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Molecular determinants underlying the production of siderophores in the human and animal pathogen Staphylococcus aureus and the contribution of siderophore production to the virulence of this bacterium have, until now, remained undefined. Here, we show that S. aureus strains RN6390 and Newman produce siderophore when the cells are starved for iron. We further identified and characterized a nine-gene, iron-regulated operon, designated sbn and situated between sirABC and galE on the S. aureus chromosome, that is involved in the production of a siderophore. Mutation of the sbnE gene, in both RN6390 and Newman, eliminates the ability of these strains to produce a siderophore under iron-limited growth conditions, while introduction of multicopy sbnE into sbnE mutants complemented the inability of the mutants to produce the siderophore. sbnE mutants, in both the RN6390 and Newman backgrounds …