A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 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 δsRNA 154. Characterizing M. mazei δs R N A 154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of δsRNA 154 in regulation of nitrogen fixation by posttranscriptional regulation. © 2011 Claudia Ehlers et al.
Ehlers, C., Jäger, D., & Schmitz, R. A. (2011). Establishing a markerless genetic exchange system for Methanosarcina mazei strain Gö1 for constructing chromosomal mutants of small RNA genes. Archaea, 2011. https://doi.org/10.1155/2011/439608