Transcription-Associated Mutagenesis in Bacillus Subtilis Cells in Conditions of Stress

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Cells undergo mutagenic events under non-dividing conditions, thereby adding growth-independent processes to evolution. This prediction is highly debated because of the implication that the organism directs the accumulation of beneficial mutations. One way beneficial mutants may accumulate in a stochastic manner is by transcription-associated mutagenesis. In this project, the investigators seek to test the hypothesis that transcription de-repression and formation of transcription-induced DNA secondary structures contribute to the generation of adaptive mutations. Aim 1 of the project is to establish whether a decrease in the level of transcription correlates with a reduction in mutation frequency in a gene under selection. The experimental system is a leucine auxotrophic Bacillus subtilis strain that expresses a leuC427 mutant allele. This mutant gene is governed by an effector (S-adenosyl-methionine, SAM)-responsive riboswitch control mechanism such that increasing levels of SAM lead to incrementally greater repression of the leuC417 gene. The expectation is that a decrease in transcription will proportionally lower mutant accumulation and escape from leucine starvation. Aim 2 seeks to confirm the related hypothesis that transcription-induced secondary DNA stem-loop structures are particularly prone to mutagenesis, thereby enhancing the mutant accumulation under selection. To test this hypothesis, the investigators will create inducible thiF and argF alleles that differ only in the potential to form stable stem-loop structures to measure their relative accumulation of mutants under inducing and non-inducing conditions. The expectation is that the number of Thi+ and Arg+ revertants decreases significantly in the strains carrying the alleles with destabilized stem-loop structures (low mutability) compared to those with stable secondary structures (high mutability). Together, these specific aims will provide potential novel insights into the molecular mechanism of stress-induced adaptive mutagenesis, and consequently provide a better understanding of the molecular processes that have been at the core of evolution.Broader ImpactsThis work will promote and educate the public in the process of evolution. In addition,this study will advance and provide opportunities in science for students in a state that historically has been under-funded for science initiatives and education. The sponsoring state has a growing population of minorities. These objectives will be accomplished by implementing lecture programs offered to local high schools. This effort will be implemented and coordinated with the local high school district. Lastly, this research program will also provide much needed science hands-on experience to college students.