期刊
ARCHAEA-AN INTERNATIONAL MICROBIOLOGICAL JOURNAL
卷 2011, 期 -, 页码 -出版社
HINDAWI LTD
DOI: 10.1155/2011/439608
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资金
- Deutsche Forschungsgemeinschaft (DFG) [SPP 1258]
A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gol using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA(154). Characterizing M. mazei Delta sRNA(154) under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA(154) in regulation of nitrogen fixation by posttranscriptional regulation.
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