Investigation on Bacterial Signaling through Generation of a ppGpp Biosensor

Guanosine tetraphosphate (ppGpp) is a bacterial signaling molecule involved in activating the stringent response, a cellular reaction to environmental stress that downregulates cell division and metabolism processes to conserve nutrients. The stringent response is implicated in some instances of antibiotic persistence, so broadening the current understanding of ppGpp signaling is useful. This thesis seeks to generate a ppGpp biosensor that will bind ppGpp and emit fluorescent light in its presence, which will allow for improved research into the pathways and functions of the signaling molecule. To generate a novel ppGpp biosensor, I converted a biosensor previously used to detect cyclic di-GMP (a different signaling molecule) to contain a binding site transformed to now bind specifically with ppGpp. The genetic sequence for the cyclic di-GMP binding site was replaced with the ppGpp hydrolase domain which has a specific affinity for ppGpp; however, hydrolase activity would provide unwanted breakdown of the ppGpp, so it is mutated further to neutralize hydrolase activity. The desired outcome of this thesis results in a biosensor with a binding site that has a specific and sufficient binding affinity for the ppGpp molecule. Using this, we can determine how ppGpp levels are regulated in bacteria under conditions of stress, and how this signaling molecule is related to the survival of bacteria in response to antibiotic treatment