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

我们首先证明，创伤弧菌M2799利用需氧肌动蛋白作为外源铁载体在铁限制条件下的生长，伴随着76 kDa的铁抑制性外膜蛋白的生产增强。随后，应用的毛皮滴定测定方法的M2799基因组文库，然后进一步克隆的候选基因周围的区域，导致鉴定的8.4 kb的好氧肌动蛋白利用基因簇，包括5个基因排列在三个不同的转录单位。操纵子结构的前三个基因编码与大肠杆菌FhuCDB同源的蛋白质，其包含异羟肟酸盐型铁载体的ATP结合盒转运系统。单个基因的第二个单位的蛋白质产物，命名为iutR，与GntR家族的转录抑制因子共享氨基酸序列同源性。另一个单基因的第三个单元编码与大肠杆菌pColV IutA同源的蛋白质，IutA是铁需氧菌素的外膜受体。引物延伸分析表明，第一和第三个单位的转录是铁调节通过潜在的Fur盒样序列检测在其启动子区域。然而，在iutR的启动子区没有发现Fur盒样序列，与该基因的组成型表达一致。中断iutR导致76 kDa的铁的需氧肌动蛋白受体的生产增强，即使在铁限制条件下的需氧肌动蛋白的情况下，这表明该基因编码的iutA的转录阻遏物。据我们所知，这是iutA同源物的第一个例子，其转录通过阻遏蛋白进行调节。

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.