A markerless genetic exchange system was successfully established in Methanosarcina mazei strain G？1 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 sRNA154. Characterizing M. mazei under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation. 1. Introduction Methanosarcina mazei strain G？1 belongs to the methylotrophic methanogenic Archaea and, due to its role in methane production, is of high ecological relevance [1]. 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. Using Methanosarcina acetivorans, Metcalf and coworkers developed a so-called markerless exchange method using the hpt gene encoding hypoxanthine phosphoribosyltransferase as a counterselectable marker [10]. A hpt strain which shows resistance towards the toxic purine analog 8-aza-2,6-diaminopurine (8-ADP) can be used for counterselection following integration of an nonreplicable plasmid containing the wild-type hpt gene and the desired mutation with flanking regions for recombination. The complete plasmid is integrated into the site of the desired mutation (pop-in) in the chromosome by a single homologous recombination event, making the strain sensitive to 8-ADP and allowing selection
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