Shigella flexneri, a causative agent of bacterial dysentery, possesses two predicted iron-sulfur cluster biosynthesis systems called Suf and Isc. S. flexneri strains containing deletion mutations in the entire suf operon (UR011) or the iscSUA genes (UR022) were constructed. Both mutants were defective in surviving exposure to oxidative stress. The suf mutant showed growth that was comparable to that of the parental strain in both iron-replete and iron-limiting media; however, the isc mutant showed reduced growth, relative to the parental strain, in both media. Although the suf mutant formed wild-type plaques on Henle cell monolayers, the isc mutant was unable to form plaques on Henle cell monolayers because the strain was noninvasive. Expression from both the suf and isc promoters increased in iron-limiting media and in the presence of hydrogen peroxide. Iron repression of the suf promoter was mediated by Fur, and increased suf expression in iron-limiting media was enhanced by the presence of IscR. Iron repression of the isc promoter was mediated by IscR. Hydrogen peroxide-dependent induction of suf expression, but not isc expression, was mediated by OxyR. Furthermore, IscR was a positive regulator of suf expression in the presence of hydrogen peroxide and a negative regulator of isc expression in the absence of hydrogen peroxide. Expression from the S. flexneri suf and isc promoters increased when Shigella was within Henle cells, and our data suggest that the intracellular signal mediating this increased expression is reduced iron levels.As a facultative intracellular pathogen, Shigella flexneri spends a significant portion of its life cycle within the epithelial cells lining the human colon. Invasion of and intracellular survival/replication of the bacteria within these epithelial cells requires the ability to sense the environment and initiate an appropriate metabolic strategy during infection. Global analysis of Shigella transcription during epithelial cell infection indicated that a variety of metabolic genes, including the suf genes, are precisely regulated when Shigella is intracellular (16,27).The Shigella flexneri suf and isc loci encode predicted ironsulfur (Fe-S) cluster biosynthesis systems. Iron-sulfur clusters are essential for a wide variety of biological processes, including redox reactions, substrate binding and activation, iron storage, protein structure, and regulation of gene expression (11). The S. flexneri isc locus contains iscR, iscS, iscU, iscA, hscB, hscA, and fdx and is highly conserved with the Escherichia coli isc locus. E. coli IscS catalyzes the desulfurization of L-cysteine for the recruitment of S for Fe-S cluster formation (4). IscU and IscA are predicted to form scaffolds for Fe-S cluster assembly based on similarities with Azobacter vinelandii NifU and IscA Nif (1, 11, 13). The chaperones HscB and HscA aid in Isc-mediated Fe-S protein maturation, although the specifics are not entirely clear (for a review, see reference 11). Deletion of the isc locus in E. coli reduced the gro...