scholarly journals Characteristics of N2 fixation in Mo-limited batch and continuous cultures of Azotobacter vinelandii

1984 ◽  
Vol 224 (3) ◽  
pp. 853-862 ◽  
Author(s):  
R R Eady ◽  
R L Robson
Keyword(s):  
N2 Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Defined Medium ◽  
Pulse Labelling ◽  
Mofe Protein ◽  
Fe Protein ◽  
Limiting Nutrient ◽  
Mo Content

Steady-state chemostat cultures of Azotobacter vinelandii were established in a simple defined medium that had been chemically purified to minimize Mo and that contained no utilizable combined N source. Growth was dependent on N2 fixation, the limiting nutrient being the Mo contaminating the system. The Mo content of the organisms was at least 100-fold lower than that of Mo-sufficient cultures, and they lacked the characteristic g = 3.7 e.p.r. feature of the MoFe-protein of nitrogenase. A characteristic of nitrogenase activity in vivo in Mo-limited populations was a disproportionately low activity for acetylene reduction, which was 0.3 to 0.1 of that expected from the rate of N2 reduction. Acetylene was also a poor substrate in comparison with protons as a substrate for nitrogenase, and did not markedly inhibit H2 evolution, in contrast with Mo-sufficient populations. In batch cultures in similar medium or ‘spent’ chemostat medium inoculated with Mo-limited organisms, the addition of Mo elicited a biphasic increased growth response at concentrations as low as 2.5 nM, provided that sufficient Fe was supplied. In this system V did not substitute for Mo, and Mo-deficient cultures ceased growth at a 25-fold lower population density compared with cultures supplemented with Mo. Nitrogenase component proteins could not be unequivocally detected by visual inspection of fractionated crude extracts of Mo-limited organisms. 35SO42-pulse-labelling studies also showed that the rate of synthesis of the MoFe-protein component of nitrogenase was too low to be quantified. However, the Fe-protein of nitrogenase was apparently synthesized at high rates. The discussion includes an evaluation of the possibility that A. vinelandii possesses an Mo-independent N2-fixation system.

scholarly journals Nitrogenase of Klebsiella pneumoniae: kinetics of formation of the transition-state complex and evidence for an altered conformation of MoFe protein lacking a FeMoco centre

1997 ◽  
Vol 326 (3) ◽  
pp. 637-640 ◽  
Author(s):  
Faridoon K. YOUSAFZAI ◽  
Robert R. EADY
Keyword(s):  
Klebsiella Pneumoniae ◽  
Transition State ◽  
Nitrogenase Activity ◽  
Active Species ◽  
Slow Phase ◽  
Mofe Protein ◽  
Fe Protein ◽  
Catalytically Active ◽  
Mo Content ◽  
Kinetics Of

We have investigated the kinetics of inactivation of Mo-nitrogenase isolated from Klebsiella pneumoniae when it forms an inhibited putative transition-state complex on incubation with ADP and AlF4-. In the presence of excess Kp2 (Fe protein of the Mo-nitrogenase of K. pneumoniae), the kinetics were found to depend on the Mo content of Kp1 (the MoFe protein of Mo-nitrogenase of K. pneumoniae). The residual nitrogenase activity versus time of incubation using Kp1 preparations containing integral, i.e. one or two Mo atoms per molecule of Kp1, were essentially monophasic, but significantly different rates of inactivation were observed. In contrast, the progress curves for preparations of Kp1 with non-integral Mo content were biphasic, suggesting the presence of two discrete catalytically active species of Kp1. The best fit to the observed data was obtained with a two-exponential expression, the amplitude of which was consistent with the Mo content, provided that the fast phase of the reaction was assigned to a Kp1 species containing one, and the slow phase to a species containing two Mo atoms per α2β2 tetramer. This analysis provides the first evidence for the existence of a catalytically active Kp1 species containing a single Mo atom. These data also indicate that MoFe protein which does not have all FeMoco binding sites occupied has an altered conformation compared with a fully loaded protein, and that the Fe protein reacts with these conformations at different rates to form the stable, but inhibited transition-state complex.

scholarly journals Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity

1984 ◽  
Vol 224 (3) ◽  
pp. 961-969 ◽  
Author(s):  
T D Paul ◽  
P W Ludden
Keyword(s):  
Protein Modification ◽  
Rhodospirillum Rubrum ◽  
Adenine Nucleotide ◽  
Nitrogenase Activity ◽  
Energy Charge ◽  
Whole Cell ◽  
Fe Protein ◽  
Nucleotide Pools ◽  
Phenazine Methosulphate

Adenine nucleotide pools were measured in Rhodospirillum rubrum cultures that contained nitrogenase. The average energy charge [([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP])] was found to be 0.66 and 0.62 in glutamate-grown and N-limited cultures respectively. Treatment of glutamate-grown cells with darkness, ammonia, glutamine, carbonyl cyanide m-chlorophenylhydrazone, or phenazine methosulphate resulted in perturbations in the adenine nucleotide pools, and led to loss of whole-cell nitrogenase activity and modification in vivo of the Fe protein. Treatment of N-limited cells resulted in similar changes in adenine nucleotide pools but not enzyme modification. No correlations were found between changes in adenine nucleotide pools or ratios of these pools and switch-off of nitrogenase activity by Fe protein modification in vivo. Phenazine methosulphate inhibited whole-cell activity at low concentrations. The effect on nitrogenase activity was apparently independent of Fe protein modification.

scholarly journals Using synthetic biology to overcome barriers to stable expression of nitrogenase in eukaryotic organelles

2020 ◽  
Vol 117 (28) ◽  
pp. 16537-16545 ◽  
Author(s):  
Nan Xiang ◽  
Chenyue Guo ◽  
Jiwei Liu ◽  
Hao Xu ◽  
Ray Dixon ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Klebsiella Oxytoca ◽  
Plant Mitochondria ◽  
Eukaryotic Cells ◽  
Mofe Protein ◽  
The Stability ◽  
Biological Nitrogen ◽  
Nif Proteins

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction ofnifgenes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein fromAzotobacter vinelandiiandKlebsiella oxytocawere found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, fromK. oxytocathat confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast,Oryza sativa,Nicotiana tabacum, andArabidopsis thalianainEscherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.

scholarly journals Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase

1988 ◽  
Vol 256 (2) ◽  
pp. 429-432 ◽  
Author(s):  
R W Miller ◽  
R R Eady
Keyword(s):  
Low Temperature ◽  
Electron Flux ◽  
Specific Activity ◽  
Nitrogenase Activity ◽  
Azotobacter Chroococcum ◽  
Effect Of Temperature ◽  
High Electron ◽  
Mofe Protein ◽  
Fe Protein ◽  
Vanadium Nitrogenase

A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.

scholarly journals Characterization of the DraT/DraG System for Posttranslational Regulation of Nitrogenase in the Endophytic Betaproteobacterium Azoarcus sp. Strain BH72

2009 ◽  
Vol 191 (11) ◽  
pp. 3726-3735 ◽  
Author(s):  
Janina Oetjen ◽  
Barbara Reinhold-Hurek
Keyword(s):  
Blot Analysis ◽  
Protein Modification ◽  
Nitrogenase Activity ◽  
Posttranslational Regulation ◽  
Fe Protein ◽  
Grass Endophyte ◽  
Genes Encoding ◽  
Microaerobic Conditions ◽  
First Time

ABSTRACT DraT/DraG-mediated posttranslational regulation of the nitrogenase Fe protein by ADP-ribosylation has been described for a few diazotrophic bacteria belonging to the class Alphaproteobacteria. Here we present for the first time the DraT/DraG system of a betaproteobacterium, Azoarcus sp. strain BH72, a diazotrophic grass endophyte. Its genome harbors one draT ortholog and two physically unlinked genes coding for ADP-ribosylhydrolases. Northern blot analysis revealed cotranscription of draT with two genes encoding hypothetical proteins. Furthermore, draT and draG2 were expressed under all studied conditions, whereas draG1 expression was nitrogen regulated. By using Western blot analysis of deletion mutants and nitrogenase assays in vivo, we demonstrated that DraT is required for the nitrogenase Fe protein modification but not for the physiological inactivation of nitrogenase activity. A second mechanism responsible for nitrogenase inactivation must operate in this bacterium, which is independent of DraT. Fe protein demodification was dependent mainly on DraG1, corroborating the assumption from phylogenetic analysis that DraG2 might be mostly involved in processes other than the posttranslational regulation of nitrogenase. Nitrogenase in vivo reactivation was impaired in a draG1 mutant and a mutant lacking both draG alleles after anaerobiosis shifts and subsequent adjustment to microaerobic conditions, suggesting that modified dinitrogenase reductase was inactive. Our results demonstrate that the DraT/DraG system, despite some differences, is functionally conserved in diazotrophic proteobacteria.

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