Compiling a versatile toolbox for inducible gene expression in .

is a model organism, providing a platform to explore methanoarchaeal regulation mechanisms on the transcriptional and translational level. This study investigates and evaluates various molecular tools to allow inducible gene expression in . (i) The TetR/TetO system was utilized to induce expression of a designed antisense RNA directed against sRNA allowing to increase transcripts of asRNA (500-fold), resulting in a significant decrease of sRNA levels (tetracycline-induced knockdown mutant). Strong reduction of sRNA was further confirmed in the knockdown mutant by up to 50-fold decreased transcript levels of the genes , and , the stability of which is increased by sRNA. (ii) For translational regulation, an RNA thermometer was designed and first-ever utilized in an archaeon, inserted into the 5'-untranslated region of a reporter gene, which showed enhanced protein expression upon a temperature shift from 30°C to 40°C. (iii) The long 5'-UTR of a trimethylamine (TMA)-inducible polycistronic mRNA was evaluated and studied as a potential genetic tool for induced gene expression on the translational level. However, we discovered TMA-dependent regulation occurs most likely on the transcript level. (iv) A new selection marker (nourseothricin resistance) was established for using the streptothricin acetyltransferase gene. Taken together, our findings provide a foundation for future exploration of genetic regulation and inducible gene expression in and other methanoarchaea, advancing genetic studies in these organisms and enhancing their potential for biotechnology applications.