title: Role of GlnK in NifL-mediated regulation of NifA activity in Azotobacter vinelandii.
abstract: In several diazotrophic species of Proteobacteria, P(II) signal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH(4)(+) by several mechanisms. In Azotobacter vinelandii, expression of nifA, encoding the nif-specific activator, is constitutive, and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control. In vitro and genetic evidence suggests that the nitrogen response involves the P(II)-like GlnK protein and GlnD (uridylyltransferase/uridylyl-removing enzyme), which reversibly uridylylates GlnK in response to nitrogen limitation. Here, the roles of GlnK and GlnK-UMP in A. vinelandii were studied to determine whether the Nif (-) phenotype of glnD strains was due to an inability to modify GlnK, an effort previously hampered because glnK is an essential gene in this organism. A glnKY51F mutation, encoding an unuridylylatable form of the protein, was stable only in a strain in which glutamine synthetase activity is not inhibited by NH(4)(+), suggesting that GlnK-UMP is required to signal adenylyltransferase/adenylyl-removing enzyme-mediated deadenylylation. glnKY51F strains were significantly impaired for diazotrophic growth and expression of a nifH-lacZ fusion. NifL interacted with GlnK and GlnKY51F in a yeast two-hybrid system. Together, these data are consistent with those obtained from in vitro experiments (Little et al., EMBO J., 19:6041-6050, 2000) and support a model for regulation of NifA activity in which unmodified GlnK stimulates NifL inhibition and uridylylation of GlnK in response to nitrogen limitation prevents this function. This model is distinct from one proposed for the related bacterium Klebsiella pneumoniae, in which unmodified GlnK relieves NifL inhibition instead of stimulating it.
authors.affiliation: Department of Plant Pathology, The University of Arizona, Tucson, AZ 85721, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Rudnick
authors.forename: Christopher
authors.initials: C
authors.lastname: Kunz
authors.forename: Malkanthi K
authors.initials: MK
authors.lastname: Gunatilaka
authors.forename: Eric R
authors.initials: ER
authors.lastname: Hines
authors.forename: Christina
authors.initials: C
authors.lastname: Kennedy
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000235:genetics; D009586:Nitrogen Fixation / Q000235:genetics*; D009713:Nucleotidyltransferases / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D013489:Suppression, Genetic; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D020798:Two-Hybrid System Techniques; D014542:Uridine Monophosphate / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C113488:GlnK protein, Azorhizobium caulinodans; C061338:NifA protein, Bacteria; D014157:Transcription Factors; C084296:nifL protein, Bacteria; D000249:Adenosine Monophosphate; D014542:Uridine Monophosphate; D009713:Nucleotidyltransferases; D005974:Glutamate-Ammonia Ligase