bSiderophores, which are produced to overcome iron deficiency, are believed to be closely related to the adaptability of bacteria. The high-siderophore-yielding Pseudomonas sp. strain HYS simultaneously secretes the fluorescent siderophore pyoverdine and another nonfluorescent siderophore that is a major contributor to the high siderophore yield. Transposon mutagenesis revealed siderophore-related genes, including the two-component regulators GacS/GacA and a special cluster containing four open reading frames (the nfs cluster). Deletion mutations of these genes abolished nonfluorescent-siderophore production, and expression of the nfs cluster depended on gacA, indicating that gacS-gacA may control the nonfluorescent siderophore through regulation of the nfs cluster. Furthermore, regulation of the nonfluorescent siderophore by GacS/GacA involved the Gac/Rsm pathway. In contrast, inactivation of GacS/GacA led to upregulation of the fluorescent pyoverdine. The two siderophores were secreted under different iron conditions, probably because of differential effects of GacS/GacA. The global GacS/GacA regulatory system may control iron uptake by modulating siderophore production and may enable bacteria to adapt to changing iron environments.