Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis

The Gram-positive microorganismBacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2′-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels onB. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strainB. subtilis YB955 (hisC952 metB5 leu427 ) in which expression of thenrdEF operon was modulated by isopropyl-β-d -thiogalactopyranoside (IPTG). Moreover, genetic inactivation ofytcG , predicted to encode a repressor (NrdR) ofnrdEF in this strain, dramatically increased the expression levels of a transcriptionalnrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression ofnrdEF promoted growth-dependent mutagenesis and SPM. We also found thatnrdEF expression was induced by H2 O2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starvedB. subtilis cells increase the levels of RNR, which have a direct impact on SPM.IMPORTANCE Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allowBacillus subtilis to escape from growth-limited conditions.