Mechanisms of a Natural Bacterial Biosensor Using RT-DNA

The J. David Gladstone Institutes, San Francisco CA

Investigators

Abstract

This project aims to determine how bacterial immune systems known as retrons defend cell populations against viruses. Retrons are unique, and poorly understood, immune systems that use reverse-transcribed DNA to sense bacterial viruses and set off an immune response. This project will use quantitative approaches to gain insight into the molecular mechanisms of retron systems. The knowledge gained will fill in critical gaps in our understanding of natural microbial ecosystems, such as our own human microbiome, in addition to industrial processes that leverage bacteria. Retron components also hold important potential for use in biomanufacturing and biomedicine. Thus, the fundamental studies of the retron in this project will inform future development of advanced biotechnologies. The Broader Impacts of this research include training for underrepresented students transitioning from two and four year colleges and working with those students to develop retron discovery kits using 3D printed parts and simple, do-it-yourself electronics, as well as the plans and protocols for anyone who wants to build this as an experiment or lesson plan. The central question in this work is how a fragment of DNA that is produced by cells can serve as the key element of a phage biosensor. Work will progress through parallel investigations into (1) the sequence and structure of the reverse transcribed DNA, (2) the protein interactome of the retron, and (3) the population dynamics of retron expression in a native host. All of these investigations will use quantitative, high-throughput approaches, including synthesized variant libraries, quantitative mass spectrometry, and multiplexed single cell analysis. When completed, these experiments will answer (1) how the reverse transcribed DNA interacts with the protein components of the retron to neutralize them in the absence of phage, (2) how those protein components link into the broader proteome of the cell to effect a biological response, and (3) how a population of cells balances the costs and benefits of using a self-toxic system to defend against phage infection. 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.