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

• Published in: Bio-protocol 2017-12, Vol.7 (23), p.e2634-e2634
• Main Authors: Castro-Cerritos, Karla Viridiana, Villegas-Negrete, Norberto, Ramírez-Ramírez, Norma, Robleto, Eduardo A, Pedraza-Reyes, Mario
• Format: Article
• Language: English
• Subjects: Biology; Methods
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/BioProtoc.2634

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 YB955, which measures the reversions rates to the chromosomal auxotrophies and (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 , 2014 ; Gómez- Marroquín , 2016 ). In a recent study (Castro- Cerritos , 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 YB955 in which expression of the operon was modulated by isopropyl-β-D-thiogalactopyranoside (IPTG) (Castro- Cerritos , 2017 ). The conditions to determine mutation frequencies conferring amino acid prototrophy in three genes ( ) 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 cells.