Global transcriptional analysis of Methanosarcina mazei strain Gö1 under different nitrogen availabilities

Abstract

Certain archaeal species can fix molecular nitrogen under nitrogen limiting conditions although little is known about this process at either the genetic or molecular level. To address this on a genome-wide scale, transcriptional analysis was performed on the model methanogen Methanosarcina mazei strain Gö1 using DNA-microarrays. The genomic expression patterns for cells grown under nitrogen fixing conditions versus nitrogen sufficiency (10 mM ammonium) revealed that approximately 5% of all genes are differentially expressed. Besides a small set of genes previously known to be up-regulated under nitrogen limitation, 14 additional genes involved in nitrogen metabolism were identified plus 10 genes encoding potential transcriptional regulators, 13 genes involved in carbon metabolism, 3 genes in general stress response, 8 putative transporter genes, and an additional 21 genes with unknown function. Quantitative reverse transcriptase PCR experiments confirmed the differential expression of a subset of these genes. Promoter analysis revealed a palindromic DNA motif centered nearby the transcriptional start point for several genes up-regulated under nitrogen limitation. A bioinformatics study demonstrated the presence of this motif in the up-stream region of 52 genes genome-wide, the majority of which showed nitrogen dependent differential transcription. We therefore hypothesize that this DNA element is involved in nitrogen control in M. mazei where it may act as a binding site for a regulatory protein.

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

“…1 illustrates TSS identification and nitrogen source-dependent transcription regulation of glnA 1 (encoding glutamine synthetase). Nitrogenresponsive regulation of glnA 1 was previously shown by quantitative RT-PCR analysis (25) and is verified here by Northern blot probing (Fig. 1).…”

“…Of the cDNAs, 26.4% (NF) and 31.6% (NS) mapped to intergenic regions (IGRs) of the genome in the enriched (ϩ) libraries, as compared with 21% and 28%, respectively, in the cognate nonenriched (Ϫ) libraries, indicating depletion of processed rRNAs and tRNAs in favor of IGR transcripts. Furthermore, a strong enrichment of cDNAs clustering toward the TSS of several mRNA genes analyzed in previous studies was observed [e.g., of grpE and glnA 1 (24,25)]; the 5Ј ends of the grpE and glnA 1 cDNA clusters exactly match the previously identified TSS (Table S1). Fig.…”

“…M. mazei strain Gö 1 was grown under anaerobic conditions at 37°C with an atmosphere of 80% N 2 plus 20% CO2 in 70-mL sealed tubes in minimal medium that contained 150 mM methanol plus 40 mM acetate as carbon source and 10 mM ammonium chloride (N sufficiency); for nitrogenfixing conditions the gas atmosphere served as sole nitrogen source (25). Cultures were grown until cells reached a turbidity of 0.18 -0.21 (N fixation) or 0.5-0.6 (N sufficiency) at 600 nm, which corresponds to the respective midexponential growth phase.…”

“…Cells were harvested at 4°C and RNA isolated by phenol extraction followed by DNase I treatment (49). Before cDNA construction, differential transcription of the glnK 1 gene was verified by quantitative RT-PCR as described previously (25).…”

Global Transcriptomic and Proteomic Responses of Dehalococcoides ethenogenes Strain 195 to Fixed Nitrogen Limitation

“…This PII homolog is expected to regulate the ammonium transporter (DET1125) and the GS-GOGAT pathway (25). The upregulation of all three PII proteins should allow cells to respond immediately to changes in ammonium concentrations, and the differential upregulation of PII proteins is commonly observed for the Bacteria and Archaea under conditions of nitrogen stress (31,51,52).…”