Determining Mutations the Suppresses R-loop Formation in Bacillus subtilis

Abstract

Maintaining genome integrity is important for all living cells. A stable genome ensures that cells can perform their functions and minimize the occurrence of mutations that could lead to dysfunctional cellular pathways. Transcription has been known to cause instability in genomes by promoting R-loop formation. Stable R-loops can cause replication stress, which is a hallmark of cancer, and are lethal to living cells. However, the molecular mechanisms that lead to stable R-loop formation are unclear. Here, I hypothesize that mutations can arise in B. subtilis that suppress R-loop formation and prevent R-loop-induced death. To test this hypothesis, we used a strain that expresses an inducible, engineered gene construct that generates R-loops, that has previously been shown to induce cell death. The strain was then grown in the absence of the inducer, and cells were subsequently plated on media containing the inducer of the construct. Mutants that survive R-loop formation and still express the engineered were then chosen and subjected to whole genome sequencing. In the preliminary analysis, we identified multiple strains that were able to survive the induction of the engineered construct. Using a survival assay, we confirmed the expression of the construct and the survival of the mutants compared to the ancestral strain. Through this, we were able to identify a novel mutation in the gene encoding Sigma Factor A, which is an essential transcription factor involved in the initiation of transcription. Our results suggest that transcription initiation or early elongation events play a critical role in driving toxic R-loop formation. Future studies are needed to map R-loop formation to better understand how SigA impacts the distribution of R-loops genome-wide. Furthermore, future structural studies will allow us to identify how this SigA variant associates with RNA polymerase and/or promoter sequences.