Open AccessImplementation of a loss-of-function system to determine growth and stress-associated mutagenesis in Bacillus subtilis
Author(s) -
Norberto Villegas-Negrete,
Eduardo A. Robleto,
Armando Obregón-Herrera,
Ronald E. Yasbin,
Mario Pedraza-Reyes
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0179625
Subject(s) - bacillus subtilis , mutagenesis , function (biology) , loss function , biology , genetics , computational biology , chemistry , mutation , microbiology and biotechnology , bacteria , phenotype , gene
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.