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A repertoire of sophisticated genetic tools has significantly enhanced studies of Methanosarcina genera, yet the lack of multiple positive selectable markers has limited the types of genetic experiments that can be performed. In this study, we report the development of an additional positive selection system for Methanosarcina that utilizes the antibiotic nourseothricin and the Streptomyces rochei streptothricin acetyltransferase (sat) gene, which may be broadly applicable to other groups of methanogenic archaea. Nourseothricin was found to inhibit growth of four different methanogen species at concentrations ≤300 μg/ml in liquid or on solid media. Selection of nourseothricin resistant transformants was possible in two genetically tractable Methanosarcina species, M. acetivorans and M. barkeri, using the sat gene as a positive selectable marker. Additionally, the sat marker was useful for constructing a gene deletion mutant strain of M. acetivorans, emphasizing its utility as a second positive selectable marker for genetic analyses of Methanosarcina genera. Interestingly, two human gut-associated methanogens Methanobrevibacter smithii and Methanomassillicoccus luminyensis were more sensitive to nourseothricin than either Methanosarcina species, suggesting the nourseothricin-sat gene pair may provide a robust positive selection system for development of genetic tools in these and other methanogens., (© FEMS 2019.)