Development of genetic methods and construction of a chromosomal glnK1 mutant in Methanosarcina mazei strain Gö1

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.

In vitroproof of direct regulation of glutamine synthetase by GlnK proteins in the extreme halophilic archaeonHaloferax mediterranei

“…To date, and as far as we know, no PII homologue from a halophilic organism has been studied and the only known representatives from the archaeal domain is M. mazei PII proteins [8,15,16].…”

The Homotetrameric Phosphoseryl-tRNA Synthetase from Methanosarcina mazei Exhibits Half-of-the-sites Activity

“…An experimental genetic system has been established for this mesophilic methanogen, permitting correlative in vivo studies (24). Further, M. mazei (and Methanosarcina barkeri) possess both redundant pathways for synthesis of CystRNA Cys and three distinct tRNA Cys isoacceptors in their genomes (25,26), suggesting that the properties of SepRS from these organisms may be of particular interest.…”

Establishing a Markerless Genetic Exchange System forMethanosarcina mazeiStrain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes

“…Plasmid DNA was transformed into E. coli according to the method of Inoue et al [14] and into M. mazei using liposome-mediated transformation as described recently [8, 15]. …”

“…In order to provide the hpt gene of pRS311 with a strong archaeal promoter, the known p mcr promoter of Methanococcus voltae [9] was cloned upstream of the gene. This was achieved by amplifying p mcr with the primers pmcr BamHI and pmcr XhoI using pRS207 [8] as template. The PCR product was cloned into TOPO-TA-cloning vector pDRIVE (Qiagen, Hilden, Germany) yielding plasmid pRS269.…”

“…It serves as an archaeal model for investigating nitrogen stress responses, salt adaptation, methane production from different substrates, energy metabolism, as well as analyzing the role of small RNAs as regulatory elements in stress responses [2–7]. Although it only grows under strictly anaerobic conditions, the organism is genetically tractable and single colonies can be obtained on agar plates, a general requirement for genetic studies [8, 9]. However, genetic manipulation is restricted due to the fact that puromycin is the only selectable marker commercially available for methanoarchaea, which complicates generation of multiple mutations or even complementation experiments.…”