Interplay between different SHP/Rgg quorum sensing systems that control production of post-translationally modified peptides in Streptococcus thermophilus

In Bacillota , the Rap-Rgg-NprR-PlcR-PrgX-AimR (RRNPPA) superfamily of regulators mediates quorum sensing via autoinducing peptides (AIPs). AIPs are secreted, processed and reimported before interacting with their cognate regulators, modulating the latter’s activity. Within RRNPPA families, short hydrophobic peptides (SHPs) interact with Rgg transcriptional regulators. A widely used bacterium in the dairy industry, Streptococcus thermophilus , possesses an unusually high number of Rgg regulators (mean of 5 and max of 10 per genome). These regulators mainly control the expression of operons involved in the production of ribosomally synthesized and post-translationally modified peptides (RiPPs). Because SHP/Rgg systems display similarities at the amino acid level, we investigated potential crosstalk (i.e. cross-activation) among systems. We focused on five SHP/Rgg systems located upstream of operons involved in producing RiPPs that are modified by radical SAM enzymes as well as a putative RiPP that is modified by a ThiF-like adenylyltransferase/cyclase enzyme. Using genetic approaches (reporter fusions into different genetic backgrounds) and analytical chemistry (liquid chromatography tandem mass spectrometry), we found three key results: (1) each Rgg regulator activates the transcription of its proximal RiPP operon, (2) cross-activation also occurs, where one operon is co-regulated by two distinct Rgg regulators and (3) a single Rgg regulator can be activated by multiple SHPs. A particularly novel finding is that one of the five operons is expressed under the control of two independent quorum-sensing pathways: an SHP/Rgg system and the ComR/S competence system. The result is the production of a newly identified RiPP, named ThiF-like RiPP 6405 , that undergoes post-translational modifications catalysed by a ThiF-like adenylyltransferase/cyclase. Overall, our study has revealed that individual SHP/Rgg systems function autonomously but that their activity is further shaped by crosstalk involving both SHP- and DNA-binding. These findings underscore that these systems should be thoroughly characterized at the strain level to allow precise, targeted modifications.