Regulation of nitrogen fixation in Klebsiella pneumoniae: NifL, one protein transducing two environmental signals to the nif-transcriptional activator NifA

Klebsiella pneumoniae, NifL modulates the activity of the transcriptional activator NifA in response to combined nitrogen or external molecular oxygen. We recently showed that K. pneumoniae NifL is a flavoprotein which apparently senses oxygen through a redox-sensitive, conformational change. We characterized the redox properties of NifL synthesized in Escherichia coli in the presence of different nitrogen sources. FAD analyses showed that purified NifL carried FAD as cofactor independent of nitrogen and oxygen availability. The redox potential of NifL synthesized in the presence of ammonium was 277 ±5 mV at pH 8.0 and 25° C, as determined by reduction with dithionite or with enzymatic reduction by xanthine oxidase in the presence of methyl viologen as redox mediator. When synthesized under nitrogen-limiting conditions, NifL showed a redox potential of - 274 ±6 mV at pH 8.0 and 25° C. Fully reduced NifL fractions, synthesized under either condition listed above, reoxidized rapidly in the presence of molecular oxygen. These results indicate that for NifL synthesized in E. coli, the redox potential of the NifL-bound FAD is not influenced by the nitrogen source. The two NifL fractions differed, however, in that a non-flavin specific absorbance at 420 nm was found only in NifL synthesized in the presence of ammonium. By monitoring the inhibition of NifA-mediated expression of K. pneumoniae o(nifH-lacZ) fusions in different genetic backgrounds we have now studied the oxygen regulation of NifL activity in E. coli and K. pneumoniae strains. 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--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(s) whose product(s) function to relieve NifL inhibition by reducing the FAD cofactor under oxygen-limiting conditions. Potentially, these Fnr-dependent gene products could be membrane-bound components of the anaerobic electron transport chain; consequently, in this study we have examined the localization of NifL within the cell under various growth conditions. In K. pneumoniae grown under oxygen and nitrogen-limited conditions approximately 55 % of the total NifL protein was found in the membrane fraction. However, when the cells were grown under aerobic conditions or shifted to nitrogen sufficiency significantly less membrane-associated NifL was detectable (less than 10 % of total NifL). These findings suggest that a significant higher membrane affinity of NifL under derepressing conditions may create a spatial gap between NifL and its target protein NifA impairing inhibition of NifA by NifL. Localization analysis of the primary nitrogen sensor GlnK further showed that under nitrogen-limited conditions but independent of oxygen presence 15 - 20 % of the total GlnK is membrane associated. A shift to nitrogen sufficiency resulted in rapid degradation of the cytosolic GlnK fraction, whereas the membrane associated GlnK fraction displayed a significantly longer half-life. These findings suggest that proteolytic cleavage of cytosolic GlnK may be the mechanism for restoration of NifL activity upon a shift to nitrogen sufficiency.The inhibitory function of Klebsiella pneumoniae NifL on NifA transcriptional activity in vitro increases upon addition of ATP and ADP, when NifL was synthesized under nitrogen sufficiency (NifLNH4). Interestingly, NifLNH4 also hydrolyzes ATP (2500 mU/mg). In contrary, NifL synthesized under nitrogen limitation is not affected by adenine nucleotides and exhibits no ATP hydrolyzing activity. These major differences indicate that stimulation of the inhibitory function and ATP-hydrolysis depend on a specific NifL conformation induced by ammonium. We hypothesize that the presence of ammonium alters the conformation of NifL, enabling NifL to use the energy of ATP-hydrolysis to form NifL-NifA complexes more efficiently.