Genetic screen for regulatory mutations in Methanococcus maripaludis and its use in identification of induction-deficient mutants of the euryarchaeal repressor NrpR.
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.
Lie
Thomas J
TJ
Department of Microbiology, University of Washington, Box 357242, Seattle, WA 98195-7242, USA. leighj@u.washington.edu
Leigh
John A
JA
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*
D019843:Archaeal Proteins; D007656:Ketoglutaric Acids; D012097:Repressor Proteins; D009584:Nitrogen