In streptococci, intracellular quorum sensing pathways are based on quorum-sensing systems that are responsible for peptide secretion, maturation, and reimport. These peptides then interact with Rgg or ComR transcriptional regulators in the Rap, Rgg, NprR, PlcR, and PrgX (RRNPP) family, whose members are found in Gram-positive bacteria. Short hydrophobic peptides (SHP) interact with Rgg whereas ComS peptides interact with ComR regulators. To date, in Streptococcus thermophilus, peptide secretion, maturation, and extracellular fate have received little attention, even though this species has several (at least five) genes encoding Rgg regulators and one encoding a ComR regulator. We studied pheromone export in this species, focusing our attention on PptAB, which is an exporter of signaling peptides previously identified in Enterococcus faecalis, pathogenic streptococci and Staphylococcus aureus. In the S. thermophilus strain LMD-9, we showed that PptAB controlled three regulation systems, two SHP/Rgg systems (SHP/Rgg₁₃₅₈ and SHP/Rgg₁₂₉₉), and the ComS/ComR system, while using transcriptional fusions and that PptAB helped to produce and export at least three different mature SHPs (SHP₁₃₅₈, SHP₁₂₉₉, and SHP₂₇₉) peptides while using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using a deep sequencing approach (RNAseq), we showed that the exporter PptAB, the membrane protease Eep, and the oligopeptide importer Ami controlled the transcription of the genes that were located downstream from the five non-truncated rgg genes as well as few distal genes. This led us to propose that the five non-truncated shp/rgg loci were functional. Only three shp genes were expressed in our experimental condition. Thus, this transcriptome analysis also highlighted the complex interconnected network that exists between SHP/Rgg systems, where a few homologous signaling peptides likely interact with different regulators.

In Bacillota, quorum sensing can be mediated by RRNPP regulators that are activated by autoinducing peptides (AIPs). In this study, we derived a hidden Markov model profile from a 3D-informed alignment to establish RRNPP repertoires for 527 genomes of streptococci in the salivarius group and identified probable AIPs. The salivarius group encompasses Streptococcus salivarius and Streptococcus vestibularis , which are part of the normal human oral microflora, and Streptococcus thermophilus , one of the most widely used bacteria in the dairy industry. We observed a large amount of plasticity in these repertoires, as well as profound differences among species. Notably, S. salivarius displayed an accumulation of ComR regulators, while S. thermophilus displayed an accumulation of Rgg regulators. The latter family included SHP-associated Rgg regulators, systems in which SHPs serve as AIPs; most of these regulators control the production of post-translationally modified peptides (RaS-RiPPs). Their level of richness contrasts with the genome reduction that accompanied S. thermophilus ’ adaptation to milk. We then used liquid chromatography-high resolution tandem mass spectrometry to analyze the activity of the eight most common SHP/Rgg systems by characterizing the SHPs and RaS-RiPPs found in the supernatants. We detected four SHPs and one RaS-RiPP that have never been seen before in S. thermophilus , and we showed that seven of the eight SHP/Rgg systems were functional. Finally, by simultaneously monitoring the amounts of both the SHPs and RaS-RiPPs, we demonstrated that the fates of these two peptide types differed during growth. SHP presence in the supernatant was transient, a pattern likely related to the peptides’ signaling role. IMPORTANCE: Streptococcus thermophilus possesses an unusually high number of Rgg regulators, which are activated by SHP pheromones that control the production of RaS-RiPPs, peptides with cyclization motifs and growth inhibition properties. We conducted an in silico analysis of regulator repertoires across a wide range of strains; a subsequent experimental study revealed that the majority of the SHP/Rgg systems were functional. Employing an optimized liquid chromatography-high resolution tandem mass spectrometry protocol, we were able to better detect and follow SHP and RaS-RiPP accumulation. While RaS-RiPPs accumulated during growth, SHPs were only transiently present in the extracellular environment. This observation suggests that we could manipulate quorum sensing by adding SHPs to the growth medium and highlights the need to study the functions of the RaS-RiPPs.

Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium’s three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP₁₃₅₈) and a transcriptional regulator (Rgg₁₃₅₈) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP₁₃₅₈ inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus, whose myriad applications include the delivery of therapeutic peptides or proteins.

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.