Mechanisms of non-canonical retron phage defense and expanded biotechnological applications

The J. David Gladstone Institutes, San Francisco CA

Investigators

Abstract

Retrons are bacterial immune systems that sense when a cell is infected by a phage and respond by killing the infected cell to spare the larger bacterial population. This project will investigate exactly how retrons accomplish this act of sensing and responding to phage invasion. Not only does this provide new knowledge of the natural world, but understanding these mechanisms gives researchers new ways to build tools that modify cells for research or industrial applications. This team takes that additional step to build molecular technologies from retron parts for genome engineering, and broadens access to these technologies by making and disseminating versions that work across many different bacterial species. Finally, this project also broadens participation in science by providing summer laboratory experiences for students who started in two-year community colleges, but are seeking to transfer to four-year universities to pursue degrees in science and engineering. The team is particularly interested in a new type of retron that they discovered in previous NSF-supported work, which does not follow the canonical retron mechanism. The canonical retron mechanism involves a three-part operon: (1) a non-coding RNA (ncRNA), (2) a reverse transcriptase (RT) that generates a single-stranded reverse-transcribed DNA (RT-DNA) from the ncRNA, and (3) an effector protein that is toxic to the host bacterium. When a phage infects a cell, phage-encoded proteins trigger the retron system by modifying the RT-DNA, releasing the toxic effector, and leading to death of the infected cell. The novel system employs a longer non-coding RNA than a typical retron and does not constitutively produce the RT-DNA sensor, yet still provides defense against a range of phages. The team will use a range of genetic experiments, biochemistry, phage assays, and sequencing to uncover the mechanism of this exotic retron and expand our understanding of bacterial immunity. Retrons have also been used to produce reverse transcribed recombineering donors for genome editing in E. coli. This project will additionally expand the range of those tools to get the broadest possible benefit for research and industrial engineering, focusing on gram positive bacterial species. 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.