title: Regulation of nif expression in Methanococcus maripaludis: roles of the euryarchaeal repressor NrpR, 2-oxoglutarate, and two operators.
abstract: The methanogenic archaean Methanococcus maripaludis can use ammonia, alanine, or dinitrogen as a nitrogen source for growth. The euryarchaeal nitrogen repressor NrpR controls the expression of the nif (nitrogen fixation) operon, resulting in full repression with ammonia, intermediate repression with alanine, and derepression with dinitrogen. NrpR binds to two tandem operators in the nif promoter region, nifOR(1) and nifOR(2). Here we have undertaken both in vivo and in vitro approaches to study the way in which NrpR, nifOR(1), nifOR(2), and the effector 2-oxoglutarate (2OG) combine to regulate nif expression, leading to a comprehensive understanding of this archaeal regulatory system. We show that NrpR binds as a dimer to nifOR(1) and cooperatively as two dimers to both operators. Cooperative binding occurs only with both operators present. nifOR(1) has stronger binding and by itself can mediate the repression of nif transcription during growth on ammonia, unlike the weakly binding nifOR(2). However, nifOR(2) in combination with nifOR(1) is critical for intermediate repression during growth on alanine. Accordingly, NrpR binds to both operators together with higher affinity than to nifOR(1) alone. NrpR responds directly to 2OG, which weakens its binding to the operators. Hence, 2OG is an intracellular indicator of nitrogen deficiency and acts as an inducer of nif transcription via NrpR. This model is upheld by the recent finding (J. A. Dodsworth and J. A. Leigh, submitted for publication) in our laboratory that 2OG levels in M. maripaludis vary with growth on different nitrogen sources.
authors.affiliation: Department of Microbiology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Lie
authors.forename: Gwendolyn E
authors.initials: GE
authors.lastname: Wood
authors.forename: John A
authors.initials: JA
authors.lastname: Leigh
mesh_terms: D000409:Alanine / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D019848:Gene Expression Regulation, Archaeal / Q000187:drug effects; D019766:Genes, Archaeal / Q000235:genetics*; D007656:Ketoglutaric Acids / Q000494:pharmacology; D017017:Methanococcus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008957:Models, Genetic; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D009586:Nitrogen Fixation / Q000235:genetics; D009875:Operator Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D007656:Ketoglutaric Acids; D012097:Repressor Proteins; D004247:DNA; D000409:Alanine