Membrane association of Klebsiella pneumoniae NifL is affected by molecular oxygen and combined nitrogen

In the diazotroph Klebsiella pneumoniae, NifL and NifA regulate transcription of the nitrogen fixation genes in response to molecular oxygen and combined nitrogen. We recently showed that Fnr is the primary oxygen sensor. Fnr transduces the oxygen signal towards the negative regulator NifL by activating genes whose products reduce the FAD moiety of NifL under anoxic conditions. These Fnr-dependent gene products could be membrane-bound components of the anaerobic electron transport chain. Consequently, in this study we 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 aerobically or shifted to nitrogen sufficiency, less than 10% of total NifL was membrane-associated. In contrast to NifL, NifA was located in the cytoplasm under all growth conditions tested. Further studies using K. pneumoniae mutant strains showed that, under derepressing conditions but in the absence of either the primary oxygen sensor Fnr or the primary nitrogen sensor GlnK and the ammonium transporter AmtB, NifL was located in the cytoplasm and inhibited NifA activity. These findings suggest that under nitrogen- and oxygen-limitation, a significantly higher membrane affinity of NifL might create a spatial gap between NifL and its cytoplasmic target protein NifA, thereby impairing inhibition of NifA by NifL. Localization of GlnK further showed that, under nitrogen-limited conditions but independent of the presence of oxygen, 15-20% of the total GlnK is membrane-associated.