RUI: Regulon identification for metalloregulatory transcription factors in the extremophile, Thermus thermophilus HB8

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 technique, restriction endonuclease protection, selection and amplification (REPSA), to identify the preferred DNA-binding motifs of eight uncharacterized T. thermophilus HB8 MTFs. These MTFs include members from the ferric uptake regulator (FUR), mercury resistance operon regulator (MerR) and arsenic resistance operon regulator (ArsR) superfamilies of bacterial transcription factors. The discovered motifs will be mapped to the T. thermophilus HB8 genome, and transcription regulatory networks will be validated using in vitro and in vivo analyses. The research will be conducted by undergraduate students in the PI’s laboratory and as part of a new course-based undergraduate research experience (CURE) that will serve as a laboratory component to a biochemistry course at the institution. Understanding DNA-protein interactions is a learning objective for the biochemistry course; thus, students will make strong connections between the concepts learned in lectures and applied in CURE experiments. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria. NSF Award ID: 2540489 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: John Barrows | Institution: Reinhardt College, WALESKA, GA | Award Amount: $332,976 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2540489 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2540489.html