Tomislav Volosen Uta Gutbier Ramon Korn Juliane Korp Tobias Göttsche Linda Schuster Carolin Pohl Cindy Rau Diana Wolf ^* Kai Ostermann ^*

• Technical University Dresden, Dresden, Germany

Understanding communication among microorganisms through the array of signal molecules and establishing controlled signal transfer between different species is a major goal of the future of biotechnology and controlled multi-species bioreactor cultivations will open a wide range of applications. In this study, we used two quorum sensing peptides from Bacillus subtilis -namely the competence and sporulation factor (CSF) and regulator of the activity of phosphatase RapF (PhrF)to establish a controlled interkingdom communication system between pro-and eukaryotes. For this purpose, we engineered Bacillus subtilis to act as a reporter capable of detecting the CSF and PhrF peptides heterologously produced by the yeast Saccharomyces cerevisiae. The reporter strain included the ComA-dependent srfAA promoter fused to the bioluminescence or fluorescence reporter gene(s) to monitor promoter activity that was measured in a multi-mode microplate reader. First measurements of srfAA promoter activity showed a specific response of the reporter strain to the peptides CSF and PhrF. On this basis a systematic mutagenesis of genes that modulate the activity of ComA in the reporter strain resulted in an increased activity of the promoter and thereby higher sensitivity to the heterologously produced CSF/PhrF. Robustness of the signal transfer was further confirmed in cocultivation studies in both liquid and solid media. The reporter strain exhibited an up to 5-fold increase in promoter activity in the presence quorum sensing peptides producing cells of S. cerevisiae. In summary, a quorum sensing peptide driven interkingdom crosstalk between yeast and bacteria was successfully established, which might serve as a basis for controlled protein expression in cocultivations, establishing biological sensor-actuator systems or study cell-cell interaction and metabolite exchange in bioreactors cultivations.