ABSTRACTLabile [4Fe-4S]2+clusters found at the active sites of many dehydratases are susceptible to damage by univalent oxidants that convert the clusters to an inactive [3Fe-4S]1+form. Bacteria repair damaged clusters in a process that does not requirede novoprotein synthesis or the Isc and Suf cluster assembly pathways. The current study investigates the participation of the bacterial frataxin ortholog CyaY and the YggX protein, which are proposed to play roles in iron trafficking and iron-sulfur cluster repair. Previous reports found that individual mutations incyaYoryggXwere not associated with phenotypic changes inEscherichia coliandSalmonella entericaserovar Typhimurium, suggesting that CyaY and YggX might have functionally redundant roles. However, we have found that individual mutations incyaYoryggXconfer enhanced susceptibility to hydrogen peroxide inSalmonella entericaserovar Typhimurium. In addition, inactivation of thestm3944open reading frame, which is located immediately upstream ofcyaYand which encodes a putative inner membrane protein, dramatically enhances the hydrogen peroxide sensitivity of acyaYmutant. Overexpression of STM3944 reduces the elevated intracellular free iron levels observed in anS. Typhimuriumfurmutant and also reduces the total cellular iron content under conditions of iron overload, suggesting that thestm3944-encoded protein may mediate iron efflux. Mutations incyaYandyggXhave different effects on the activities of the iron-sulfur cluster-containing aconitase, serine deaminase, and NADH dehydrogenase I enzymes ofS. Typhimurium under basal conditions or following recovery from oxidative stress. In addition,cyaYandyggXmutations have additive effects on 6-phosphogluconate dehydratase-dependent growth during nitrosative stress, and acyaYmutation reducesSalmonellavirulence in mice. Collectively, these results indicate that CyaY and YggX play distinct supporting roles in iron-sulfur cluster biosynthesis and the repair of labile clusters damaged by univalent oxidants.Salmonellaexperiences oxidative and nitrosative stress within host phagocytes, and CyaY-dependent maintenance of labile iron-sulfur clusters appears to be important forSalmonellavirulence.