Methanosarcina mazei and related mesophilic archaea are the only organisms fermenting acetate, methylamines, and methanol to methane and carbon dioxide, contributing significantly to greenhouse gas production. The biochemistry of these metabolic processes is well studied, and genome sequences are available, yet little is known about the overall transcriptional organization and the noncoding regions representing 25% of the 4.01-Mb genome of M. mazei. We present a genome-wide analysis of transcription start sites (TSS) in M. mazei grown under different nitrogen availabilities. Pyrosequencing-based differential analysis of primary vs. processed 5 ends of transcripts discovered 876 TSS across the M. mazei genome. Unlike in other archaea, in which leaderless mRNAs are prevalent, the majority of the detected mRNAs in M. mazei carry long untranslated 5 regions. Our experimental data predict a total of 208 small RNA (sRNA) candidates, mostly from intergenic regions but also antisense to 5 and 3 regions of mRNAs. In addition, 40 new small mRNAs with ORFs of <30 aa were identified, some of which might have dual functions as mRNA and regulatory sRNA. We confirmed differential expression of several sRNA genes in response to nitrogen availability. Inspection of their promoter regions revealed a unique conserved sequence motif associated with nitrogen-responsive regulation, which might serve as a regulator binding site upstream of the common IIB recognition element. Strikingly, several sRNAs antisense to mRNAs encoding transposases indicate nitrogen-dependent transposition events. This global TSS map in archaea will facilitate a better understanding of transcriptional and posttranscriptional control in the third domain of life.Methanosarcina mazei strain Gö1 is a representative methaneproducing archaeon of ecologic significance because of its role in biogenic methane production in various anaerobic habitats on Earth (1). The genome sequences of M. mazei and its close relatives Methanosarcina acetivorans and Methanosarcina barkeri have recently become available and have revealed an unexpected low proportion of coding region (74.2% in M. acetivorans, 75.15% in M. mazei, and 79.2% in M. barkeri) (2-4). The biochemical basis of methanogenesis has been analyzed in considerable detail (5, 6). In contrast, little is known about global regulatory networks that ensure survival in periods of nutrient starvation or stress in this important group of archaea. More than 50 predicted transcriptional regulators were annotated in the genome of M. mazei. 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.Bes...

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.

No abstract

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.