DescriptionIron-sulfur (FeS) cluster containing proteins are essential to fundamental cellular processes like central metabolism and respiration. Organisms unable to make FeS clusters show metabolic abnormalities such as amino acid auxotrophies, poor growth when Fe is limiting, and resistance to certain antibiotics. Bacillus subtilis strains lacking ylaN show similar phenotypes, therefore we tested the hypothesis that the protein YlaN functions in the maturation of FeS proteins. Our results showed that a Staphylococcus aureus strain lacking ylaN displayed reduced growth in iron-limited media and when grown in the presence of the divalent metal chelator 2,2’-dipyridyl. The ΔylaN strain grew better than wild-type in the presence of streptonigrin, which is an antibiotic that requires Fe for killing. The ΔylaN strain also had decreased activity of the FeS cluster dependent enzyme aconitase. Purified YlaN prevented free Fe from binding to 1,10-phenanthroline. Taken together, these results show that YlaN has a role in regulating free Fe in S. aureus which is important in the maturation of FeS proteins.