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 Impacts This 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.
Intellectual merit: The generation of mutations is central to the concept of evolution and how organisms produce genetic diversity. The production of genetic diversity is important because it allows organisms to be fit to changing environments (e.g. escape from starvation, become resistant to growth inhibitors and to gain the ability to invade host cells). On the other hand, producing genetic variants in multicellular organisms may lead to disruption of how cells and tissues are organized. The overall goal of this project concerned the study of a novel cell process that produces mutations. Historically, the mechanisms that produce mutations have been described as dependent upon cell growth and division; a mutation is a consequence of making mistakes during synthesis of DNA. However, there is also evidence that cells generate mutations in growth-arrested or growth-limiting conditions. While the evidence for the production of this type of mutations is clear, the mechanisms of how these events take place remains largely unknown. One process that generates mutations in cells experiencing growth-limiting conditions and the study subject of this award is the phenomenon of transcription-mediated mutagenesis (when genes are being made into products). Our experiments showed that stressed cells (like those observed when cells experience starvation or the treatment with growth inhibitors) accumulate mutations in genes whose products alleviate stress. Furthermore, the experiments under the purview of this award also elucidated how this type of mutations is generated. This mutagenic mechanism starts by the acquisition of damage to DNA during the synthesis of products in response to stress. Stressed cells then proceed to repair lesions in DNA in an error-prone manner. Three elements that differentiate how stressed cells produce mutations differently from growing or non-stressed cells elucidated by this research are: 1) stressed cells activate or synthesize fewer genes than growing cells, 2) lesions that lead to the formation of mutations in stressed cells accumulate at a higher rate in the fraction of genes being produced than in growing cells, and 3) stressed cells produce factors that introduce mutations during DNA repair. Broader impacts The broader implications of this award were several: 1) Scientific community - A total of 10 peer-reviewed publications disseminated the results of this award. These publications showed and elucidated factors that are important for the production mutations in stressed cells. 21 posters or talks were presented in the subject studied in this award. 2) Mentoring - a total of 21 undergraduates (11 female, 9 underrepresented minority) and 2 high school students were mentored and presented posters or talks on the research they conducted in the PI lab. Several of these undergrads have gone to graduate school and health related schools. One graduate student conducted her dissertation under the auspice of this award and is currently teaching microbiology at a small college in Wyoming. Three of the 21 undergrads joined the graduate program at UNLV and are under the supervision of the PI; Two of them received graduate assistantships for their dissertation projects (one is sponsored by Nevada-NASA EPSCoR and the other is sponsored by NSF-GRFP). 3) Collaborative research – this award allowed the PI to collaborate with Dr. Mario Pedraza-Reyes of the University of Guanajuato. This collaborative research resulted in several publications and cross-mentoring of students in both campuses. 4) Outreach and academic activities – This award integrated or foster the following activities. One, the research sponsored by this award integrated with an NSF-REU site (the PI is the Co PI of the REU site award - DBI1005223). Two, the PI of this award is also the PI/treasurer of the Conference on Prokaryotic Biology (Funded by NSF - MCB1305076). The objective of this conference is to provide opportunities for students to present their research and interact with top-notch scientists in the field of microbiology in a small setting. Most of the funds for this conference are to sponsor student travel. Three, the PI presented recruiting talks for the Nevada Pipeline for Undergraduates. These talks were delivered to community college students of Nevada and promoted participating in summer research and attending graduate school. Four, students conducting research sponsored by this award presented posters at the Festival of Communities (an open house initiative by UNLV that invites the citizens of Las Vegas to find out what UNLV does beyond the classroom). Lastly, the PI is a McNair Scholar Mentor and is the faculty mentor for the student chapter of the regional ASM. The mentor and students in this association are tasked with disseminating the importance of microbiology in the community and k-12 schools. This is done by conducting demos at schools and museums and bringing seminar speakers to the School of Life Sciences at UNLV.
