Quorum sensing regulators AphA and HapR differentially regulate fluvibactin biosynthesis in Vibrio fluvialis

IntroductionSiderophores are secondary metabolites with high affinity and specificity for iron and play a crucial role in iron acquisition. Vibrio fluvialis, an emerging foodborne opportunistic pathogen of increasing concern, produces the catechol siderophore fluvibactin. Quorum sensing (QS) is a process of bacterial communication vital for many biological processes. To date, the genes responsible for fluvibactin biosynthesis and the role of quorum sensing (QS) in its regulation remain unclear.MethodsIn this study, we characterized the gene cluster involved in fluvibactin biosynthesis and investigated how the QS regulators AphA at low cell density (LCD) and HapR at high cell density (HCD) regulate this process. Gene expression, promoter activity, and protein–DNA interactions were analyzed using qPCR, reporter assays, electrophoretic mobility shift assays (EMSA), DNase I footprinting, and 5′ rapid amplification of cDNA ends (RACE). Also, animal experiments were conducted with a mutant which fails to biosynthesize fluvibactin.ResultsWe identified a fluvibactin biosynthesis gene cluster consisting of 16 genes organized into 9 transcriptional units, which are activated under iron-deficient conditions. Fluvibactin production was increased in the ΔaphA mutant at LCD and decreased in the ΔhapR mutant at HCD compared to the wild type. Consistently, the expression and promoter activities of representative genes (vfbH, vfbA, vfbCE and vfbB) were repressed by AphA at LCD and activated by HapR at HCD. Direct binding of AphA and HapR to the promoters of these genes was confirmed. Additionally, we demonstrated that fluvibactin production was associated with increased bacterial loads in infected mice.DiscussionThese findings provide insights into the genetic basis of fluvibactin biosynthesis and its regulation by QS in V. fluvialis. This study enhances our understanding of the pathogenicity and environmental adaptability of this organism under iron-limited conditions and contributes to the knowledge of catechol siderophores in Vibrios.