(632e) Towards Programmable Interspecies Cell-Cell Communication in Bacterial Consortia: Synthetic Homoserine Lactone-Based Quorum Sensors for Gram-Positive B. Subtilis

Bacteria use biochemical cell-to-cell communication via diffusible molecules and gene regulatory elements to bring about large-scale responses. The ability to predictably design multicellular responses in diverse bacterial communities would have a wide range of applications including efficient biomanufacturing, bioenergy conversion, novel therapeutic strategies, sustainable agriculture, and bioremediation. According to the paradigm for bacterial quorum sensing, gram-negative and gram-positive bacteria utilize different chemical classes of quorum signaling molecules, specifically homoserine lactone (HSL) versus oligopeptide signals, respectively. A universal biochemical signal for cell-cell communication would facilitate the programming of dynamic responses in diverse bacterial consortia.

Here, we present a strategy to develop synthetic HSL-mediated quorum sensors for gram-positive bacteria. In this work, we use the model gram-positive Bacillus subtilis. Challenging the bacterial quorum sensing paradigm, we show that synthetic LuxR-type quorum sensors can be engineered in B. subtilis 168. These synthetic HSL-mediated quorum sensors are comprised of the LuxR-type regulator and a synthetic cognate promoter. Starting from the inactive gram-negative bacterial promoter sequence, we show the promoter requirements to achieve HSL-mediated activation in the model gram-positive using a set of LuxR-type quorum sensors. To engineer the gram-positive RpaR quorum sensor for p-coumaric HSL, we constructed a combinatorial library of synthetic P_Rpa promoters by combining different -35 elements, -10 elements, -16 elements and transcription start sites. Further, we applied statistical design of experiments to dissect the effect of each promoter element and their interactions that contribute to quorum sensor activity in B. subtilis. The dynamic range for synthetic RpaR quorum sensor variants can exceed 300-fold in B. subtilis. Following the promoter structure of selected variants in the P_Rpa library, we engineered synthetic LuxR (~20 fold) and CinR quorum sensors (~30-fold) in B. subtilis that can achieve 20-fold and 30-fold activation, respectively. These HSL-based quorum sensing systems can be used for programmable multicellular genetic circuits between gram-negative and gram-positive bacteria and will facilitate the predictive design of customizable bacterial consortia with specified dynamic responses.