Implementation of a loss-of-function system to determine growth and stress-associated mutagenesis in Bacillus subtilis

摘要: A forward mutagenesis system based on the acquisition of mutations that inactivate thymidylate synthase gene (TMS) and confer a trimethoprim resistant (Tmpr) phenotype was developed utilized to study transcription-mediated (TMM). In addition thyA, Bacillus subtilis possesses thyB, whose expression occurs under conditions cell stress; therefore, we generated thyB- thyA+ mutant strain. Tmpr colonies this strain were produced with spontaneous mutation frequency ~1.4 × 10-9. Genetic disruption canonical mismatch (MMR) guanine oxidized (GO) repair pathways increased by ~2-3 orders magnitude. wide spectrum base substitutions as well insertion deletions in ORF thyA found phenotype. Stationary-phase-associated (SPM) assays revealed phenotype, accumulated over period ten days ~ 60 ×10-7. The further modified TMM constructing ΔthyA ΔthyB carrying an IPTG-inducible Pspac-thyA cassette. transcriptional induction generation ~3-fold compared repression. Further, Mfd GreA factors necessary for presence IPTG B. subtilis. Because facilitate transcription-coupled repair, our results suggest is mechanim produce genetic diversity highly transcribed regions growth-limited cells.