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.

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The methanogenic archaeon Methanosarcina mazei strain Gö1 has so far proven to be genetically intractable due to its low plating efficiency on solid medium and the lack of an effective transformation method. Here, we report the first significant improvement in plating efficiency (up to 10%), which was achieved by (1) selecting for a spontaneous mutant of M. mazei that shows significantly higher resistance to mechanical stress during spreading an agar plates, and (2) plating the cells in 0.5% top agar with trimethylamine as a carbon and energy source under a H2S-containing atmosphere (0.1%). Using this mutant we succeeded in establishing a liposome-mediated transformation protocol, which for the first time allowed genetic manipulation of the M. mazei Gö1 strain. We further report on the construction of the first chromosomal deletion mutant of M. mazei by means of homologous recombination. Characterization of this mutant strain revealed that M. mazei cells lacking a functional glnK1-gene exhibited a partial growth defect under nitrogen limitation when molecular nitrogen was used as the sole nitrogen source. Quantitative RT-PCR analysis, however, showed that genes involved in nitrogen assimilation or nitrogen fixation are transcribed in the glnK1 mutant as in the wild type. Thus, we propose that the archaeal GlnK1 protein is not directly involved in the transcriptional regulation of genes involved in nitrogen metabolism, but rather affects their protein products directly.