One prerequisite for the virulence of Yersinia pestis, causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the pgm locus of the Y. pestis chromosome. Several gene products within the HPI have demonstrated functions in the synthesis or transport of Ybt. Here we examine the roles of ybtU and ybtT. In-frame mutations in ybtT or ybtU yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from a Ybt-producing strain of Y. pestis. The ybtU mutant failed to express four indicator Ybt proteins (HMWP1, HMWP2, YbtE, and Psn), a pattern similar to those for other ybt biosynthetic mutants. In contrast, strains carrying mutations in ybtT or ybtS (a previously identified gene required for Ybt biosynthesis) produced all four proteins at wild-type levels under iron-deprived conditions. To assess the effects of ybtT, -U, and -S mutations on transcription of ybt genes, reporter plasmids with ybtP or psn promoters controlling lacZ expression were introduced into these mutants. Normal iron-regulated beta-galactosidase activity was observed in the ybtT and ybtS mutants, whereas a significant loss of expression occurred in the DeltaybtU strain. These results show that ybtT and ybtU genes are involved in the biosynthesis of the Ybt siderophore and that a ybtU mutation but not ybtT or ybtS mutations affects transcription from the ybtP and psn promoters.