Signal-independent activation reveals two-component regulatory networks.
Each bacterial species has specific regulatory systems to control physiology, adaptation, and host interactions. One challenge posed by this diversity is to define the evolving gene regulatory networks. This study aims to characterise two-component systems (TCS) in Streptococcus gagalactiae, the main cause of neonatal meningitis. Here we demonstrate signal-independent activation of signalling pathways by systematically targeting the conserved mechanism of phosphatase activity of the  histidine kinases of the two main TCS families. Transcriptomic analysis resolves most pathways with high resolution, encompassing specialized, connected, and global regulatory systems. The activated network notably reveals the connection between CovRS and SaeRS signaling through the adhesin PbsP, linking the main regulators of host  interactions to balance pathogenicity. Additionally, constitutive activation of the BceRS system reveals its role in cell envelope homeostasis beyond antimicrobial resistance. Overall, this study demonstrates the generalizability and versatility of TCS genetic activation to uncover regulatory logics and biological processes. Overall design: To investigate the activation of  regulatory networks by phosphatase-deficient histidine kinases, we generated 14 strains (called the HK+ mutants), with an alanine substitution of the predicted phosphatase catalytic residue or at the corresponding position. We then performed differential gene expression analysis by RNA-seq between the wild-type parental strain (BM110) and the 14 HK+ mutants. RNA are purified from exponentialy growing cultures (OD600 = 0.5) in rich media (THY) incubated at 37°C in static condition. Biological triplicate (Replicate 1, 2, and 3) are done on different days. Two wild-type controls (BM110_A and BM110_B) are included.