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

In Klebsiella pneumoniae, the flavoprotein, NifL regulates NifA mediated transcriptional activation of the N 2 -fixation (nif) genes in response to molecular O 2 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

Regulation der Stickstoffixierung in <i>Klebsiella pneumoniae</i>: die Rolle von Fnr in der Sauerstoffsignaltransduktion

In Klebsiella pneumoniae wird die Aktivität des Transkriptionsaktivators NifA durch NifL in Abhängigkeit von gebundenem Stickstoff und molekularem Sauerstoff geregelt. NifL, ein Flavoprotein, das redox-sensitiv ist, scheint, das Sauerstoffsignal über eine Konformationsänderung auf NifA zu übertragen. Kürzlich konnten wir zeigen, dass in einer K. pneumoniae fnr null Mutante NifL das Signal für Sauerstofflimitierung nicht erhält, was zu einer inhibitorischen Konformation des Proteins führt. Dies bedeutet, dass Fnr, indirekt oder direkt, in diese Signalübertragung involviert ist. Wir nehmen an, dass Fnr als primärer Sauerstoffsensor Gene der anaeroben Atmungskette kontrolliert, die wiederum Elektronen für die Reduktion des FAD-Cofaktors in NifL zur Verfügung stellen. Interessanterweise findet sich in einer unter Sauerstoff- und Stickstofflimitierung gewachsenen fnr Mutante bis zu 95% des NifL Proteins im Cytoplasma, während im Wildtyp 55% des Regulators in der Membranfraktion nachgewiesen werden können. In der Mutante wird der räumliche Abstand zwischen NifL und NifA, der letztlich NifA die Enhancer Funktion in der nif Gene Transkription ermöglicht, verhindert.Unter Sauerstofflimitation ist NifL also hauptsächlich an der Membran lokalisiert, wobei sein FAD-Cofaktor reduziert wird und NifL so in seiner nicht-inhibitorischen Konformation verweilt. Um diese Reduktion nachzuweisen, untersuchten wir die Übertragung von Elektronen von reduzierten invertierten Membranvesikeln bzw. Quinolen auf den FAD-Cofaktor von NifL. Wir konnten eindeutig zeigen, dass sowohl Vesikel als auch Quinole in der Lage sind, Elektronen von NADH auf NifL zu übertragen. Dies unterstützt die Theorie der Reduktion von NifL mit Elektronen aus Quinol-Poolen, die durch die anaerobe Atmungskette generiert werden, an der Cytoplasmamembran. Unterstützt wird diese These durch NifA-abhängige Expressionsstudien mit K. pneumoniae Stämmen, die Null-Mutationen in Oxidoreduktasen, z.B. NADH-Oxidoreduktase, der anaeroben Atmung aufweisen. In diesen Stämmen sind die Expressionslevel nahezu identisch mit denen des Wildtyp Stammes unter aeroben Bedingungen.Wir schlagen deshalb ein Model vor, in dem NifL an der cytoplasmatischen Membran durch Elektronen aus dem Quinol-Pool reduziert wird. Gespeist wird dieser Pool mit Elektronen der anaeroben Atmungskette

Characterization of GlnK(1) from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK(1)

Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M. mazei GlnK(1) is able to functionally complement an Escherichia coli glnK mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of E. coli GlnK. M. mazei GlnK(1) is encoded in the glnK(1)-amtB(1) operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal glnK(1) shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK(1) was cloned and overexpressed in E. coli, and GlnK(1) was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK(1)-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK(1) indicated significant differences compared to E. coli GlnK. First, GlnK(1) from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK(1) and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK(1) was able to complement growth of an E. coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function

Fnr Is Required for NifL-Dependent Oxygen Control of nif Gene Expression in Klebsiella pneumoniae

In Klebsiella pneumoniae, NifA-dependent transcription of nitrogen fixation (nif) genes is inhibited by NifL in response to molecular oxygen and combined nitrogen. We recently showed that K. pneumoniae NifL is a flavoprotein, which apparently senses oxygen through a redox-sensitive, conformational change. We have now studied the oxygen regulation of NifL activity in Escherichia coli and K. pneumoniae strains by monitoring its inhibition of NifA-mediated expression of K. pneumoniae ø(nifH′-′lacZ) fusions in different genetic backgrounds. Strains of both organisms carrying fnr null mutations failed to release NifL inhibition of NifA transcriptional activity under oxygen limitation: nif induction was similar to the induction under aerobic conditions. When the transcriptional regulator Fnr was synthesized from a plasmid, it was able to complement, i.e., to relieve NifL inhibition in the fnr mutant backgrounds. Hence, Fnr appears to be involved, directly or indirectly, in NifL-dependent oxygen regulation of nif gene expression in K. pneumoniae. The data indicate that in the absence of Fnr, NifL apparently does not receive the signal for anaerobiosis. We therefore hypothesize that in the absence of oxygen, Fnr, as the primary oxygen sensor, activates transcription of a gene or genes whose product or products function to relieve NifL inhibition by reducing the flavin adenine dinucleotide cofactor under oxygen-limiting conditions