The human gut microbiota is a dense and diverse community of bacteria that interact with the host and have major impacts on health and disease. Two-component systems (TCS) are the primary means by which bacteria, including those in the gut microbiome, sense and adapt to their environment. The prototypical TCS consists of a membrane-bound sensor histidine kinase (SHK) and a cytoplasmic response regulator (RR). In the presence of a specific stimulus, the SHK phosphorylates the RR, which then binds to DNA and activates a transcriptional response. One example of a gut-relevant TCS is the major virulence regulator PhoPQ, which recognizes antimicrobial peptides (AMPs) during Salmonella infection. Here, we identify altered sensitivity to surface-displayed AMPs in E. coli PhoPQ compared to Salmonella Typhimurium PhoPQ. In unpublished work, the Tabor lab has computationally identified over 1,600 uncharacterized TCSs in the genomes of 450 common human gut bacteria. We hypothesize that these gut microbiome TCSs sense a diverse array of small molecules, macromolecules, and other signals associated with gut function and gut-linked diseases. Our goal is to elucidate stimuli detected by gut microbiome TCSs and repurpose them to engineer gut bacteria that diagnose and treat disease. In particular, our lab recently developed a technique to rewire TCSs to well-characterized output promoters by modularly swapping the response regulator DNA-binding domain. Previous work in the Tabor Lab computationally selected 543 diverse TCSs from the human microbiome, synthesized the genes, and transformed the library into E. coli. Each TCS controls the expression of a barcoded mRNA and a GFP reporter gene. Here, we develop a simple computational tool to infer TCS inputs based on nearby genes and predicted regulatory relationships. Using this approach, we have identified two novel divalent metal sensing TCSs, and a candidate sensor of the microbial metabolite cadaverine. We characterize both divalent metal sensing TCSs as homologs of bacterial copper sensors with relaxed metal specificity and amino acid substitutions in metal-binding sites. We characterize specificity of our candidate cadaverine sensor and demonstrate sensitivity of the TetR-inducible gene expression system to polyamines, exposing a critical failure mode in synthetic biological circuits. We also develop a next-generation sequencing (NGS) approach to high-throughput screening of this library in response to discrete chemical ligands. We demonstrate detection of transcriptional signals from TCSs on a 5-minute time scale. We identify low dynamic range in transcriptional signals, and propose design modifications to improve signal strength for high-throughput screens using this method. This unique TCS discovery platform has implications in understanding the function of the microbiome and should enable the engineering of diagnostic and therapeutic gut bacteria for a wide range of diseases.