Insights into the NrpR regulon in Methanosarcina mazei Gö1

Abstract

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.

Deep sequencing analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability

“…A single sRNA, GlmY, has been demonstrated to be transcribed by the 54 -RNA polymerase in Escherichia coli, but independently of the global nitrogen transcription regulator NtrC (34). Consistent with nitrogen-regulated transcription of ORFs by the global nitrogen regulator NrpR in M. mazei, 2 of the verified nitrogen-regulated sRNAs contained the NrpR operator in their promoters (8,35). Furthermore, a new motif (box I), which seems to be associated with nitrogen-responsive regulation, was detected up-and downstream of the BRE and TATA-box of 12 sRNA candidates (Fig.…”

“…Strikingly, most of them seem to be closely related to bacterial proteins (2), whereas the basic components of the archaeal transcription and translation machineries generally are more similar to those of eukaryotes (7). A recent genetic study (8) discovered the first global transcriptional regulator of M. mazei, the nitrogen regulator NrpR, which was experimentally demonstrated to globally repress transcription of nitrogen fixation and assimilation genes in response to the nitrogen source.…”

The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite

“…Genome-wide expression analysis of an M. mazei NrpR1 knockout strain showed elevated transcription of 27 genes under nitrogen sufficiency conditions, including nifH, glnA1, and glnK1 (98). The M. mazei NrpR1 and NrpR2 proteins physically interact and bind as a complex to the nifH and glnK1 promoters in the presence of low 2-OG levels (99).…”

A Functional Approach To Uncover the Low-Temperature Adaptation Strategies of the Archaeon Methanosarcina barkeri

“…Two other DNA binding and repair proteins were found to be upregulated in phase 2 and phase 3 samples compared to phase 1 samples. These included a DNA regulator (Mbar_A1584) involved in transcription (42). General metabo- lism proteins identified in the LTAS study were found to be involved in diverse pathways, including amino acid and vitamin biosynthesis, glycolysis, and electron transport ( Table 3).…”