Previous studies showed that a Bacillus subtilis strain deficient in mismatch repair (MMR; encoded by the mutSL operon) promoted the production of stationary-phase-induced mutations. However, overexpression of the mutSL operon did not completely suppress this process, suggesting that additional DNA repair mechanisms are involved in the generation of stationary-phase-associated mutants in this bacterium. In agreement with this hypothesis, the results presented in this work revealed that starved B. subtilis cells lacking a functional error prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic propensity to increase the number of stationary-phase-induced revertants. These results strongly suggest that the occurrence of mutations is exacerbated by reactive oxygen species in nondividing cells of B. subtilis having an inactive GO system. Interestingly, overexpression of the MMR system significantly diminished the accumulation of mutations in cells deficient in the GO repair system during stationary phase. These results suggest that the MMR system plays a general role in correcting base mispairing induced by oxidative stress during stationary phase. Thus, the absence or depression of both the MMR and GO systems contributes to the production of stationary-phase mutants in B. subtilis. In conclusion, our results support the idea that oxidative stress is a mechanism that generates genetic diversity in starved cells of B. subtilis, promoting stationaryphase-induced mutagenesis in this soil microorganism.Adaptive or stationary-phase mutagenesis can be defined as those mutations that permit organisms to grow and divide in response to natural or artificial selection (5) and that occur in nondividing cells during prolonged nonlethal selective pressure, e.g., starvation for an essential amino acid (32). Although this type of mutagenesis was first described in Escherichia coli (7), additional examples of adaptive mutagenesis in other prokaryotes (21, 41) and in eukaryotic organisms (14) have been published. In some cases, these mutations occurred in the absence of specific selection but in response to starvation (11). Regardless of the organisms utilized and the name used, these types of mutations and the processes that generate them are of real interest with respect to evolution and the generation of diversity across all domains of life.Studies with the FЈ lac frameshift reversion construct of E. coli (32) have demonstrated that the generation of Lac ϩ stationary-phase-associated revertants required functional recombination (15), as well as component(s) of the SOS system (24, 25). Further evidence suggests that the mutations generated by this lac system during stationary phase may also be the result of amplification of the plasmid-borne gene followed by SOS-induced mutagenesis and selection (18,38). Recent studies have demonstrated that DNA double-strand-break repair, in addition to the SOS DNA damage response and the error-prone DNA polymerase, are necessary for stress-induced reversion of the E. col...

Stress-promoted mutations that occur in nondividing cells (adaptive mutations)

Reactive oxygen species (ROS) promote the synthesis of the DNA lesion 8-oxo-G, whose mutagenic effects are counteracted in distinct organisms by the DNA glycosylase MutM. We report here that in Bacillus subtilis, mutM is expressed during the exponential and stationary phases of growth. In agreement with this expression pattern, results of a Western blot analysis confirmed the presence of MutM in both stages of growth. In comparison with cells of a wild-type strain, cells of B. subtilis lacking MutM increased their spontaneous mutation frequency to Rif r and were more sensitive to the ROS promoter agents hydrogen peroxide and 1,1=-dimethyl-4,4=-bipyridinium dichloride (Paraquat). However, despite MutM's proven participation in preventing ROS-induced-DNA damage, the expression of mutM was not induced by hydrogen peroxide, mitomycin C, or NaCl, suggesting that transcription of this gene is not under the control of the RecA, PerR, or B regulons. Finally, the role of MutM in stationary-phase-associated mutagenesis (SPM) was investigated in the strain B. subtilis YB955 (hisC952 metB5 leuC427). Results revealed that under limiting growth conditions, a mutM knockout strain significantly increased the amount of stationary-phaseassociated his, met, and leu revertants produced. In summary, our results support the notion that the absence of MutM promotes mutagenesis that allows nutritionally stressed B. subtilis cells to escape from growth-limiting conditions. IMPORTANCEThe present study describes the role played by a DNA repair protein (MutM) in protecting the soil bacterium Bacillus subtilis from the genotoxic effects induced by reactive oxygen species (ROS) promoter agents. Moreover, it reveals that the genetic inactivation of mutM allows nutritionally stressed bacteria to escape from growth-limiting conditions, putatively by a mechanism that involves the accumulation and error-prone processing of oxidized DNA bases. Reactive oxygen species (ROS), including hydrogen peroxide, superoxide, and hydroxyl radicals, are produced in all aerobic organisms as side products of oxidative metabolism or following exposure to environmental agents and are normally in balance with the cellular antioxidant defenses. Oxidative stress occurs when this critical balance is disrupted because of depletion of antioxidants or excess accumulation of ROS (1). Therefore, when antioxidant cellular defenses are deficient or overwhelmed, the damaging potential of ROS increases and they target different cellular biomolecules, including, lipids, proteins, carbohydrates, and DNA (2). One of the most common events resulting from attack of DNA by the hydroxyl radical is the formation of 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G), a DNA lesion extensively studied due to its strong mutagenic and genotoxic properties (3). However, the hydroxyl radicals can also impact the deoxyribonucleotide and ribonucleotide pools, generating the oxidized precursors 8-oxo-dGTP and 8-oxo-GTP, respectively (4, 5). The former is frequently incorporated opposite adenine ...