Directed Evolution Reveals The Mechanism Of Hitrs Signaling Transduction In Bacillus Anthracis

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.Author summaryBacterial TCSs are a primary strategy for stress sensing and niche adaptation. Although individual domains and proteins of these systems have been extensively studied, systematic characterization of an entire TCS is rare. In this study, through unbiased genetic selections and rigorous biochemical analysis, we provide a detailed characterization and structure-function analysis of an entire TCS and extend our understanding of the molecular basis of signal transduction through TCSs. Moreover, this study provides a comprehensive map of point-mutations in these well-conserved signaling proteins, which will be broadly useful for studying other TCSs. The described genetic selection strategies are applicable to any TCS, providing a powerful tool for researchers interested in microbial signal transduction.