Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool

Eur J Biochem. 2003 Apr;270(7):1555-66. doi: 10.1046/j.1432-1033.2003.03520.x.


In Klebsiella pneumoniae, the flavoprotein, NifL regulates NifA mediated transcriptional activation of the N2-fixation (nif) genes in response to molecular O2 and ammonium. We investigated the influence of membrane-bound oxidoreductases on nif-regulation by biochemical analysis of purified NifL and by monitoring NifA-mediated expression of nifH'-'lacZ reporter fusions in different mutant backgrounds. NifL-bound FAD-cofactor was reduced by NADH only in the presence of a redox-mediator or inside-out vesicles derived from anaerobically grown K. pneumoniae cells, indicating that in vivo NifL is reduced by electrons derived from membrane-bound oxidoreductases of the anaerobic respiratory chain. This mechanism is further supported by three lines of evidence: First, K. pneumoniae strains carrying null mutations of fdnG or nuoCD showed significantly reduced nif-induction under derepressing conditions, indicating that NifL inhibition of NifA was not relieved in the absence of formate dehydrogenase-N or NADH:ubiquinone oxidoreductase. The same effect was observed in a heterologous Escherichia coli system carrying a ndh null allele (coding for NADH dehydrogenaseII). Second, studying nif-induction in K. pneumoniae revealed that during anaerobic growth in glycerol, under nitrogen-limitation, the presence of the terminal electron acceptor nitrate resulted in a significant decrease of nif-induction. The final line of evidence is that reduced quinone derivatives, dimethylnaphthoquinol and menadiol, are able to transfer electrons to the FAD-moiety of purified NifL. On the basis of these data, we postulate that under anaerobic and nitrogen-limited conditions, NifL inhibition of NifA activity is relieved by reduction of the FAD-cofactor by electrons derived from the reduced quinone pool, generated by anaerobic respiration, that favours membrane association of NifL. We further hypothesize that the quinol/quinone ratio is important for providing the signal to NifL.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anaerobiosis / physiology
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Cell Membrane / metabolism
  • Cytoplasm / genetics*
  • Cytoplasm / metabolism*
  • Electron Transport Complex I
  • Electrons
  • Escherichia coli / genetics
  • Flavin-Adenine Dinucleotide / metabolism
  • Formate Dehydrogenases / metabolism
  • Gene Expression Regulation, Bacterial / physiology*
  • Klebsiella pneumoniae / metabolism*
  • NAD / metabolism
  • NADH Dehydrogenase / genetics
  • NADH, NADPH Oxidoreductases / metabolism
  • Oxidation-Reduction
  • Oxygen / metabolism*
  • Quinones / metabolism*


  • Bacterial Proteins
  • Quinones
  • nifL protein, Bacteria
  • NAD
  • Flavin-Adenine Dinucleotide
  • Formate Dehydrogenases
  • NADH, NADPH Oxidoreductases
  • NADH dehydrogenase II
  • NADH Dehydrogenase
  • Electron Transport Complex I
  • Oxygen