TY - JOUR
T1 - Development of genetic methods and construction of a chromosomal glnK 1 mutant in Methanosarcina mazei strain Gö1
AU - Ehlers, Claudia
AU - Weidenbach, Katrin
AU - Veit, Katharina
AU - Deppenmeier, Uwe
AU - Metcalf, William W.
AU - Schmitz, Ruth A.
N1 - Acknowledgements We thank Gerhard Gottschalk for continuous support and helpful discussions. This work was carried out in compliance with the current laws governing genetic experimentation in the country concerned. This work was supported by the Deutsche Forschungsgemeinschaft (SCHM1052/6-1 and 6-2) and by a Ph.D. fellowship awarded to C.E. by the Fonds der Chemis-chen Industrie.
PY - 2005/7
Y1 - 2005/7
N2 - 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.
AB - 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.
KW - GlnK
KW - Liposome-meditated transformation
KW - Methanosarcina mazei strain Gö1
KW - Nitrogen control
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U2 - 10.1007/s00438-005-1128-7
DO - 10.1007/s00438-005-1128-7
M3 - Article
C2 - 15824904
AN - SCOPUS:23744474252
SN - 1617-4615
VL - 273
SP - 290
EP - 298
JO - Molecular Genetics and Genomics
JF - Molecular Genetics and Genomics
IS - 4
ER -