In all organisms, gene regulation is primarily controlled by transcription factors, proteins that bind DNA and influence gene expression (i.e., which genes are turned “on” or “off”). The activity of many transcription factors is controlled by environmental cues like nutrient availability, toxic substances, and heavy metals. As such, transcription factors can act as biological sensors that detect external signals and transmit them to generate internal cellular responses. How transcription factors recognize these cues and what genes they regulate remain important biological questions. Answers to these questions provide valuable insights into how life senses environmental stressors and which genes are important for mediating a stress response. They also provide a foundation to engineer transcription factors as biosensors in environmental or industrial biotechnology. This project will uncover the gene regulatory properties of metal-sensing transcription factors in the heat-loving bacterium, Thermus thermophilus HB8. The research will be conducted at a primarily undergraduate institution in rural Georgia, and will provide undergraduates with hands-on experience in DNA-protein biology, microbiological techniques and bioinformatics, which are valuable skills for workforce development in biotechnology. In bacteria, metalloregulatory transcription factors (MTFs) play a critical role in sensing intracellular metal ion concentrations and eliciting a genomic response. While extensively studied in mesothermic organisms, there are limited studies exploring the biological functions of thermophilic MTF homologs. T. thermophilus HB8 contains a variety of transcription factors from different MTF families; however, the regulons of many of these MTFs remain unknown. Characterizing these regulons will reveal how thermophiles adapt to fluctuating metal ion concentrations and uncover novel gene products with biotechnological potential. The project will employ an iterative selection tec