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In Klebsiella pneumoniae, the flavoprotein, NifL regulates NifA mediated transcriptional activation of the N2-fixation (nif) genes in response tomolecular O2and ammonium.We investigated the influence of membrane-bound oxidoreduc-tases on nif-regulation by biochemical analysis of purified NifLandbymonitoringNifA-mediated expressionofnifH¢-¢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.pneumoniaecells,. | Eur. J. Biochem. 270 1555-1566 2003 FEBS 2003 doi 10.1046 j.1432-1033.2003.03520.x 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 Roman Grabbe and Ruth A. Schmitz Institut fur Mikrobiologie und Genetik Georg-August Universitdt Gottingen Germany 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 oxidoreduc-tases 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. pnuimoonÙK cells indicating that in vivo NifL is reduced by electrons derived from membrane-bound oxidoreductases of the anaerobic respiratory chain. Thís mcchanism is further supported by three lines of evidence First K. ppnuuetonÙK strains carrying null mutations of fdnG or nuoCD showed significantly reduced nf-induction under derepressing conditions indicating that NifL inhibition of NifA was not relieved in the absence of formate dehydrogenase-N or NADH ubiqui-none oxidoreductase. The smme effect was vec in a heterologous Escherichia coli system carrying a ndh null allele coding for NADH dehydrogenaseII . Second tttidying nif-induction in K. liitemiioiiiae revealed that during anaerobic growth in glycerol under nitrogen-limitation the presence of the terminal electron acceptor nitrate resulted in a significant decrease of nzf-induction. The hntll ine of evidence is that reduced quinone derivatives dimethylnaphthoquinol and menadiol are able to transfer electrons to the FAD-moiety of purified NifL. On the baiís fl theee data we postulate that under anaerobic and .
