The flavin transferase ApbE flavinylates the ferredoxin:NAD+-oxidoreductase Rnf required for N2 fixation in Azotobacter vinelandii

2021 ◽  
Author(s):  
Yulia V Bertsova ◽  
Marina V Serebryakova ◽  
Alexander A Baykov ◽  
Alexander V Bogachev
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Growth Defect ◽  
Deficient Mutant ◽  
Fixed Nitrogen ◽  
Oxidoreductase Activity ◽  
Threonine Residue ◽  
Ferredoxin Reductase ◽  
Relative Contribution ◽  
Fixation System

Abstract Azotobacter vinelandii, the model microbe in nitrogen fixation studies, uses the ferredoxin:NAD+-oxidoreductase Rnf to regenerate ferredoxin (flavodoxin) acting as an electron donor for nitrogenase. However, the relative contribution of Rnf into nitrogenase functioning is unknown because this bacterium contains another ferredoxin reductase, FixABCX. Furthermore, Rnf is flavinylated in the cell, but the importance and pathway of this modification reaction also remain largely unknown. We have constructed A. vinelandii cells with impaired activities of FixABCX and/or putative flavin transferase ApbE. The ApbE-deficient mutant could not produce covalently flavinylated membrane proteins and demonstrated a markedly decreased flavodoxin:NAD+ oxidoreductase activity and significant growth defect under diazotrophic conditions. The double ΔFix/ΔApbE mutation abolished the flavodoxin:NAD+ oxidoreductase activity and the ability of A. vinelandii to grow in the absence of fixed nitrogen source. ApbE flavinylated a truncated RnfG subunit of Rnf1 by forming a phosphoester bond between FMN and a threonine residue. These findings indicate that Rnf (presumably its Rnf1 form) is the major ferredoxin-reducing enzyme in the nitrogen fixation system and that the activity of Rnf depends on its covalent flavinylation by the flavin transferase ApbE.

scholarly journals Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii.

1980 ◽  
Vol 77 (12) ◽  
pp. 7342-7346 ◽  
Author(s):  
P. E. Bishop ◽  
D. M. Jarlenski ◽  
D. R. Hetherington
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Fixation System

scholarly journals Genetic determinants of ammonia excretion in nifL mutants of Azotobacter vinelandii

2021 ◽  
Author(s):  
Florence Mus ◽  
Devanshi Khokhani ◽  
Esther Rugoli ◽  
Ray Dixon ◽  
Jean-Michel Ané ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Model Organism ◽  
Ammonia Excretion ◽  
Promoter Sequence ◽  
Genetic Determinants ◽  
Two Component System ◽  
Opposite Orientation ◽  
Fixed Nitrogen ◽  
Biological Nitrogen

Abstract The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an anti-activator that tightly controls that activity of the nitrogen fixation specific transcriptional activator, NifA, in response to redox, nitrogen, and carbon status. While several studies reported mutations in A. vinelandii nifL resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonia, knowledge about the specific determinants for this ammonia-excreting phenotype is lacking. In this work, we report that only specific disruptions of nifL lead to large quantities of ammonia accumulated in liquid culture (~ 12 mM). The ammonia excretion phenotype is solely associated with deletions of NifL domains combined with the insertion of a promoter sequence in the opposite orientation to nifLA transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonia excreted by affecting rnf1 gene expression as an additional requirement for ammonia excretion. These ammonia-excreting nifL mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines the discreet determinants that bring about A. vinelandii ammonia excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.

Nitrogen fixation: key genetic regulatory mechanisms

2005 ◽  
Vol 33 (1) ◽  
pp. 152-156 ◽  
Author(s):  
I. Martinez-Argudo ◽  
R. Little ◽  
N. Shearer ◽  
P. Johnson ◽  
R. Dixon
Keyword(s):  
Nitrogen Fixation ◽  
Transcriptional Activation ◽  
Azotobacter Vinelandii ◽  
Regulatory Mechanisms ◽  
Excess Oxygen ◽  
Binary Complex ◽  
Fixed Nitrogen ◽  
Signal Transduction Protein ◽  
Sufficient Energy ◽  
Metabolic Signal

The necessity to respond to the level of fixed nitrogen and external oxygen concentrations and to provide sufficient energy for nitrogen fixation imposes common regulatory principles amongst diazotrophs. The NifL–NifA system in Azotobacter vinelandii integrates the signals of redox, fixed-nitrogen and carbon status to regulate nif transcription. Multidomain signalling interactions between NifL and NifA are modulated by redox changes, ligand binding and interaction with the signal-transduction protein GlnK. Under adverse redox conditions (excess oxygen) or when fixed nitrogen is in excess, NifL forms a complex with NifA in which transcriptional activation is prevented. Oxidized NifL forms a binary complex with NifA to inhibit NifA activity. When fixed nitrogen is in excess, the non-covalently modified form of GlnK interacts with NifL to promote the formation of a GlnK–NifL–NifA ternary complex. When the cell re-encounters favourable conditions for nitrogen fixation, it is necessary to deactivate the signals to ensure that the NifL–NifA complex is dissociated so that NifA is free to activate transcription. This is achieved through interactions with 2-oxoglutarate, a key metabolic signal of the carbon status, which binds to the N-terminal GAF (cGMP-specific and stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coliFhlA) domain of NifA.

scholarly journals Expression of an alternative nitrogen fixation system in Azotobacter vinelandii.

1982 ◽  
Vol 150 (3) ◽  
pp. 1244-1251 ◽  
Author(s):  
P E Bishop ◽  
D M Jarlenski ◽  
D R Hetherington
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Fixation System

Nitrogen Fixation byAzotobacter vinelandiiStrains Having Deletions in Structural Genes for Nitrogenase

Science ◽  
1986 ◽  
Vol 232 (4746) ◽  
pp. 92-94 ◽  
Author(s):  
PAUL E. BISHOP ◽  
R. PREMAKUMAR ◽  
DENNIS R. DEAN ◽  
MARTY R. JACOBSON ◽  
JOHN R. CHISNELL ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Molecular Nitrogen ◽  
Phenotypic Expression ◽  
Structural Genes ◽  
Reversal Phenomenon ◽  
Molybdenum Enzyme ◽  
Phenotypic Reversal ◽  
Free Media ◽  
Fixation System

Phenotypic reversal of Nif-mutant strains to Nif+under molybdenum-deficient conditions has been cited as evidence thatAzotobacter vinelandiipossesses two nitrogen fixation systems: the conventional molybdenum-enzyme system and an alternative nitrogen-fixation system. Since explanations other than the existence of an alternative system were possible, deletion strains ofA. vinelandiilacking the structural genes for conventional nitrogenase (nifHDK) were constructed. These strains were found to grow in molybdenum-deficient nitrogen-free media, reduce acetylene (at low rates), and incorporate molecular nitrogen labeled with nitrogen-15. Thus it can be concluded that the phenotypic reversal phenomenon cannot be due to altered phenotypic expression ofnifmutations under molybdenum-deficient conditions, but is due to the existence of an alternative nitrogen-fixation system inA. vinelandiias originally proposed.

Nitrogen fixation by cell-free extracts of Klebsiella pneumoniae

1968 ◽  
Vol 14 (1) ◽  
pp. 33-38 ◽  
Author(s):  
M. C. Mahl ◽  
P. W. Wilson
Keyword(s):  
Nitrogen Fixation ◽  
Klebsiella Pneumoniae ◽  
Azotobacter Vinelandii ◽  
Ammonium Ion ◽  
Michaelis Constant ◽  
Nitrogen Gas ◽  
Aerobacter Aerogenes ◽  
Free System ◽  
Evolving System ◽  
A Cell

A cell-free system which permits nitrogen fixation by extracts of Klebsiella pneumoniae M5al (formerly Aerobacter aerogenes) has been developed. It is, essentially, that system described by Bulen and associates for Azotobacter vinelandii, utilizing ATP as a source of energy and dithionite as a source of electrons. The Michaelis constant for fixation has been estimated to be 0.12 atm. The extracts possessed an ATP-dependent hydrogen evolving system. Hydrogen evolution from these extracts was less under nitrogen than under helium in the presence of ATP. Nitrogen gas appears to be the inducer of nitrogen fixation. In the absence of N2, no induction of nitrogenase occurs. Nitrogenase is absent in cells grown on NH4+-N. There is a lag of about 13 h after the introduction of N2 gas into a culture which has depleted its supply of NH4+-N before nitrogenase can be detected. For reasons discussed in the text, this conclusion must be regarded as tentative at this time. Ammonium ion appears to prevent the synthesis of new molecules of nitrogenase without affecting the activity of those already formed.

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