Stationary-phase Mutagenesis Soft-agar Overlay Assays in Bacillus subtilis

Elucidating how a population of non-growing bacteria generates mutations improves our understanding of phenomena like antibiotic resistance, bacterial pathogenesis, genetic diversity and evolution. To evaluate mutations that occur in nutritionally stressed non-growing bacteria, we have employed the strain B. subtilis YB955, which measures the reversions rates to the chromosomal auxotrophies hisC952, metB5 and leuC427 (Sung and Yasbin, 2002). This gain-of-function system has successfully allowed establishing the role played by repair systems and transcriptional factors in stress-associated mutagenesis (SPM) (Barajas- Ornelas et al., 2014 ; Gómez- Marroquín et al., 2016 ). In a recent study (Castro- Cerritos et al., 2017 ), it was found that Ribonucleotide Reductase (RNR) was necessary for SPM; this enzyme is essential in this bacterium. We engineered a conditional mutant of strain B. subtilis YB955 in which expression of the nrdEF operon was modulated by isopropyl-β-D-thiogalactopyranoside (IPTG) (Castro- Cerritos et al., 2017 ). The conditions to determine mutation frequencies conferring amino acid prototrophy in three genes (hisC952, metB5, leuC427) under nutritional stress in this conditional mutant are detailed here. This technique could be used to evaluate the participation of essential genes in the mutagenic processes occurring in stressed B. subtilis cells.