Identification and characterization of iron-regulated genes required for utilization of a erobactin in Vibrio vulnificus

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We first demonstrated that Vibrio vulnificus M2799 utilizes aerobactin for growth as an exogenous siderophore under iron-limiting conditions, concomitant with enhanced production of the 76-kDa iron-repressible outer membrane protein. Subsequently, application of the Fur titration assay method to the M2799 genomic libraries, followed by further cloning of the regions surrounding the candidate genes, led to the identification of the 8.4-kb aerobactin utilization gene cluster comprising five genes arranged in three distinct transcriptional units. The first three genes of an operonic structure encode proteins homologous to Escherichia coli FhuCDB which comprise an ATP-binding cassette transport system for hydroxamate-type siderophores. The protein product of the second unit of a single gene, named iutR, shared amino acid sequence homology with the GntR family of transcriptional repressors. The third unit of another single gene encodes a protein homologous to E.coli pColV IutA, the outer membrane receptor for ferric aerobactin. Primer extension analysis showed that transcription of the first and third units was iron-regulated through the potential Fur box-like sequences detected in their promoter regions. However, no Fur box-like sequence was found in the promoter region of iutR, consisting with the constitutive expression of this gene. Disruption of iutR resulted in enhanced production of the 76-kDa ferric aerobactin receptor even in the absence of aerobactin under iron-limiting conditions, suggesting that the gene encodes a transcriptional repressor for iutA. To the best of our knowledge, this is the first example of an iutA homologue whose transcription is regulated via a repressor protein.