Studying the interactions between regulatory proteins of nitrogen fixation in Klebsiella pneumoniae to gain deeper insights in the molecular mechanisms of regulation
Final Report Abstract
In Klebsiella pneumoniae the nif-gene transcriptional activator NifA is controlled by the two regulators, the antagonist NifL and the PII-protein GlnK, which represents the nitrogen sensory protein for nif-gene regulation. NifL antagonizes cytoplasmic NifA in the presence of ammonium and / or molecular oxygen by direct protein interaction leading to a transcriptionally non-active NifL/NifA complex. Upon anaerobiosis and simultaneous absence of combined nitrogen, conditions under which nitrogen fixation occurs, NifL is sequestered to the cytoplasmic membrane leading to active NifA in the cytoplasm. The respective release of NifL from the NifL/NifA complex under nitrogen limiting conditions requires the nitrogen status sensing GlnK that dissociates the NifL/NifA complex by direct protein-protein interaction. Under oxygen limiting conditions, NifL is reduced at the cytoplasmic membrane by the menachinol pool resulting in a conformational change and sequestration to the cytoplasmic membrane. The aim of this project was to focus on GlnK and its protein-protein interaction with both regulatory protein partners to gain insights into the proposed dissociation of GlnK from NifL upon NifL reduction at the cytoplasmic membrane. Interaction studies between reduced NifL protein and GlnK under anaerobic conditions strongly supported that upon reduction of NifL protein GlnK is no longer binding to NifL and is not co-sequestered to the cytoplasmic membrane by NifL. The second part of this project focused on the interaction of GlnK with NifA and NifL by mutational analysis. We aimed to identify amino acids crucial for the interaction by screening for suppressor mutations in NifL or NifA using genetic backgrounds with three different glnK mutations, which result in a nif- phenotype. By generating independent mutation pools of nifA and nifL on a plasmid and establishing an efficient screening system - selecting for growth of the chromosomal glnK mutants under nitrogen and oxygen limiting conditions – 293 amino acids of NifL and NifA that are apparently essential for the interaction with GlnK were identified out of approximately 5.600 idifferent mutants. Sequence analysis showed that - in case of NifL -suppressor mutations were obtained, which are unique for the respective GlnK mutation, located over the whole protein length – with the exception of the Q-linker mutations, which were not unique for the specific GlnK mutant. Further analysing several selected GlnK*/ NifL suppressor partners in binding assays by quantitative pull down approaches, demonstrated that only few combinations were able to restore binding affinity of the two wild type proteins. This is due to several identified false positive suppressor mutations in NifL, which showed significantly less effective binding of the NifL mutant protein to NifA - also resulting in restoring the nif phenotype - and are thus not directly suppressing the respective GlnK mutation but reduce the inhibitory effect of NifL on NifA. These interaction studies are still ongoing for the complete set of identified amino acids. Once the identified crucial amino acids of NifL and NifA are confirmed, the final step is to model the interaction between GlnK and NifL and NifA taking the predicted structure into account.
