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

我们首先证明了创伤弧菌M2799在铁限制条件下利用有氧肌动蛋白作为外源铁载体生长，同时增强了76-kDa铁抑制外膜蛋白的产生。随后，将Fur滴定法应用于M2799基因组文库，随后对候选基因周围的区域进行进一步克隆，鉴定出8.4 kb的有氧肌动蛋白利用基因簇，该基因簇由五个基因组成，排列在三个不同的转录单位中。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，该蛋白质包括用于羟酸盐型铁载体的atp结合盒运输系统。单个基因第二单元的蛋白产物iutR与转录抑制因子GntR家族共享同源氨基酸序列。另一个单基因的第三个单元编码一种与大肠杆菌pColV IutA同源的蛋白质，即铁有氧肌动蛋白的外膜受体。引物延伸分析表明，第一和第三单元的转录是通过启动子区域检测到的潜在的Fur盒状序列进行铁调控的。然而，在iutR的启动子区域未发现Fur box-like序列，这与该基因的组成性表达有关。即使在限铁条件下缺乏有氧肌动蛋白，iutR的破坏也会导致76 kda的有氧肌动蛋白受体的产生增加，这表明该基因编码了iutA的转录抑制因子。据我们所知，这是iutA同源物通过抑制蛋白调控转录的第一个例子。

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.