title: Genetic screen for regulatory mutations in Methanococcus maripaludis and its use in identification of induction-deficient mutants of the euryarchaeal repressor NrpR.
abstract: NrpR is an euryarchaeal transcriptional repressor of nitrogen assimilation genes. Previous studies with Methanococcus maripaludis demonstrated that NrpR binds to palindromic operator sequences, blocking transcription initiation. The metabolite 2-oxoglutarate, an indicator of cellular nitrogen deficiency, induces transcription by lowering the affinity of NrpR for operator DNA. In this report we build on existing genetic tools for M. maripaludis to develop a screen for change-of-function mutations in a transcriptional regulator and demonstrate the use of an X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) screen for strict anaerobes. We use the approach to address the primary structural requirements for the response of NrpR to 2-oxoglutarate. nrpR genes from the mesophilic M. maripaludis and the hyperthermophilic Methanopyrus kandleri were targeted for mutagenesis. M. maripaludis nrpR encodes a protein with two homologous NrpR domains while the M. kandleri nrpR homolog encodes a single NrpR domain. Random point mutagenesis and alanine replacement mutagenesis identified two amino acid residues of M. kandleri NrpR involved in induction of gene expression under nitrogen-deficient conditions and thus in the response to 2-oxoglutarate. Mutagenesis of the corresponding regions in either domain of M. maripaludis NrpR resulted in a similar effect, demonstrating a conserved structure-function relationship between the two repressors. The results indicate that in M. maripaludis, both NrpR domains participate in the 2-oxoglutarate response. The approach used here has wide adaptability to other regulatory systems in methanogenic Archaea and other strict anaerobes.
authors.affiliation: Department of Microbiology, University of Washington, Box 357242, Seattle, WA 98195-7242, USA. leighj@u.washington.edu
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Lie
authors.forename: John A
authors.initials: JA
authors.lastname: Leigh
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019848:Gene Expression Regulation, Archaeal; D019766:Genes, Archaeal; D007656:Ketoglutaric Acids / Q000378:metabolism / Q000494:pharmacology; D017017:Methanococcus / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D007656:Ketoglutaric Acids; D012097:Repressor Proteins; D009584:Nitrogen