYP-dependent metabolism of TRI and increased CYP2E1 activity may increase susceptibility to TRI-induced toxicity in the human.     

Purification and characterization of a NADP+/NADPH-specific flavoprotein that is overexpressed in FdI- strains of Azotobacter vinelandii

Earlier studies have established that mutant strains of Azotobacter vinelandii that do not synthesize ferredoxin I (AvFdI) overexpress another protein designated Protein X (Morgan, T. V., Lundell, P. J., and Burgess, B. K. (1988) J. Biol. Chem. 263, 1370-1375). This protein has now been purified using two-dimensional gel electrophoresis as an assay. The purified protein is a monomer with M(r) approximately 29,000 which degrades slowly to a specific M(r) approximately 22,000 form when stored in solution. The native protein is bright yellow and contains noncovalently attached FAD that is reduced by either dithionite or NADPH without formation of a stable semiquinone. Titration with NADP+/NADPH gives an E0' value of approximately -327 mV versus SHE. Because this E0' is so close to that of the NADP+/NADPH couple it is not clear if Protein X is an NADPH oxidase or an NADP+ reductase in vivo. Comparison of the NH2-terminal sequence and other properties of Protein X with those of other proteins, suggests that it is likely to be related to the Escherichia coli ferredoxin NADP+ reductase (the fpr gene product), and affinity chromatography shows that Protein X binds specifically to AvFdI.     

The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii

vnfG and anfG encode the delta subunits of alternative nitrogenases 2 and 3 in Azotobacter vinelandii, respectively. As a first step towards elucidating the role of these subunits, diazotrophic growth and acetylene reduction studies were conducted on mutants containing alterations in the genes encoding these subunits. Mutants containing a stop codon (C36stop) or an in-frame deletion in anfG were unable to grow in N-free, Mo-deficient medium (Anf-). Mutants in which cysteine 36 of AnfG (a residue conserved between VnfG and AnfG) was changed to Ala or Ser were Anf+. Thus, this conserved cysteine is not essential for the function of AnfG in dinitrogenase 3. A mutant with a stop codon in vnfG (C17stop) grew after a lag of 25 h in N-free, Mo-deficient medium containing V2O5. However, a Nif- Anf- strain with this mutation was unable to grow under these conditions. This shows that the vnfG gene product is required for nitrogenase 2-dependent growth. Strains with mutations in vnfG and anfG reduced acetylene to different degrees. This indicates that the delta subunits are not required for acetylene reduction by nitrogenases 2 and 3.     

Evidence for multiple substrate-reduction sites and distinct inhibitor-binding sites from an altered Azotobacter vinelandii nitrogenase MoFe protein

The arginine-277 residue of the alpha-subunit of the nitrogenase MoFe protein was targeted for substitution because it is (i) a close neighbor of alpha-cysteine-275, which is one of only two residues anchoring the FeMo cofactor to the polypeptide, and (ii) a component of a potential channel for entry/exit of substrates/products and for accepting FeMo cofactor during MoFe-protein maturation. Several of the eight mutant strains constructed were capable of good diazotrophic growth and also contained FeMo cofactor as indicated by its biologically unique S = 3/2 EPR spectrum. These observations indicate that the positively charged alpha-arginine-277 residue is not required for acceptance of the negatively charged FeMo cofactor by the separately synthesized, cofactor-deficient, apo-MoFe protein. The wide range of nitrogen-fixation phenotypes shown by these mutant strains generally correlated well with their C2H2- and proton-reduction activities, which range from 5 to 65% of wild-type activity. One notable exception is the histidine-substituted strain, DJ788 (alpha-277His). This strain, although unable to fix N2 and grow diazotrophically, elaborates an altered alpha-277His MoFe protein that catalyzes the reduction of the alternative substrates, C2H2, HCN, HN3, and protons. These observations are best explained if multiple redox levels are available to the MoFe protein but the alpha-277His MoFe protein is incapable of reaching the more-reduced redox levels required for nitrogen fixation. Under nonsaturating CO concentrations, the alpha-277His MoFe-protein-catalyzed reduction of C2H2 showed sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity among two C2H4-evolving sites and indicates the presence of three sites, which can be simultaneously occupied, on the MoFe protein. Similar kinetics were not observed for alpha-277His MoFe-protein-catalyzed reduction of either HCN or HN3 with nonsaturating CO levels, indicating that these substrates are unlikely to share common binding sites with C2H2. Further, CN- did not induce cooperativity in C2H2 reduction and, therefore, CO and CN- are unlikely to share a common binding site. These changed substrate specificities, reinforced by changes in the FeMo-cofactor-derived S = 3/2 EPR spectrum, clearly indicate the importance of the alpha-277 residue in catalysis and the delicate control exerted on the properties of bound FeMo cofactor by its polypeptide environment.     

Chemical modification by trinitrobenzenesulfonate of a lipid and proteins of intracytoplasmic membranes isolated from Chromatium vinosum and Azotobacter vinelandii

1. The structure of intracytoplasmic membranes of a photosynthetic bacterium chromatium vinosum and a nitrogen-fixing bacterium Azotobacter vinelandii was studied by chemical modification of amino groups of phosphatidylethanolamine and proteins with trinitrobenzenesulfonate. 2. Almost all the constituents of intracytoplasmic membranes of C. vinosum were solubilized in a mixture of chloroform, methanol and trichloroacetic acid. One-third of proteins in the intracytoplasmic membranes of C. vinosum was found solubilized in a mixture of chloroform and methanol. By using a column chromatography with Sephadex LH-20 in organic solvents, the unmodified as well as the trinitrophenylated proteins and also the trinitrophenylated phosphatidylethanolamine were separated from the other colored substances. 3. In the chemical modification of the intracytoplasmic membrane preparations, 30% of phosphatidylethanolamine and 15% of protein amino groups in C. vinosum and 45% of phosphatidylethanolamine and 20% of protein amino groups in A. vinelandii were estimated to be exposed to the aqueous phase. In the single-layered liposomes composed of phosphatidylethanolamine and phosphatidylglycerol with a ratio of 2:1, 40% of phosphatidylethanolamine were estimated to be exposed to the aqueous phase.     

Pyridine nucleotide transhydrogenase from Azotobacter vinelandii. Improved purification, physical properties and subunit arrangement in purified polymers

1. Pyridine nucleotide transhydrogenase from Azotobacter vinelandii was purified with a scaled-up procedure. In a typical purification 500 ml cell-free extract from 200 g cells is loaded on an Ado-2',5'-P2--Sepharose 4B affinity column (20 ml bed volume). After washing, the enzyme is desorbed with 2'AMP at neutral pH and further purified by Sephadex G-200 gel chromatography. The enzyme (10--12 mg) is obtained in 40--60% yield and is homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The homogeneity of the purified enzyme is also apparent from electron microscopy studies, where the enzyme appears as a polydisperse set of polymers without contaminating structures and from fluorescence lifetime studies by the method of single-photon counting. The flavin fluorescence appears to decay with a single lifetime tau = 2.5 ns. The polymeric nature of transhydrogenase can be aptly demonstrated by density gradient centrifugation in the presence of KBr. After centrifuging for 50 h at 160 000 X g and 10 degrees C the enzyme is concentrated in a narrow fluorescent band with buoyant density rho b = 1.305 g cm-3. 3. The arrangement of subunits in the transhydrogenase polymer has been derived from optical diffraction studies of electron micrographs. The polymers are built up from a linear assembly of tetramers. Four subunits are placed in a rhomb with sides of 13.5 mm and an angle of 45 degrees (135 degrees) between the sides. A second tetramer is located staggered on top of the first one. Since a variety of other studies have indicated that the polymers dissociate into octamers under alkaline conditions [Voordouw, G. et al. (1979 Eur. J. Biochem. 98,447--454] we conclude that this smallest functional unit is build up from two tetramers.