tailieunhanh - Báo cáo khoa học: Towards understanding the nitrogen signal transduction for nif gene expression in Klebsiella pneumoniae

In the diazotrophKlebsiella 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 ammo-nium and molecular oxygen. | ỊFEBS Journal Towards understanding the nitrogen signal transduction for nif gene expression in Klebsiella pneumoniae Jens Gloer Robert Thummer Heike Ullrich and Ruth A. Schmitz Institut fur Allgemeine Mikrobiologie Christian-Albrechts-Universitat Kiel Germany Keywords GlnK Klebsiella pneumoniae nif gene transcriptional control NifA NifL Correspondence R. A. Schmitz Institut fur Allgemeine Mikrobiologie Christian-Albrechts-Universitat Kiel Am Botanischen Garten 1-9 24118 Kiel Germany Fax 49 431 8802194 Tel 49 431 8804334 E-mail rschmitz@ Website http mikrobio Received 22 July 2008 revised 17 October 2008 accepted 22 October 2008 doi 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. pneumoniae DglnK 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