Salmonella typhimurium strains containing deletions of oxyR, a positive regulator of defenses against oxidative stress, show 10-to 55-fold higher frequencies of spontaneous mutagenesis compared to otherwise isogenic oxyR+ control strains. The high spontaneous-mutation frequency in oxyR deletion strains is decreased by a factor of 3 when the strains are grown anaerobically. oxyR deletion strains show an increase in small deletion mutations and at least three of the six possible base-substitution mutations (TA to A-T, COG to T-A, and C-G to A-T). However, the largest increase in mutation frequency is observed for T-A to A-T transversions (40-to 146-fold), the base-substitution mutation most frequently caused by chemical oxidants. The introduction into oxyR deletion strains of multicopy plasmids carrying the oxyRregulated genes for catalase (kaIG) or alkyl hydroperoxide reductase (ahp) results in overexpression of the respective enzyme activities and decreases the number of spontaneous mutants to wild-type levels. The introduction into oxyR deletions of a plasmid carrying the gene for superoxide dismutase (sodA) decreases the mutation frequency by a factor of 5 in some strain backgrounds. Strains that contain a dominant oxyR mutation and overexpress proteins regulated by oxyR show lower spontaneous-mutation frequencies by a factor of 2. These results indicate that oxyR and oxyR-regulated genes play a significant role in defense against spontaneous oxidative DNA damage. The role of oxidative damage to DNA in "spontaneous" mutagenesis is discussed.Spontaneous mutations could arise by several different mechanisms. These include depurination, deamination, and other forms of DNA damage and repair, as well as recombination and errors in DNA replication (1). Among the potential sources of DNA damage, active oxygen species have become recognized as being of biological importance (2-7). Studies have shown that oxygen metabolism influences DNA damage in mammals (6, 7) and bacteria. Hartman et al. (8) showed that Salmonella typhimurium strains grown under aerobic conditions had increased frequencies of small deletion and frameshift mutations relative to strains grown anaerobically, while the frequency of G-C to AT transitions was depressed under the aerobic conditions. Recently, Farr et al. (9) showed that Escherichia coli mutants that lack both the manganese-and iron-containing superoxide dismutases (sodA sodB double mutants) have enhanced spontaneousmutation frequencies compared to wild-type strains during aerobic growth. In addition, several E. coli DNA repair mutants-recA xth double mutants (10), certain polA mutants (11), and polA recB double mutants (12)-form filaments and die aerobically, though they grow normally under anaerobic conditions.We recently have identified and characterized a gene, oxyR, that positively controls a regulon involved in cellular defenses against oxidative stress in both S. typhimurium and E. coli (13,14). A dominant mutant, oxyRi, is resistant to hydrogen peroxide and constitutively...