The methanogenic archaeon Methanosarcina mazei strain Gö1 contains two homologues of NrpR, the transcriptional repressor of nitrogen assimilation genes recently discovered and characterized in Methanococcus maripaludis. Insertion of a puromycin-resistance conferring cassette into MM1085 encoding a single NrpR domain with an N-terminal helix-turn-helix domain (NrpRI) lead to a significant reduction of the lag-phase after a shift from nitrogen sufficiency to nitrogen limitation. Consistent with this finding, loss of NrpRI resulted in significantly increased transcript levels of genes involved in nitrogen fixation or nitrogen assimilation though growing under nitrogen sufficiency as demonstrated by quantitative reverse transcriptional PCR analysis. Genome-wide analysis using DNA-microarrays confirmed that transcript levels of 27 ORFs were significantly elevated in the M. mazei MM1085::pac mutant under nitrogen sufficiency, including genes known to be up-regulated under nitrogen limitation (e.g., nifH, glnA(1), glnK(1)), and 17 additional genes involved in metabolism (4), encoding a flagella related protein (1) and genes encoding hypothetical proteins (12). Using cell extracts of Escherichia coli expressing MM1085 fused to the maltose binding protein (MBP-NrpRI) and employing promoter binding studies by DNA-affinity chromatography demonstrated that MBP-NrpRI binds specifically to the nifH-promoter. Deletion of various bases in the promoter region of nifH confirmed that the regulatory element ACC-N(7)-GGT is required for specific binding of NrpRI to the promoter.

We report here on the formation of a complex between the two NrpR homologs present in Methanosarcina mazei Go¨1 and their binding properties to the nifH and glnK 1 promoters. Reciprocal co-chromatography demonstrated that NrpRI forms stable complexes with NrpRII (at an NrpRI : NrpRII molar ratio of 1 : 3), which are not affected by 2-oxoglutarate. Promoter-binding, analyses using DNA-affinity chromatography and electrophoretic gel mobility shift assays, verified that NrpRII is not able to bind to either the nifH promoter or the glnK 1 promoter except when in complex with NrpRI. Specific binding of NrpRI to the nifH and glnK 1 promoters was shown to be highly sensitive to 2-oxoglutarate, regardless of whether only NrpRI, or NrpRI in complex with NrpRII, bound to the promoter. Finally, strong interactions between NrpRII and the general transcription factors TATA-binding proteins (TBP) 1-3 and the general transcription factor TFIIB (TFB) were demonstrated, interactions which are also sensitive to 2-oxoglutarate. On the basis of these findings we propose the following: under nitrogen sufficiency NrpRII binds from solution to either the nifH promoter or the glnK 1 promoter by simultaneously contacting NrpRI and TBP plus TFB, resulting in full repression of transcription; whereas, under nitrogen limitation, increasing 2-oxoglutarate concentrations significantly decrease the binding of NrpRI to the operator as well as the binding of NrpRII to TBP and TFB, ultimately allowing recruitment of RNA polymerase to the promoter.