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

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Original research article

A brief version of this protocol appeared in:
Journal of Bacteriology
Feb 2017


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.

Keywords: Bacillus subtilis, Ribonucleotide reductase, Amino acid starvation, Stress-associated mutagenesis


Around 270 genes, including dnaA, dnaB, dnaC, involved in DNA replication as well as the nrdEF operon, which encodes RNR, are considered essential for B. subtilis growth (Kobayashi et al., 2003). Here we describe a protocol that has been applied to understand the role of this enzyme in modulating events of mutagenesis in nutritionally stressed non-growing cells of strain B. subtilis YB955 (hisC952, lecuC427, metB5). To this end, we implemented a genetic system that represses the expression of the essential nrdEF operon (non-permissive condition) while limiting two of the essential amino acids to avoid transient and weak phenotypes produced by variants of less efficient enzymes (Pybus et al., 2010). These restriction conditions allowed us first inquiring whether RNR influences mutagenesis in a population of non-growing (but viable) B. subtilis cells. In a second stage, a soft agar overlay, which provides permissive conditions for growth, allows detection of prototrophic colonies only if a mutation that restores at least one amino acid prototrophy has occurred. Whereas this protocol has been employed here with RNR, it can be potentially extended to study additional genes that are essential for metabolism as well as DNA replication and transcription. In addition, conditions may be adjusted to other bacterial species or selective markers.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Castro-Cerritos, K. V., Villegas-Negrete, N., Ramirez-Ramirez, N., Robleto, E. A. and Pedraza-Reyes, M. (2017). Stationary-phase Mutagenesis Soft-agar Overlay Assays in Bacillus subtilis. Bio-protocol 7(23): e2634. DOI: 10.21769/BioProtoc.2634.

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