Nif regulon

Nif regulon
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Klebsiella pneumoniae is a coliform bacterium that is capable of nitrogen fixation under anaerobic and microaerophilic conditions. Nitrogen fixation is regulated by nif regulon, which is a set of seven operons which includes 17 nif genes.The nif regulon is situated between the his and the Shi-A operon of the bacterium.


Nitrogen Fixation

Nitrogen fixation is catalysed by the nitrogenase enzyme.Nitrogenase is made up of two soluble proteins :component I and II. Component I known as MoFe protein or nitrogenase contains 2 Mo atoms, 28 to 34 non-heme Fe atoms, and 26 to 28 acid-labile sulfides. Component I is composed of two copies each of two subunits (α and β);each subunit’s stability depends on the other in vivo.Component II known as Fe protein or nitrogenase reductase is composed of two copies of a single subunit. This protein has four non-heme Fe atoms and four acid-labile sulfides. Substrate binding and reduction take place oncomponent I.The role of component II is to supply electrons,one at a time to component I.Nitrogen fixation requires ATP due to high activation energy. ATP is not hydrolyzed to ADP until component II transfers an electron to component I. 21-25 ATPs are required for each N2 fixed.

The Nif Regulon

The nif regulon comprises of 7 operons: nifRLA , nifJ, nifHDK ,nifEN, nifUSVM, nifWF, nifBQ .

nifRLA operon: The tight expression regulation of the nitrogen fixation (nif) genes is mediated by the products of the nifRLA operon. NifA activates transcription of nif genes by the alternative form of RNA-polymerase, s54-holoenzyme.NifL is a negative regulatory gene which inhibits the activation of other nif genes by nifA protein. NifR is a repressor binding site , between the promoter of the nifRLA operon and the nifL gene. No protein coded by nifR gene has been found.

nifHDK operon:It comprises of three structural genes viz nifK nif D and nifH. nifK encodes for B-subunit of Component 1 of nitrogenase. nifD encodes for alpha subunit of component 1 of nitrogenase. nifH encodes for component 2 of nitrogenase.

nifEN and nifBQ operons: This comprises of nifE, nifN, nifB and nifQ genes which are responsible for formation of a functional Mo-Fe protein. (Mo-Fe-co catalytic site for nitrogenase.) nifQ is not absolutely essential.

nifJ operon:The nifJ gene encodes for the pyruvate-flavodoxin-oxidoreductase protein.This enzyme is involved in electron transfer to nitrogenase.

nifUSVM operon: The nifS, nifV and nifM genes encode for a protein that is required to process component II. Function of the nifU gene is undetermined.

nifWF operon: The function of nifW is undetermined. The nifF gene mediates electron transfer from nifJ protein to Fe protein of nitrogenase.



The action site of O2 is the nifL protein which is basically a flavoprotein with FAD as the redox sensing cofactor. Fnr(fumarate nitrate reduction regulator) is the signal transduction molecule which transduces the oxygen status to the nifL protein. In the absence of O2, the nifL protein is in its reduced form (FADH2 as the cofactor ) and is unable to inhibit the action of nifA protein. In the O2 presence, oxidized nifL (FAD as the cofactor) inhibits nifA protein and there by turns off all the other operons.


The presence of ammonium ions in large amounts in the environment inhibits the transcription of nitrogenase and all the other nif genes. NH4+ acts as a co repressor to the glutamine synthetase by modifying it covalently(adenylylation). This modifies enzyme binds to the nifR region of the nifRLA operon and prevents the transcription of the genes, nifL and nifA. So, there is no initiation of transcription of the other genes by the σ-RNA polymerase.

Glnk protein

In the nitrogen limiting growth conditions, the inhibition of nifA protein by the nifL protein is prevented by the antagonizing action of Glnk protein on the nifL protein.

A homologue of the NifL-NifA regulatory gene system has not been found among the eukaryotes. However Entamoeba histolytica was found to possess a simplified and non-redundant NIF (nitrogen fixation)-like system for the Fe- S cluster formation, composed of only a catalytic component, NifS, and a scaffold component, NifU. EhNifS and EhNifU were found to be necessary and sufficient for Fe-S clusters of non-nitrogenase Fe-S proteins to form under anaerobic conditions. This is the first demonstration of the presence and biological significance of the NIF-like system in eukaryotes.


[1] [2] [3] [4] [5]

  1. ^ WINSTON JL BRILL “Biochemical Genetics of Nitrogen Fixation” MICROBIOLOGICAL REVIEWS, Sept. 1980, p. 449-467
  2. ^ GARY P. ROBERTS AND WINSTON J. BRILL,”Gene-Product Relationships of the nif Regulon of Klebsiella Pneumonia”, JOURNAL OF BACTERIOLOGY, Oct. 1980, p. 210-216
  3. ^ Maria Milenkov,Robert Thummer,Jens Glo¨er,Joachim Gro¨tzinger,Sascha Jung and Ruth A. Schmitz,Insights into Membrane Association of Klebsiella pneumoniae NifL under Nitrogen-Fixing Conditions from Mutational Analysis, JOURNAL OF BACTERIOLOGY, Feb. 2011, p. 695–705
  4. ^ Ruth A. Schmitz*, Kai Klopprogge, and Roman Grabbe,Regulation of Nitrogen Fixation in Klebsiella pneumoniae and Azotobacter vinelandii: NifL, Transducing Two Environmental Signals to the nif Transcriptional Activator NifA,J. Mol. Microbiol. Biotechnol. (2002) 4(3): 235–242
  5. ^ Janet Deistung and Roger N.F.Thorneley,Electron Transfer to nitrogenase,Biochem J.(1986)239,69-75

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