title: Towards understanding the nitrogen signal transduction for nif gene expression in Klebsiella pneumoniae.
abstract: In the diazotroph Klebsiella pneumoniae, the nitrogen sensory protein GlnK mediates the cellular nitrogen status towards the NifL/NifA system that regulates transcription of the nitrogen fixation genes in response to ammonium and molecular oxygen. To identify amino acids of GlnK essential for this signal transduction by protein-protein interaction, we performed random point mutagenesis by PCR amplification under conditions of reduced Taq polymerase fidelity. Three thousand two hundred mutated glnK genes were screened to identify those that would no longer complement a K. pneumoniaeDeltaglnK strain for growth under nitrogen fixing conditions. Twenty-four candidates resulting in a Nif(-) phenotype were identified, carrying 1-11 amino acid changes in GlnK. Based on these findings, as well as structural data, several single mutations were introduced into glnK by site-directed mutagenesis, and the Nif phenotype and the respective effects on NifA-mediated nif gene induction was monitored in K. pneumoniae using a chromosomal nifK'-'lacZ fusion. Single amino acid changes resulting in significant nif gene inhibition under nitrogen limiting conditions were located within the highly conserved T-loop (A43G, A49T and N54D), the body of the protein (G87V and K79E) and in the C-terminal region (I100M, R103S, E106Q and D108G). Complex formation analyses between GlnK (wild-type or derivatives) and NifL or NifA in response to 2-oxoglutarate indicated that: (a) besides the T-loop, the C-terminal region of GlnK is essential for the interaction with NifL and NifA and (b) GlnK binds both proteins in the absence of 2-oxoglutarate, whereas, in the presence of 2-oxoglutarate, NifA is released but NifL remains bound to GlnK.
authors.affiliation: Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Glöer
authors.forename: Robert
authors.initials: R
authors.lastname: Thummer
authors.forename: Heike
authors.initials: H
authors.lastname: Ullrich
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Schmitz
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D004579:Electron Transport; D015964:Gene Expression Regulation, Bacterial; D014644:Genetic Variation; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008958:Models, Molecular; D009584:Nitrogen / Q000378:metabolism; D009586:Nitrogen Fixation / Q000235:genetics; D011487:Protein Conformation; D015398:Signal Transduction / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; C061338:NifA protein, Bacteria; D014157:Transcription Factors; C084296:nifL protein, Bacteria; D009584:Nitrogen