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.

7-Hydroxytropolone (7-HT) is a symmetrical seven-membered heteroatomic ring with a carboxyl group and two hydroxyl groups and was recently reported to be an iron scavenger of HYS. Cluster 1 includes 12 genes related to the synthesis of 7-HT; among these genes, those for two regulators, Orf1 and Orf12, were predicted to regulate 7-HT biosynthesis and to be LysR-type transcriptional regulators (LTTRs) and TetR/AcrR family transcriptional regulators, respectively. Data from real-time quantitative PCR and β-galactosidase and classical siderophore assays indicated that the transcription levels of and , as well as those of crucial genes to , were repressed under high-iron conditions. The deletion of and led to an absence of 7-HT and a decrease in expression. Orf1 and Orf12 were essential for the production of 7-HT through These two regulators are regulated by the Gac/Rsm system; Orf1 facilitates the expression of Orf12, and Orf12 concomitantly stimulates the expression of to synthesize 7-HT. The overexpression of Orf12 decreased 7-HT yields, possibly through decreased expression. This work thus outlines a complex mechanism regulating the biosynthesis of the iron scavenger 7-HT in HYS. The synergy between Orf1 and Orf12 ensures that 7-HT acts as an iron chelator despite being toxic to bacteria and provides new ideas for the novel regulation of dual-functional secondary metabolism and research on 7-HT and its derivates in other bacteria. A complex regulation mechanism including two regulators, LysR and TetR/AcrR, in the biosynthesis of the novel iron scavenger 7-hydroxytropolone (7-HT) was verified in HYS. The coaction of LysR Orf1 and TetR/AcrR Orf12 may balance the toxicity and iron chelation of 7-HT in HYS to overcome iron deficiency, as well as improve the bacterial competitiveness under iron-scarce conditions because of the toxicity of 7-HT toward other bacteria, making the accurate regulation of 7-HT biosynthesis indispensable. This regulation mechanism may be ubiquitous in the group but may better explain the group's strong adaptability.

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