A proposed role for the Azotobacter vinelandii NfuA protein as an intermediate iron-sulfur cluster carrier

Iron-sulfur clusters ([Fe-S] clusters) are assembled on molecular scaffolds and subsequently used for maturation of proteins that require [Fe-S] clusters for their functions. Previous studies have shown that Azotobacter vinelandii produces at least two [Fe-S] cluster assembly scaffolds: NifU, required for the maturation of nitrogenase, and IscU, required for the general maturation of other [Fe-S] proteins. A. vinelandii also encodes a protein designated NfuA, which shares amino acid sequence similarity with the C-terminal region of NifU. The activity of aconitase, a [4Fe-4S] cluster-containing enzyme, is markedly diminished in a strain containing an inactivated nfuA gene. This inactivation also results in a null-growth phenotype when the strain is cultivated under elevated oxygen concentrations. NifU has a limited ability to serve the function of NfuA, as its expression at high levels corrects the defect of the nfuA-disrupted strain. Spectroscopic and analytical studies indicate that one [4Fe-4S] cluster can be assembled in vitro within a dimeric form of NfuA. The resultant [4Fe-4S] cluster-loaded form of NfuA is competent for rapid in vitro activation of apo-aconitase. Based on these results a model is proposed where NfuA could represent a class of intermediate [Fe-S] cluster carriers involved in [Fe-S] protein maturation.     

Molecular weights of nitrogenase components from Azotobacter vinelandii.

Meniscus depletion sedimentation equilibrium ultracentrifuge experiments were performed on purified MoFe and Fe proteins of $\text{Azotobacter vinelandii}$. The MoFe protein was found to have a molecular weight of 245,000, using an experimentally confirmed partial specific volume of 0.73. The MoFe protein formed one band on sodium dodecyl sulfate gel electrophoresis and had a subunit molecular weight of 56,000. The subunit molecular weight from ultracentrifuge experiments in 8 M urea was 61,000. The molecular weight of the Fe protein was calculated to be 60,500 in meniscus depletion experiments. Similar experiments in 8 M urea solvent indicated a subunit molecular weight of 30,000. A subunit molecular weight of 33,000 was obtained from sodium dodecyl sulfate gel electrophoresis experiments.     

Activity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster.

The Azotobacter vinelandii genes encoding the nitrogenase structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). In this study various A. vinelandii mutant strains which contain defined deletions within the nitrogenase structural genes were isolated and studied. Mutants deleted for the nifD or nifK genes were still able to accumulate significant amounts of the unaltered MoFe protein subunit as well as active Fe protein. Extracts of such nifD or nifK deletion strains had no MoFe protein activity. However, active MoFe protein could be reconstituted by mixing extracts of the mutant strains. These results establish an approach for the purification of the individual MoFe protein subunits. Mutants lacking either or both of the MoFe protein subunits were still able to synthesize the iron-molybdenum cofactor (FeMo-cofactor), indicating that in A. vinelandii the FeMo-cofactor is preassembled and inserted into the MoFe protein. In contrast, a mutant strain lacking both the Fe protein and the MoFe protein failed to accumulate any detectable FeMo-cofactor. The further utility of specifically altered A. vinelandii strains for the study of the assembly, structure, and reactivity of nitrogenase is discussed.     

Hydrogen-oxidizing electron transport components in nitrogen-fixing Azotobacter vinelandii.

Membranes from N2-fixing Azotobacter vinelandii were isolated to identify electron transport components involved in H2 oxidation. We found direct evidence for the involvement of cytochromes b, c, and d in H2 oxidation by the use of H2-reduced minus O2-oxidized absorption difference spectra. Carbon monoxide spectra showed that H2 reduced cytochrome d but not cytochrome o. Inhibition of H2 oxidation by cyanide was monophasic with a high Ki (135 microM); this was attributed to cytochrome d. Cyanide inhibition of malate oxidation showed the presence of an additional, low Ki (0.1 microM cyanide) component in the membranes; this was attributed to cytochrome o. However, H2 oxidation was not sensitive to this cyanide concentration. Chlorpromazine (at 160 microM) markedly inhibited malate oxidation, but it did not greatly inhibit H2 oxidation. Irradiation of membranes with UV light inhibited H2 oxidation. Adding A. vinelandii Q8 to the UV-damaged membranes partially restored H2 oxidation activity, whereas addition of UV-treated Q8 did not increase the activity. 2-n-Heptyl-4-hydroxyquinoline-N-oxide inhibited both H2 and malate oxidation.     

Selective oxidative destruction of iron-sulfur clusters. Ferricyanide oxidation of Azotobacter vinelandii ferredoxin I

The destructive oxidation of aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I [(7Fe)FdI] by Fe(CN)3-6 is examined using low-temperature magnetic circular dichroism (MCD) and EPR. The results demonstrate that oxidation of the [3Fe-3S] cluster occurs only after essentially complete destruction of the [4Fe-4S] cluster. It is therefore feasible by controlled Fe(CN)3-6 oxidation to obtain a partially metallated form of FdI, (3Fe)FdI, containing only a [3Fe-3S] cluster. The MCD and EPR data demonstrate that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in the native protein.     

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.     

Phospholipids of Azotobacter vinelandii

Analyses of resting cells of Azotobacter vinelandii revealed that numerous phospholipids were present that did not concentrate in the membranous R(3) fraction which carried out electron transport function.     

Ultrastructure of Azotobacter vinelandii

Vegetative cells and cysts of Azotobacter vinelandii 12837 were prepared for electron microscopy by several methods assumed to preserve structural details destroyed by techniques previously reported in the literature. Examination of large numbers of cells and cysts by these methods revealed four structural details not reported previously: intine fibrils, intine vesicles, intine membrane, and microtubules. The intine fibrils form a network in the gel-like homogeneous matrix of the CC2 layer. Intine vesicles which seem to originate in the cell wall complex of the central body are seen in the intine and exine of cysts. Analogous structures are found on vegetative cells. The intine is divided into two chemically distinct areas by the two-layered intine membrane. Microtubules, previously reported only in vegetative cells, were found in cysts.     

Hydrogen-mediated enhancement of hydrogenase expression in Azotobacter vinelandii.

Azotobacter vinelandii cultures express more H2 uptake hydrogenase activity when fixing N2 than when provided with fixed N. Hydrogen, a product of the nitrogenase reaction, is at least partly responsible for this increase. The addition of H2 to NH4+-grown wild-type cultures caused increased whole-cell H2 uptake activity, methylene blue-dependent H2 uptake activity of membranes, and accumulation of hydrogenase protein (large subunit as detected immunologically) in membranes. Both rifampin and chloramphenicol inhibited the H2-mediated enhancement of hydrogenase synthesis. Nif- A. vinelandii mutants with deletions or insertions in the nif genes responded to added H2 by increasing the amount of both whole-cell and membrane-bound hydrogenase activities. Nif- mutant strain CA11 contained fourfold more hydrogenase protein when incubated in N-free medium with H2 than when incubated in the same medium containing Ar. N2-fixing wild-type cultures that produce H2 did not increase hydrogenase protein levels in response to added H2.     

Genetic analysis of the transcriptional arrangement of Azotobacter vinelandii alginate biosynthetic genes: identification of two independent promoters

The study of alginate biosynthesis, the exopolysac charide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, might lead to different bio-technological applications. Here we report the cloning of A. vinelandii algA, the gene coding for the bifunctional enzyme phosphomannose isomerase-guano-sine diphospho-D-mannose pyrophosphorylase (PMI-GMP). This gene was selected by the complementation for xanthan gum production of Xanthomonas campestris pv. campestris xanB mutants, which lack this enzymatic activity. The complementing cosmid clones selected, besides containing algA, presented a gene coding for an alginate lyase activity (algL), and some of them also contained algD which codes for GDP-mannose dehydrogenase. We present here the characterization of the A. vinelandii chromosomal region comprising algD and its promoter region, algA and algL, showing that, as previously reported for P. aeruginosa, A. vinelandii has a cluster of the biosynthetic alginate genes. We provide evidence for the presence of an algD-independent promoter in this region which transcribes at least algL and algA, and which is regulated in a manner that differs from that of the algD promoter.