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

A forward mutagenesis system based on the acquisition of mutations that inactivate the thymidylate synthase gene (TMS) and confer a trimethoprim resistant (Tmp r ) 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. Tmp r 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 Tmp r 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 Tmp r phenotype. Stationary-phase-associated mutagenesis (SPM) assays revealed that colonies with a Tmp r phenotype, accumulated over a period of ten days with a frequency of ~ 60 ×10 −7 . The Tmp r system was further modified to study TMM by constructing a Δ thyA Δ thyB strain carrying an IPTG-inducible P spac-thyA cassette. In conditions of transcriptional induction of thyA , the generation of Tmp r colonies increased ~3-fold compared to conditions of transcriptional repression. Further, the Mfd and GreA factors were necessary for the generation of Tmp r 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.