Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii.

The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions. It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake. The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity. An alternative proposal is given.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.     

Co-ordination and fine-tuning of nitrogen fixation in Azotobacter vinelandii

Nitrogen fixation by the free-living organism Azotobacter vinelandii can occur through the activity of three different systems that are genetically distinct but mechanistically related. A combination of bioinformatic and biochemical-genetic studies has revealed that at least 82 different genes are likely to be associated with the formation and regulation of these systems. Studies performed over many years have established that cross-talk occurs between the various nitrogen fixation systems, and that expression and fine-tuning of their activities are integrated with overall cellular physiology. Martinez-Noel and co-workers now report another newly discovered aspect of the process. Evidence is presented to suggest that a nitrogen fixation-specific paralogue of ClpX is used to control the accumulation of proteins involved in formation of a metal-sulphur cluster that provides a nitrogenase active site. The intriguing aspect of this work is that it indicates that the nitrogen fixation-associated ClpX must recruit ClpP, for which a paralogue is not duplicated within any of the nitrogen fixation regions of the genome, to achieve its function related to nitrogen fixation. Inspection of the A. vinelandii genome indicates that such recruitment of cellular housekeeping components is a common feature used to integrate nitrogen fixation with global cellular physiology.     

Plasmid transformation of Azotobacter vinelandii OP

Azotobacter vinelandii OP which had been naturally induced to competence by growth in iron- and molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif+). Plasmid- and chromosomal-DNA-mediated transformation events were competitive, magnesium-dependent, 42 degrees C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT210 transformed cells equally well whereas EcoRI- or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10- to 50-fold when pKT210 contained an insert fragment of A. vinelandii nif DNA, indicating that A. vinelandii possessed a homology-facilitated transformation system. However, all transformants failed to maintain the plasmid-encoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HincII-digested plasmid DNA containing the cloned A. vinelandii nif-10 marker.     

Nitrogenase in synchronized Azotobacter vinelandii OP.

Azotobacter vinelandii OP was synchronized by the continuous phased culture technique. The nitrogenase (nitrogen:(acceptor)oxidoreductase)(EC 1.7.99.2) activity of the culture was determined continuously within the fermentor by acetylene reduction. Addition of NH4+ in excess of 5 x 10(-3)M to the culture lowered nitrogenase activity immediately. Other sources of fixed nitrogen had no immediate effect on nitrogenase activity, but nitrogenase synthesis decreased in the cell cycle following the one in which the fixed nitrogen was added.     

Expression of an alternative nitrogen fixation system in Azotobacter vinelandii.

Nitrogenase activities were determined from maximum acetylene reduction rates for mutant strains of Azotobacter vinelandii which are unable to fix N2 in the presence of molybdenum (Nif-) but undergo phenotypic reversal to Nif+ under conditions of Mo deficiency. The system responsible for N2 fixation under these conditions is thought to be an alternative N2 fixation system (Bishop et al., Proc. Natl. Acad. Sci. U.S.A. 77:7342-7346, 1980). Phenotypic reversal of Nif- strains to Nif+ strains was also observed in N-free medium without Mo but with either V or Re. Two protein patterns were found on two-dimensional gels of proteins from the extracts of wild-type cells cultured in N-free medium without Mo and with or without V or Re. The expression of each protein pattern in the wild-type strain of A. vinelandii seemed to depend upon the physiological state of the N2-fixing culture. Electron paramagnetic resonance experiments were conducted on whole cells of A. vinelandii grown under conditions of Mo deprivation in the absence of fixed N. No g = 3.65 signal (an electron paramagnetic resonance signal characteristic of the Mo-containing component of nitrogenase) was detectable in these cells, regardless of whether V or Re was present during growth of these cells, These results are discussed from the perspective that the well-known effect of V on N2 fixation by A. vinelandii may involve an alternative N2 fixation system.     

Regulation of Nitrogen Fixation in Azotobacter vinelandii OP and in an Apparently Partially Constitutive Mutant

Methylamine and 2-methylalanine appeared to act as co-repressors of nitrogenase in Azotobacter vinelandii OP. They inhibited the growth of this organism on molecular nitrogen but not on nitrate, ammonia, or Casamino Acids; they prevented the formation of nitrogenase by cells transferred from repression to induction conditions; and they did not inhibit the activity of nitrogenase in vitro. A mutant of strain OP, selected on the basis of its relative resistance to methylalanine, appeared partially constitutive because nitrogenase in this strain was less sensitive to repressors than was the enzyme in the wild-type strain.     

Isolation and characterization of pyruvate carboxylase from Azotobacter vinelandii OP

Pyruvate carboxylase has been detected in, and partially purified from, cell-free extracts of Azotobacter vinelandii OP. The best preparations obtained have specific activities in the range of 4 units/mg and appear approximately 15% pure when analyzed by polyacrylamide gel electrophoresis. The partially purified enzyme is activated by both univalent and divalent cations, contains one or more functional biotinyl residues, and exhibits apparent Michaelis constants for the substrates (pyruvate, Mg-ATP^2?, and HCO₃^?) which are in the same range as those observed for other pyruvate carboxylases. However, A. vinelandii pyruvate carboxylase is fully active in the absence of added acetyl-coenzyme A and is insensitive to inhibition by dicarboxylic acids such as l-aspartate, l-glutamate, and α-ketoglutarate. The molecular weight of the catalytically active species is obtained as 296,000.The level of pyruvate carboxylase is highest in extracts of A. vinelandii grown on pyruvate or l-lactate as sole carbon source and this level is further enhanced on addition of succinate to the medium. The enzyme is absent from cells grown on succinate and is present at intermediate levels in cells grown on sucrose, glucose, glycerol, or acetate. In contrast, the level of phosphoenolypyruvate carboxylase in these extracts is essentially independent of the carbon source. These data suggest that pyruvate carboxylase in A. vinelandii is induced by pyruvate or some closely related metabolite.     

Bioavailability of mineral-associated trace metals as cofactors for nitrogen fixation by Azotobacter vinelandii

Life on Earth depends on N₂-fixing microbes to make ammonia from atmospheric N₂ gas by the nitrogenase enzyme. Most nitrogenases use Mo as a cofactor; however, V and Fe are also possible. N₂ fixation was once believed to have evolved during the Archean-Proterozoic times using Fe as a cofactor. However, δ¹⁵N values of paleo-ocean sediments suggest Mo and V cofactors despite their low concentrations in the paleo-oceans. This apparent paradox is based on an untested assumption that only soluble metals are bioavailable. In this study, laboratory experiments were performed to test the bioavailability of mineral-associated trace metals to a model N₂-fixing bacterium Azotobacter vinelandii. N₂ fixation was observed when Mo in molybdenite, V in cavansite, and Fe in ferrihydrite were used as the sole sources of cofactors, but the rate of N₂ fixation was greatly reduced. A physical separation between minerals and cells further reduced the rate of N₂ fixation. Biochemical assays detected five siderophores, including aminochelin, azotochelin, azotobactin, protochelin, and vibrioferrin, as possible chelators to extract metals from minerals. The results of this study demonstrate that mineral-associated trace metals are bioavailable as cofactors of nitrogenases to support N₂ fixation in those environments that lack soluble trace metals and may offer a partial answer to the paradox.