title: 2-Oxoglutarate and the PII homologues NifI1 and NifI2 regulate nitrogenase activity in cell extracts of Methanococcus maripaludis.
abstract: Summary Post-translational regulation of nitrogen fixation, or switch-off, in the methanogenic archaeon Methanococcus maripaludis does not involve detectable covalent modification of the dinitrogenase reductase as in some bacteria, and the genes encoding the PII homologues NifI(1) and NifI(2) are both required, indicating a novel mechanism. To further understand the mechanism of switch-off, we assayed nitrogenase activity in cell extracts from wild-type and nifI mutant strains in the absence or presence of potential signals of nitrogen status. Activity in extracts from a DeltanifI(1)nifI(2) strain was sixfold higher than in extracts from wild-type cells. Addition of 2-oxoglutarate to wild-type extracts enhanced activity up to fivefold, a level similar to that observed in DeltanifI(1)nifI(2) extracts. 2-Oxoglutarate did not affect activity in DeltanifI(1)nifI(2) or single nifI mutant extracts. Furthermore, extracts from genetically complimented nifI mutants regained wild-type characteristics, indicating an in vitro correlation with in vivo effects. Extraction and quantification of 2-oxoglutarate indicated concentrations 10-fold higher in nitrogen-fixing cells than in switched-off and ammonium-grown cells. We propose a model for switch-off where the NifI proteins have an inhibitory effect on nitrogenase activity that is counteracted by high levels of 2-oxoglutarate, which acts as a signal of nitrogen limitation.
authors.affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Dodsworth
authors.forename: Nathaniel C
authors.initials: NC
authors.lastname: Cady
authors.forename: John A
authors.initials: JA
authors.lastname: Leigh
mesh_terms: D000114:Acetylene / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D019848:Gene Expression Regulation, Archaeal; D005812:Genes, Switch; D007656:Ketoglutaric Acids / Q000378:metabolism*; D017017:Methanococcus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D009154:Mutation; D009586:Nitrogen Fixation / Q000235:genetics; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins
chemical_list: D019843:Archaeal Proteins; D007656:Ketoglutaric Acids; C423625:NifI1 protein, Methanococcus maripaludis; C423626:NifI2 protein, Methanococcus maripaludis; D051922:PII Nitrogen Regulatory Proteins; D009591:Nitrogenase; D000114:Acetylene