Role of Mfd and GreA in Bacillus subtilis Base Excision Repair-Dependent Stationary-Phase Mutagenesis

Abstract

We report that the absence of an oxidized guanine (GO) system or the apurinic/apyrimidinic (AP) endonucleases Nfo, ExoA, and Nth promoted stress-associated mutagenesis (SAM) in <i>Bacillus subtilis</i> YB955 (<i>hisC952 metB5 leuC427</i>). Moreover, MutY-promoted SAM was Mfd dependent, suggesting that transcriptional transactions over nonbulky DNA lesions promoted error-prone repair. Here, we inquired whether Mfd and GreA, which control transcription-coupled repair and transcription fidelity, influence the mutagenic events occurring in nutritionally stressed <i>B. subtilis</i> YB955 cells deficient in the GO or AP endonuclease repair proteins. To this end, <i>mfd</i> and <i>greA</i> were disabled in genetic backgrounds defective in the GO and AP endonuclease repair proteins, and the strains were tested for growth-associated and stress-associated mutagenesis. The results revealed that disruption of <i>mfd</i> or <i>greA</i> abrogated the production of stress-associated amino acid revertants in the GO and <i>nfo exoA nth</i> strains, respectively. These results suggest that in nutritionally stressed <i>B. subtilis</i> cells, spontaneous nonbulky DNA lesions are processed in an error-prone manner with the participation of Mfd and GreA. In support of this notion, stationary-phase Δ<i>ytkD</i> Δ<i>mutM</i> Δ<i>mutY</i> (referred to here as ΔGO) and Δ<i>nfo</i> Δ<i>exoA</i> Δ<i>nth</i> (referred to here as ΔAP) cells accumulated 8-oxoguanine (8-OxoG) lesions, which increased significantly following Mfd disruption. In contrast, during exponential growth, disruption of <i>mfd</i> or <i>greA</i> increased the production of His⁺, Met⁺, or Leu⁺ prototrophs in both DNA repair-deficient strains. Thus, in addition to unveiling a role for GreA in mutagenesis, our results suggest that Mfd and GreA promote or prevent mutagenic events driven by spontaneous genetic lesions during the life cycle of <i>B. subtilis</i><b>IMPORTANCE</b> In this paper, we report that spontaneous genetic lesions of an oxidative nature in growing and nutritionally stressed <i>B. subtilis</i> strain YB955 (<i>hisC952 metB5 leuC427</i>) cells drive Mfd- and GreA-dependent repair transactions. However, whereas Mfd and GreA elicit faithful repair events during growth to maintain genome fidelity, under starving conditions, both factors promote error-prone repair to produce genetic diversity, allowing <i>B. subtilis</i> to escape from growth-limiting conditions.