Compiling a versatile toolbox for inducible gene expression in Methanosarcina mazei

Methanosarcina mazei 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 M. mazei. (i) The TetR/TetO system was utilized to induce expression of a designed antisense RNA directed against sRNA₁₅₄ allowing to increase transcripts of asRNA₁₅₄ (500-fold), resulting in a significant decrease of sRNA₁₅₄ levels (tetracycline-induced knockdown mutant). Strong reduction of sRNA₁₅₄ was further confirmed in the knockdown mutant by up to 50-fold decreased transcript levels of the genes nifH, glnK , and glnA , the stability of which is increased by sRNA₁₅₄. (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 M. mazei using the streptothricin acetyltransferase gene. Taken together, our findings provide a foundation for future exploration of genetic regulation and inducible gene expression in M. mazei and other methanoarchaea, advancing genetic studies in these organisms and enhancing their potential for biotechnology applications.