title: 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.
abstract: 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.
authors.affiliation: Institut für Mikrobiologie und Genetik, Georg-August Universität Göttingen, Germany.
authors.forename: Roman
authors.initials: R
authors.lastname: Grabbe
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Schmitz
mesh_terms: D000693:Anaerobiosis / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics* / Q000378:metabolism*; D042967:Electron Transport Complex I; D004583:Electrons; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D007711:Klebsiella pneumoniae / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000235:genetics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011809:Quinones; C084296:nifL protein, Bacteria; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D005560:Formate Dehydrogenases; D009247:NADH, NADPH Oxidoreductases; C088908:NADH dehydrogenase II; D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I; D010100:Oxygen