Bacterial two-component systems (TCSs) are the largest family of signal transduction pathways that enable bacteria to sense a diversity of stimuli including small peptides, environmental pollutants, and light. Canonical TCSs are composed of a transmembrane sensor histidine kinase (SK) that converts stimulus detection into phosphorylation of a cognate response regulator (RR). Upon phosphorylation, the cytoplasmic RR binds target output promoters, hereby modulating gene expression. TCSs are valuable sensors for synthetic biology due to their diverse sensing capabilities and straightforward transduction of detected stimulus into transcriptional regulation. TCSs are also emerging targets for novel therapeutic development due to their extensive role in regulating bacterial virulence and antibiotic resistance. Although TCSs are exciting sensors for synthetic biology and targets for therapeutic applications, most TCSs remain difficult to harness for applications and study due to output promoters that are unknown, subject to cross- regulation, or silent in heterologous hosts. In the first portion of my work, I develop a method to overcome the hurdles in characterizing and utilizing TCSs as biosensors. Through the framework of synthetic biology, I demonstrate that the two largest families of RR DNA binding domains (DBDs) can be interchanged with remarkable flexibility, enabling the corresponding TCSs to be rewired to synthetic output promoters. In collaboration with Kristina Daeffler, we exploit this plasticity to eliminate cross-regulation and in collaboration with Brian Landry, we un-silence a gram-negative TCS in a gram-positive host and engineer a sensor with over 1,300-fold activation. In collaboration with Kathryn Brink, we also apply DBD swapping to screen uncharacterized Shewanella oneidensis TCSs in Escherichia coli, leading to the discovery of a novel pH sensor. In the second portion of my work, I demonstrate a method for identifying inhibitors of a virulence regulating TCS. This work focuses on the methicillin-resistant Staphylococcus aureus (MRSA) virulence regulating TCS SaeRS. I first heterologously express saeRS in Bacillus subtilis to remove the native, confounding regulation of the TCS and its output promoter. I then screen heterologous SaeRS against a diverse 1,593 small molecule library to identify inhibitors of its signaling. A lead compound emerged from the screen as an effective inhibitor of SaeRS signaling, leading to decreased exoprotein secretion and virulence from treated MRSA similar to MRSA lacking saeRS. My work described herein should accelerate 1) fundamental TCS studies and the engineering of a large family of biosensors with diverse applications and 2) the discovery of new anti-virulence compounds.