Investigating a Potential Link Between Prophage Activation and the DNA Damage Response in the E. coli Long-Term Evolution Experiment

The Long-Term Evolution Experiment (LTEE) is an open-ended endeavor to understand evolution through one of the simplest and quickest-replicating forms of life: Escherichia coli. Cultured in the lab since 1988, the 12 populations of E. coli in the LTEE have reached over 75,000 generations and accumulated many beneficial mutations in their genomes. One area of interest is a prophage from the P2/186 phage family called RybbB. Most prophage regions found in the genome of the ancestor of the LTEE are not functional, but RybbB was shown to be inducible by DNA damage. Additionally, past genome sequencing data showed evidence of more than one copy, on average, of the prophage region’s DNA per cell in the ancestor and clonal isolates from early generations, but not in later generations of all 12 LTEE populations. This extra sequencing coverage, as well as the presence of reads supporting a new junction in the DNA suggested that the prophage was stochastically replicating in these strains. However, the reason for the apparent decrease in the prophage copy number in later generations is unknown. I performed experiments to understand these unexplained changes in prophage activation. First, I measured prophage copy number in early strains of the LTEE using quantitative PCR (qPCR). Second, I used a reporter plasmid to test whether changes in the SOS DNA damage response evolved during the LTEE that might have affected stochastic prophage activation. Unlike in prior results, I found that Rybb*B prophage copy number remains relatively consistent throughout the early LTEE strains except for the ancestor which seems to have a higher prophage copy number. I also found that the SOS reporter is induced by azidothymidine similarly in strains from 5000 generations or earlier but does not appear to activate in strains from 10000 generations.