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A Complex Mechanism Involving LysR and TetR/AcrR That Regulates Iron Scavenger Biosynthesis in Pseudomonas donghuensis HYS

ABSTRACT 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 Pseudomonas donghuensis 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 orf1 and orf12, as well as those of crucial genes orf6 to orf9, were repressed under high-iron conditions. The deletion of orf1 and orf12 led to an absence of 7-HT and a decrease in orf6-orf9 expression. Orf1 and Orf12 were essential for the production of 7-HT through orf6-orf9. These two regulators are regulated by the Gac/Rsm system; Orf1 facilitates the expression of Orf12, and Orf12 concomitantly stimulates the expression of orf6-orf9 to synthesize 7-HT. The overexpression of Orf12 decreased 7-HT yields, possibly through decreased orf6-orf9 expression. This work thus outlines a complex mechanism regulating the biosynthesis of the iron scavenger 7-HT in P. donghuensis 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. IMPORTANCE 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 Pseudomonas donghuensis HYS. The coaction of LysR Orf1 and TetR/AcrR Orf12 may balance the toxicity and iron chelation of 7-HT in P. donghuensis 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 Pseudomonas putida group but may better explain the group's strong adaptability.