| Summary: | Two-component signaling systems are found in bacteria, fungi and plants. They mediate many of the physiological responses of these organisms to their environment and display several conserved biochemical and structural features. This thesis identifies a potential functional role for two commonly found architectures in two-component signaling system, the split kinases and phosphate sink, which suggests that by enabling switch-like behaviors they could underlie physiological decision making. I report that split histidine kinases, where autophosphorylation and phosphotransfer activities are segregated onto distinct proteins capable of complex formation, enable ultrasensitivity and bistability. By employing computer simulations and analytical approaches, I show that the specific biochemical features of split kinases “by design” enable higher nonlinearity in the system response compared to conventional two-component systems and those using bifunctional (but not split) kinases. I experimentally show that one of these requirements, namely segregation of the phosphatase activity only to the free form of one of the proteins making up the split kinase, is met in proteins isolated from Rhodobacter sphaeroides. While the split kinase I study from R. sphaeroides is specifically involved in chemotaxis, other split kinases are involved in diverse responses. Genomics studies suggest 2.3% of all chemotaxis kinases, and 2.8% of all kinases could be functioning as split kinases. Combining theoretical and experimental approaches, I show that the phosphate sink motif found in microbial and plant TCSs allows threshold behaviors. This motif involves a single histidine kinase that can phosphotransfer reversibly to two separate response regulators and examples are found in bacteria, yeast and plants. My results show that one of the response regulators can act as a “sink” or “buffer” that needs to be saturated before the system can generate significant responses. This sink, thereby allows the generation of a signal threshold that needs to be exceeded for there to be significant phosphoryl group flow to the other response regulator. Thus, this system can enable cells to display switch-like behavior to external signals. Using an analytical approach, I identify mathematical conditions on the system parameters that are necessary for threshold dynamics. I find these conditions to be satisfied in both of the natural systems where the system parameters have been measured. Further, by in vitro reconstitution of a sample system, I experimentally demonstrate threshold dynamics for a phosphate-sink containing two-component system. This study provides a link between these architectures of TCSs and signal-response relationship, thereby enabling experimentally testable hypotheses in these diverse two-component systems. These findings indicate split kinases and phosphate as a microbial alternative for enabling ultrasensitivity and bistability - known to be crucial for cellular decision making. By demonstrating ultrasensitivity, threshold dynamics and their mechanistic basis in a common class of two-component system, this study allows a better understanding of cellular signaling in a diverse range of organisms and will open the way to the design of novel threshold systems in synthetic biology. Thus, I believe that this study will have broad implications not only for microbiologists but also systems biologists who aim to decipher conserved dynamical features of cellular networks.
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