Error-Prone Processing of Apurinic/Apyrimidinic (AP) Sites by PolX Underlies a Novel Mechanism That Promotes Adaptive Mutagenesis in Bacillus subtilis

In growing cells, apurinic/apyrimidinic (AP) sites generated spontaneously or resulting from the enzymatic elimination of oxi-dized bases must be processed by AP endonucleases before they compromise cell integrity. Here, we investigated how AP sites and the processing of these noncoding lesions by the AP endonucleases Nfo, ExoA, and Nth contribute to the production of mu-tations (hisC952,metB5, and leuC427) in starved cells of the Bacillus subtilis YB955 strain. Interestingly, cells from this strain that were deficient for Nfo, ExoA, and Nth accumulated a greater amount of AP sites in the stationary phase than during expo-nential growth. Moreover, under growth-limiting conditions, the triple nfo exoA nth knockout strain significantly increased the amounts of adaptive his,met, and leu revertants produced by the B. subtilis YB955 parental strain. Of note, the number of sta-tionary-phase-associated reversions in the his,met, and leu alleles produced by the nfo exoA nth strain was significantly de-creased following disruption of polX. In contrast, during growth, the reversion rates in the three alleles tested were significantly increased in cells of the nfo exoA nth knockout strain deficient for polymerase X (PolX). Therefore, we postulate that adaptive mutations in B. subtilis can be generated through a novel mechanismmediated by error-prone processing of AP sites accumu-lated in the stationary phase by the PolX DNA polymerase. The genetic alterations that allow organisms to escape fromgrowth-limiting conditions in response to natural or artificial selection during prolonged nonlethal selective pressure are re-ferred to as adaptive or stationary-phase mutagenesis (1). Thi