Quantifying the Prevalence and Phenotypic Consequences of Transcriptional Irreversibility in Bacteria

A key question in biology is how genetically identical cells achieve varied appearances and behaviors. These distinct cell states are realized by variations in gene expression within certain cells in the population; different genes are turned “on” or “off” in these cells. These variations in gene expression can lead to different phenotypes within a population of genetically identical cells. For example, variations in gene expression can lead some cells in a population of genetically identical bacteria to become antibiotic tolerant while other cells in the population remain suspectable to antibiotics. This project will address how variations in gene expression lead to important phenotypic changes in bacteria. To complement the research, an interactive series of lessons on mathematics in biology will be developed for high school students. These lessons will be distributed through a series of teacher workshops.

Irreversibility, hysteresis, and multistability in cell state have been quantitatively studied in a handful of specific bacterial systems — B. subtilis sporulation, the lac repressor, and the lysis-lysogeny switch are now classic examples. Here, the investigators seek to expand the understanding of these concepts to the genomic scale: the investigators will examine the time scales of reversibility, and the prevalence of irreversibility, following transient repression of all genes with known function in E. coli. To accomplish this, the investigators will develop a new reagent for light-inducible transient gene repression called LIT-CRISPRi. They will use a combination of experimental data and theory to establish expectations for the time scale of transcriptional, translational, and growth rate recovery following transient gene repression in the fully reversible case (in the absence of hysteresis) and characterize behavior for one well-studied irreversible case (the lac operon). They will then use these tools to characterize E. coli’s growth rate dynamics before, during, and after transient gene repression for a genome scale library of LIT-CRISPRi knockdowns under different environmental conditions. From these data, the investigators will examine the distribution of growth rate recovery times, identify cases of irreversibility or exceptionally long timescale recovery (quasi-irreversibility), and further validate these phenotypes through secondary experiments. Finally, they will use RNAtag-Seq to perform time-resolved transcriptomics before, during, and after transient gene repression for several genes exhibiting (quasi-)irreversible dynamics identified by our screen. Taken together, the work will establish fundamental expectations for the time scales of cellular adaptation and irreversibility following gene repression.

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.

Award Number: 2624903
Principal Investigator: Kimberly Reynolds
Funds Obligated: $337,841
State: MD