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PubMed1996

Comparison of isotope exchange, H2 evolution, and H2 oxidation activities of Azotobacter vinelandii hydrogenase.

Azotobacter vinelandii hydrogenase was purified aerobically with a 35% yield. The purified enzyme catalyzed H2 oxidation at much greater velocity than H2 evolution. There was a large difference in activation energy for the two reactions. EA was 10 kcal/mol for H2 oxidation and 22 kcal/mol for evolution. This difference in activation energies between the two reactions means that the ratio of oxidation velocity to evolution velocity drops from 70 at 33 degrees C to 8 at 48 degrees C. With D2 and H2O as substrates, both membranes and purified enzyme produced only H2 and no HD in the isotope exchange reaction. The velocity of isotope exchange was equal to the velocity of H2 evolution from reduced methyl viologen, indicating that the two reactions share the same rate-limiting step. D2 and H2 inhibited H2 evolution, but D2 did not inhibit isotope exchange. We conclude that H2 and D2 do not inhibit H2 evolution by competing with H+ for the active site of the reduced enzyme. The Km for D2 in isotope exchange is 40-times greater than its Km in D2 oxidation. The difference in Km cannot be accounted for by differences in kcat. We propose that redox environment regulates hydrogenase's affinity for D2 (and likely H2 as well).

Aerobiosis↗
PubMed1996

Posttranslational regulation of nitrogenase activity by fixed nitrogen in Azotobacter chroococcum.

Using anti-(Fe protein) antibody raised against the Fe protein of the photosynthetic bacterium Rhodospirillum rubrum, it was found that the Fe protein component of nitrogenase (EC 1.18.2.1) from Azotobacter chroococcum cells subjected to an ammonium shock, and hence with an inactive nitrogenase, appeared as a doublet in Western blot analysis of cell extracts. The Fe protein incorporated [32P]phosphate and [3H]adenine in response to ammonium treatment, and L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase (L-glutamate: ammonia ligase (ADP forming), EC 6.3.1.2), prevented Fe protein from inhibition and radioisotope labelling. These results support that A. chroococcum Fe protein is most likely ADP-ribosylated in response to ammonium. After ammonium treatment, when in vivo activity was completely inhibited, Fe-protein modification was still increasing. This suggests the existence of another mechanism of nitrogenase inhibition faster than Fe-protein modification. When ammonium was intracellularly generated instead of being externally added, as occurs with the short-term nitrate inhibition of nitrogenase activity observed in A. chroococcum cells simultaneously fixing molecular nitrogen and assimilating nitrate, a covalent modification of the Fe protein was likewise demonstrated.

Adenosine Diphosphate Ribose↗
PubMed1989

Evidence for a dissimilatory plasmid in Azotobacter chroococcum.

Dissimilation of 2,4-dichlorophenoxyacetic acid in Azotobacter chroococcum is plasmid mediated. This dissimilatory plasmid designated pMSB1, was effectively cured by mitomycin C. The plasmid was transferred to another strain of Az. chroococcum at a frequency of 2.4 x 10(-3) transconjugants/donor cell. The cured cells did not utilize 2,4-D and its intermediates and lacked the plasmid DNA.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1992

Characterization of two genes (hupD and hupE) required for hydrogenase activity in Azotobacter chroococcum.

In Azotobacter chroococcum the hydrogenase structural genes (hupSL) cover about 2.8 kb of a 15-kb region associated with hydrogen-uptake (Hup) activity. Two other genes in this region, hupD and hupE, were located 8.9 kb downstream of hupL and were shown to be essential for hydrogenase activity by insertion mutagenesis. A fragment of DNA beginning 3.4 kb downstream of hupL was able to complement the hupE mutant, supporting earlier evidence for a promoter downstream of hupSL. Hybridization experiments showed that hupD and hupE share some similarity with a region of Alcaligenes eutrophus DNA which is apparently involved in the formation of catalytically active hydrogenase. The hupD gene encodes a 379-amino acid, 41.4-kDa polypeptide while hupE codes for a 341-amino acid, 36.1-kDa product. The predicted amino acid sequences of the hupD and hupE genes are homologous to the Escherichia coli hypD and hypE gene products, respectively. A polar mutation in hupD had no effect on beta-galactosidase activity in a strain also carrying a hupL-lacZ fusion, indicating that hupD and hupE are probably not involved in regulating hydrogenase structural gene expression.

Amino Acid Sequence↗
PubMed1985

Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster.

DNA fragments coding for the structural genes for Azotobacter vinelandii nitrogenase have been isolated and sequenced. These genes, nifH, nifD and nifK, code for the iron (Fe) protein and the alpha and beta subunits of the molybdenum-iron (MoFe) protein, respectively. They are arranged in the order: promoter:nifH:nifD:nifK. There are 129 nucleotides separating nifH and nifD and 101 nucleotides separating nifD and nifK. The amino acid (aa) sequences deduced from the nucleotide sequences are discussed in relation to the prosthetic group-binding regions of the nifHDK-encoded polypeptides.

Amino Acid Sequence↗
PubMed1990

Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii.

The Azotobacter vinelandii [NiFe]hydrogenase-encoding structural genes were isolated from an A. vinelandii genomic cosmid library. Nucleotide (nt) sequence analysis showed that the two genes, hoxK and hoxG, which encode the small and large subunits of the enzyme, respectively, form part of an operon that contains at least one other gene. The hoxK gene encodes a polypeptide of 358 amino acids (aa) (39,209 Da). The deduced aa sequence encodes a possible 45-aa N-terminus extension, not present in the purified A. vinelandii hydrogenase small subunit, which could be a cellular targeting sequence. The hoxG gene is downstream form, and overlaps hoxK by 4 nt and encodes a 602-aa polypeptide of 66,803 Da. The hoxK and hoxG gene products display homology to aa sequences of hydrogenase small and large subunits, respectively, from other organisms. The hoxG gene lies 16 nt upstream from a third open reading frame which could encode a 27,729-Da (240-aa) hydrophobic polypeptide containing 53% nonpolar and 11% aromatic aa. The significance of this possible third gene is not known at present.

Amino Acid Sequence↗
PubMed1992

The Azotobacter vinelandii recA gene: sequence analysis and regulation of expression.

The nucleotide (nt) sequence of the Azotobacter vinelandii recA gene (Av-recA) was determined and compared with the recA sequences from Pseudomonas aeruginosa (Pa-recA), a soil bacterium, and Escherichia coli (Ec-recA), an enteric bacterium. The Av-recA gene and the deduced aa sequence were found to be more similar to their Pa-recA counterparts than to the Ec-recA gene and protein. Expression of Av-recA was found to be autoregulatory. Unlike Ec-recA and Pa-recA, however, expression of Av-recA was weakly enhanced upon DNA damage. In E. coli, expression of an Av-recA::lacZ fusion was poor, but its autoregulation was similar to that of Ec-recA. Av-recA expression, however, could not induce the repair system response in E. coli.

Amino Acid Sequence↗
PubMed1993

The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hupA, hupB, hupY and hupC.

The Azotobacter chroococcum chromosome contains a region spanning about 14 kb associated with hydrogen-uptake (Hup) activity. The small and large subunits of the hydrogenase are encoded by the structural genes hupS and hupL. Two other genes, hupD and hupE, are located 8.9 kb downstream from hupL and are required for the formation of a catalytically active hydrogenase. In this study, we determined the nucleotide sequence of a 3.8-kb region immediately upstream from hupD. This revealed four additional closely linked ORFs which we designated hupA, hupB, hupY and hupC; these genes potentially encode polypeptides with predicted masses of 12.6, 33.3, 80.4 and 9.0 kDa, respectively. This cluster of genes was shown to be essential for hydrogenase activity by insertion mutagenesis using antibiotic-resistance gene cassettes and a Tn5 derivative carrying a promoterless lacZ gene. A 10.5-kb fragment of DNA beginning 3.4 kb downstream from hupL, and including the sequenced region, was able to complement hupA and hupY mutants, supporting earlier evidence for a promoter downstream from hupSL. The deduced amino acid sequences of hupA, hupB and hupC are homologous to the Escherichia coli hypA, hypB and hypC gene products, respectively. Of particular interest is the fact that there is no homologue of the hupY gene product in the E. coli hyp operon. Mutations in hupY or hupB had little effect on beta-galactosidase activity in a strain also carrying a hupL::lacZ fusion, showing that hupY and hupB are not major factors in regulating the transcription of the hydrogenase structural genes.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of the Azotobacter vinelandii recF gene.

The recF gene from Azotobacter vinelandii (Av) has been cloned by complementation in an Escherichia coli (Ec) recF mutant. The sequence of 1568 bp has been determined and analyzed. It showed an open reading frame of 1092 nt coding for a 364-amino-acid (aa) polypeptide. The comparison of the deduced aa sequence of the recF of Av with those of other bacteria has elicited the presence of the four conserved domains thought to be essential for RecF function. A transcriptional fusion of a DNA fragment containing the promoter sequence of recF with the lacZ gene of Ec was constructed and 3-4-fold enhancement of promoter activity was observed upon UV induction.

Amino Acid Sequence↗
PubMed1990

Utilization of aromatic substances by Azotobacter chroococcum.

Azotobacter chroococcum utilized catechol, protocatechuic acid and gentisic acid as sole carbon source. Rothera's test indicated the ortho cleavage of phenols. The cells displayed high levels of dioxygenase activity. Catechol was cleaved by the ortho pathway mediated by catechol 1,2-dioxygenase. Protocatechuate and gentisic acid were also cleaved by the ortho pathway and cells grown in protocatechuate and gentisic acid displayed protocatechuate, 3,4-dioxygenase and gentisate dioxygenase activity, respectively.

Azotobacter↗
PubMed2005

Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein.

The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length lambdaCI of the pBT bait vector and also NifD-K (fusion) with the N-terminal alpha-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the beta-beta interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the alpha-RNAP of the pTRG vector and lambdaCI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the beta-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the beta-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the beta-subunits of the native MoFe protein in BacterioMatch Two-Hybrid System is comparable to the extent of protein-protein interaction observed between the NifD-K fusion proteins in the same system further supports this idea.

Azotobacter vinelandii↗
PubMed2004

Growth of Azotobacter vinelandii in a solid-state fermentation of technical lignin.

Azotobacter vinelandii was cultured on technical lignin, derived from Kraft pulping processes, for biofertilizer production in solid-state fermentation. The effects of the ratio of technical lignin to corn straw, initial water content, and material bed depth on the microorganisms were studied in detail. At 30 degrees C, technical lignin to corn straw at the ratio of 1:0.75, the bed depth of 5 cm, and 67% moisture content, A. vinelandii was grown and reached 4.2 x 10(10) cfu g(-1) dry rot after 36 h.

Azotobacter vinelandii↗
PubMed2004

Reduction of nitrogenase Fe protein from Azotobacter vinelandii by dithionite: quantitative and qualitative effects of nucleotides, temperature, pH and reaction buffer.

Oxidized Fe protein from Azotobacter vinelandii (Av2(0)) was reduced by dithionite (DT) in the absence and presence of nucleotides, over the temperature range 10-40 degrees C, over the pH range 7-8, and in various buffers--inorganic phosphate, TES, HEPES, and Tris. The reduction of each species of Fe protein--Av2(0), Av2(0)(MgATP)2, and Av2(0)(MgADP)2--was resolved into at least three exponential phases, with relative amplitudes of each phase varying over the range of experimental conditions, suggesting a dynamic population shift of kinetically distinct species. The rapid phase of Av2(0) reduction predominated at low temperature and pH, and in Tris buffer; rapid Av2(0)(MgATP)2 reduction was favored at high temperature and pH, and in phosphate buffer; and Av2(0)(MgADP)2 reduction was favored under more physiologically relevant conditions of 20 degrees C, pH 7.5, and in phosphate buffer. The rates of reduction of Fe protein species did not change with buffer, but temperature and pH do have an effect on the rates. With the appropriate constants, an empirically derived equation estimates the rate of Fe protein reduction at any temperature and pH within the limits 10-40 degrees C and pH 7-8, for a given species of Fe protein, and a given phase of the reaction. At 23.0 degrees C and pH 7.4, the rate of the dominant phase of Av2(0) reduction is 1.9 x 10(8) M(-1) s(-1). Under the same conditions, the rates of the two dominant phases of Av2(0)(MgATP)2 reduction are 1.2 x 10(6) and 1.5 x10 (5) M(-1) s(-1); and the rate of the dominant phase of Av2(0)(MgADP)2 reduction is 3.5 x 10(6) in M(-1) s(-1). Thermodynamic activation parameters for each phase of reduction were calculated. No breaks in the Arrhenius plots for any Fe protein species were observed.

Adenine Nucleotides↗
PubMed2005

Kinetic studies of iron deposition catalyzed by recombinant human liver heavy and light ferritins and Azotobacter vinelandii bacterioferritin using O2 and H2O2 as oxidants.

The discrepancy between predicted and measured H(2)O(2) formation during iron deposition with recombinant heavy human liver ferritin (rHF) was attributed to reaction with the iron protein complex [Biochemistry 40 (2001) 10832-10838]. This proposal was examined by stopped-flow kinetic studies and analysis for H(2)O(2) production using (1) rHF, and Azotobacter vinelandii bacterial ferritin (AvBF), each containing 24 identical subunits with ferroxidase centers; (2) site-altered rHF mutants with functional and dysfunctional ferroxidase centers; and (3) recombinant human liver light ferritin (rLF), containing no ferroxidase center. For rHF, nearly identical pseudo-first-order rate constants of 0.18 s(-1) at pH 7.5 were measured for Fe(2+) oxidation by both O(2) and H(2)O(2), but for rLF, the rate with O(2) was 200-fold slower than that for H(2)O(2) (k = 0.22 s(-1)). A Fe(2+)/O(2) stoichiometry near 2.4 was measured for rHF and its site altered forms, suggesting formation of H(2)O(2). Direct measurements revealed no H(2)O(2) free in solution 0.5-10 min after all Fe(2+) was oxidized at pH 6.5 or 7.5. These results are consistent with initial H(2)O(2) formation, which rapidly reacts in a secondary reaction with unidentified solution components. Using measured rate constants for rHF, simulations showed that steady-state H(2)O(2) concentrations peaked at 14 muM at approximately 600 ms and decreased to zero at 10-30 s. rLF did not produce measurable H(2)O(2) but apparently conducted the secondary reaction with H(2)O(2). Fe(2+)/O(2) values of 4.0 were measured for AvBF. Stopped-flow measurements with AvBF showed that both H(2)O(2) and O(2) react at the same rate (k = 0.34 s(-1)), that is faster than the reactions with rHF. Simulations suggest that AvBF reduces O(2) directly to H(2)O without intermediate H(2)O(2) formation.

Azotobacter vinelandii↗
PubMed2005

Growth of Azotobacter chroococcum in chemically defined media containing p-hydroxybenzoic acid and protocatechuic acid.

Growth and utilization of different phenolic acids present in olive mill wastewater (OMW) by Azotobacter chroococcum were studied in chemically defined media. Growth and utilization of phenolic acids were only detected when the microorganism was cultured on p-hydroxybenzoic acid at concentration from 0.01% to 0.5% (w/v) and protocatechuic acid at concentration from 0.01% to 0.3% (w/v) as sole carbon sources suggesting that only these phenolic compounds could be utilized as a carbon source by A. chroococcum. Moreover when culture media were added with a mixture of 0.3% of protocatechuic acid and 0.3% p-hydroxybenzoic acid, the microorganism degradated in first place protocatechuic acid and once the culture medium was depleted of this compound, the degradation of p-hydroxybenzoic acid commenced very fast.

Azotobacter↗
PubMed2004

2.6 A resolution crystal structure of the bacterioferritin from Azotobacter vinelandii.

The crystal structure of the bacterioferritin from Azotobacter vinelandii has been determined at 2.6 A resolution. Both the low occupancy of one iron ion in the dinuclear iron center and the deviation of its adjacent residue His130 from the center suggest migration of the iron ion from the dinuclear iron site to the inner nucleation site. The concerted movement of His130 and Glu47 may admit a dynamic gating mechanism for shift of the oxidized iron ion. Ba2+ binding to the fourfold channel implicates that the channel bears Fe2+ conductivity and selectivity to provide a route for iron access to the inner cavity during core formation.

Azotobacter vinelandii↗