Regulation of nitrogen fixation in Klebsiella pneumoniae: Nitrogen and oxygen signal perception by the negative regulator NifL

In the diazotroph Klebsiella pneumoniae, the nif specific transcriptional activator NifA is inhibited by the flavoprotein NifL in response to molecular oxygen and ammonium. We demonstrated that complexes between NifL and NifA (approximately 1:1 ratio) are formed, when synthesized in the presence of oxygen and/or ammonium. In contrast, under oxygen- and nitrogen-limitation, significant but fewer NifL/NifA-complexes (approximately 1 %) are found in the cytoplasm because the majority of NifL is sequestered to the cytoplasmic membrane. These findings indicate that inhibition of NifA in the presence of oxygen and/or ammonium occurs via direct NifL interaction and formation of those inhibitory NifL/NifA-complexes appears to be directly and exclusively dependent on the localization of NifL in the cytoplasm. To get deeper insights into the mechanism of sequestration of the reduced regulator NifL to the cytoplasmic membrane we analyzed whether the electrons required directly derived from the reduced menaquinone pool. By using an artificial membrane system containing purified components of the anaerobic respiratory chain of Wolinella succinogenes the reduction of NifL was studied. In an in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK6) or 8-methyl-menaquinone (MMK6) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation and monitored using UV-vis spectra. The resulting reduction rates, which were determined using equal amounts of NifL, elucidated that reduction of NifL is directly dependent on both, formate dehydrogenase and menaquinones incorporated into the liposomes. These findings strongly indicate that electrons are directly transferred from menaquinones to the FAD cofactor of NifL. Further we demonstrated that reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance to previous findings in vivo. On the basis of these experiments we propose that the redox state of the menaquinone pool is the redox signal for nif-regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions. In order to discover amino acids which are essential for membrane association or FAD binding of NifL we performed a random mutagenesis of the nifL-gene yielding amino acid substitutions in the regulatory protein NifL. Overall, 11.500 clones out of three independently generated pools carrying plasmids containing randomly mutated nifL-genes were screened for mutants conferring a nif - - phenotype under nitrogen fixing conditions. After several independent growth experiments with molecular nitrogen as sole nitrogen source we identified and confirmed 26 mutants showing no nitrogen fixation at all and 41 mutants showing significantly reduced nitrogen fixation. By sequence analyses we identified amino acid substitutions located mainly within the N-terminal domain, which is considered to interact with the cytoplasmic membrane and which contains the FAD cofactor, and selected parts of these mutations for separation by site directed mutagenesis to clarify their functions. For the transduction of the nitrogen signal to the nif-regulatory system the nitrogen sensory protein GlnK is required. We observed evidence that GlnK forms trimeric complexes with NifL and NifA under nitrogen limitation. The binding of GlnK to NifL/NifA was specific, however, the amount of GlnK within these complexes was small. Finally, two lines of evidence were obtained that under anaerobic conditions but in the presence of ammonium additional NtrC-independent GlnK synthesis inhibited the formation of stable inhibitory NifL/NifA-complexes. Thus, we propose that the NifL/NifA/GlnK-complex reflects a transitional structure and hypothesize that under nitrogen-limitation, GlnK interacts with the inhibitory NifL/NifA-complex, resulting in its dissociation.