ABSTRACTSalmonella entericaserovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety ofdammutantSalmonellavaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Sincedammutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-APr) isolates in systemic tissues of mice infected withdammutantSalmonella. Such 2-APrderivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of naïve mice. These 2-APrderivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parentaldammutant strain. Additionally, introduction of amutHnull mutation intodammutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required forSalmonellasystemic survival. These data suggest that restoration of intraperitoneal virulence ofdammutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.