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 o(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. In diazotrophic proteobacteria, transcription of the nitrogen fixation (nif) genes is mediated by the nif-specific activator protein NifA, a member of a family of activators that functions with s 54 (2, 4). Both the expression and the activity of NifA can be regulated in response to the oxygen and/or combined nitrogen status of the cells; the mechanisms of the regulation differ with the organism. In Klebsiella pneumoniae and Azotobacter vinelandii, NifA transcriptional activity is regulated by a second regulatory protein, NifL. This negative regulator of the nif genes inhibits the transcriptional activation by NifA in response to combined nitrogen and/or external molecular oxygen. The translationally coupled synthesis of the two regulatory proteins, immunological studies, complex analyses, and studies using the two-hybrid system in Saccharomyces cerivisiae imply that the inhibition of NifA activity by NifL apparently occurs via direct protein-protein interaction (5, 11, 21, 26). The mechanism by which nitrogen is sensed in K. pneumoniae and A. vinelandii is currently the subject of extensive studies. Very recently, He et al. (10), and Jack et al. (15) provided evidence that in K. pneumoniae, the second PII protein, GlnK, is required for relief of NifL inhibition under nitrogen-limiting conditions. This indicates that GlnK regulates NifL inhibition of NifA in response to the nitrogen status of the cells by interacting with NifL or NifA. In both organisms, K. pneumoniae and A. vinelandii, the negative regulator NifL is a flavoprotein with an N-terminally bound flavin adenine dinucleotide (FAD) as a prosthetic group