Villegas-Negrete Norberto, Robleto Eduardo A, Obregón-Herrera Armando, Yasbin Ronald E, Pedraza-Reyes Mario
Department of Biology, Division of Natural and Exact Sciences, University of Guanajuato, Guanajuato, Mexico.
School of Life Sciences, University of Nevada, Las Vegas, Nevada, United States of America.
PLoS One. 2017 Jul 11;12(7):e0179625. doi: 10.1371/journal.pone.0179625. eCollection 2017.
A forward mutagenesis system based on the acquisition of mutations that inactivate the thymidylate synthase gene (TMS) and confer a trimethoprim resistant (Tmpr) phenotype was developed and utilized to study transcription-mediated mutagenesis (TMM). In addition to thyA, Bacillus subtilis possesses thyB, whose expression occurs under conditions of cell stress; therefore, we generated a thyB- thyA+ mutant strain. Tmpr colonies of this strain were produced with a spontaneous mutation frequency of ~1.4 × 10-9. Genetic disruption of the canonical mismatch (MMR) and guanine oxidized (GO) repair pathways increased the Tmpr frequency of mutation by ~2-3 orders of magnitude. A wide spectrum of base substitutions as well as insertion and deletions in the ORF of thyA were found to confer a Tmpr phenotype. Stationary-phase-associated mutagenesis (SPM) assays revealed that colonies with a Tmpr phenotype, accumulated over a period of ten days with a frequency of ~ 60 ×10-7. The Tmpr system was further modified to study TMM by constructing a ΔthyA ΔthyB strain carrying an IPTG-inducible Pspac-thyA cassette. In conditions of transcriptional induction of thyA, the generation of Tmpr colonies increased ~3-fold compared to conditions of transcriptional repression. Further, the Mfd and GreA factors were necessary for the generation of Tmpr colonies in the presence of IPTG in B. subtilis. Because GreA and Mfd facilitate transcription-coupled repair, our results suggest that TMM is a mechanim to produce genetic diversity in highly transcribed regions in growth-limited B. subtilis cells.
开发并利用了一种基于获得使胸苷酸合成酶基因（TMS）失活并赋予甲氧苄啶抗性（Tmpr）表型的突变的正向诱变系统，以研究转录介导的诱变（TMM）。除了thyA外，枯草芽孢杆菌还拥有thyB，其表达在细胞应激条件下发生；因此，我们构建了一个thyB - thyA +突变株。该菌株的Tmpr菌落以约1.4×10 -9的自发突变频率产生。经典错配（MMR）和鸟嘌呤氧化（GO）修复途径的基因破坏使Tmpr突变频率增加了约2 - 3个数量级。发现thyA开放阅读框中的广泛碱基替换以及插入和缺失都赋予了Tmpr表型。与静止期相关的诱变（SPM）分析表明，具有Tmpr表型的菌落在十天内积累，频率约为60×10 -7。通过构建携带IPTG诱导型Pspac - thyA盒的ΔthyAΔthyB菌株，对Tmpr系统进行了进一步修饰以研究TMM。在thyA转录诱导的条件下，与转录抑制条件相比，Tmpr菌落的产生增加了约3倍。此外，Mfd和GreA因子对于枯草芽孢杆菌中存在IPTG时Tmpr菌落的产生是必需的。由于GreA和Mfd促进转录偶联修复，我们的结果表明TMM是在生长受限的枯草芽孢杆菌细胞中高度转录区域产生遗传多样性的一种机制。
