Researchers Reveal Insights into Bacterial Genome Evolution
By Andy Flick, Evolutionary Studies Scientific Coordinator
In a new study led by graduate student Carl Stone and his advisor, assistant professor of biological sciences, Megan Behringer, the team studied the effects of DNA repair pathways on genome-wide methylated DNA adenines (6mA). 6mA is a modification to DNA such that a methyl group is added on a nitrogen of adenine. This modification can influence various biological processes, including gene expression, DNA replication, pathogenesis and repair mechanisms.
According to Behringer, “E. coli uses 6mA to guide its mismatch repair system. When the DNA replication machinery makes a mistake and accidentally adds the wrong base causing a mismatch between the base pair, mismatch repair will replace the erroneous base with the correct one before it becomes a permanent mutation. There’s a problem, though, how does the repair machinery know which base in the pair is the original base from the parent strand and which is the newly-added incorrect base? Well, when the DNA replication machinery synthesizes the new DNA strand, the newly added bases are not immediately methylated so during this time the parent strand is methylated but not the daughter strand.”
This methylation allows the mismatch repair enzymes to correctly identify the parent strand to correct the other, mismatched base. Disabling mismatch repair enzymes, surprisingly, is not a lethal process to E. coli – it simply increases the volume of mutations or the mutation burden. As the primary purpose of 6mA is to guide mismatch repair, what happens to the methylations when the mismatch repair pathway is removed?
The team hypothesized that if they removed mismatch repair in E. coli and evolved the strains for several generations, they would see a reduction in these methylations.
Behringer followed up, “bacteria are very efficient, they don’t want to waste anything; if they can shuttle that carbon somewhere else, they will. What we found neat was that we saw exactly that in these lines that we evolved. There was a global reduction in 6mA. That led us to our next question, are there specific bases that lose their methylation or is it a random process?”
The team found that for the most part the reduction in 6mA was more of a random process consistent with genetic drift. However, 6mA is involved in other processes such as gene expression, so the team doesn’t expect it to disappear completely.
One application of this work is understanding the mutation process better. This process includes not just base mutations but epimutations as well. Epimutations occur when things like DNA methylations are changed and can lead to abnormal gene expression and even disease. Tracking 6mA mutations may provide a way to create a finer timescale to track evolution, which may already be the case in the field of plant evolution studies. Behringer expressed hope that using the tractability of 6mA evolution may be able to provide insights into future work in phylogenetics and systematics.
A second application comes in studying virulence of pathogens. Epimutations may increase or decrease the probability of turning on or off certain virulence factors, in other words, increasing or decreasing the probability of the pathogen becoming harmful.
Going forward, the team would like to work out more of the details for the epimutations they studied here. Specifically, they want to know if there are regions in the genome, or hotspots, that are likely to experience epimutations. Ideally, the team could tie these epimutations to particular gene expression events to be able to better predict what might happen on a system-wide scale.
This was Stone’s first publication and Behringer was proud of his work and commented, “this was one of the first, if not the first, genome-wide 6ma study. Carl wrote a program to do differential methylomics using 6ma, that’s huge! It’s exciting because this represents a whole new way to think about molecular evolution.”
Behringer has also received a pair of honorable distinctions over the past few years. Related to this research, she received an Early Career Program award from the army and was featured in their 2022 Year in Review Success Stories. In early 2022, she was awarded the Steven and Bunny Fayne Dean’s Faculty Fellowship in Biological Sciences that lasts until 2024.
Citation: Differential adenine methylation analysis reveals increased variability in 6mA in the absence of methyl-directed mismatch repair. Stone CJ, Boyer GF, Behringer MG. 2023. mBio. Early Access: e01289-23.
Funding Statement: This work was supported by Army Research Office grants W911NF-21-1-0161 (M.G.B.), National Institutes of Health grant F32GM123703 (M.G.B.), and start-up funding from Vanderbilt University and the Vanderbilt Center for Infection, Immunology, and Inflammation. High-performance computing resources were provided by the Research Computing Core at Arizona State University and the Advanced Computing Center for Research and Education (ACCRE) at Vanderbilt University.