H-038. Identification of the Novel Fur-Binding Site and Its Role in Gene Expression Controlled by Fur in Vibrio vulnificus Genome

H-J. Lee, K-H. Lee;
Hankuk Univ. Foreign Studies, Yongin, Kyunggi-Do, REPUBLIC OF KOREA.

Bacterial ability to acquire iron is essential to maintain growth as well as to elicit virulence, which is mainly regulated by a transcriptional regulator, ferric uptake regulator (Fur). In the previous report (Lee et al. 2007 J Bacteriol 189:2629), fur gene expression in Vibrio vulnificus was shown to be activated by a direct binding of iron-free Fur to the upstream region of the fur gene, which includes two direct repeats of 5'-AAATTGT-3'. These finding led us to question if this novel Fur-binding site is unique in autoregulation of fur expression, or it is widely distributed in V. vulnificus genome. Thus, in this study, the V. vulnificus-DNA microarray was utilized to define the Fur-regulon. Total 288 orfs were differentially expressed in Δfur; expression of 148 orfs was significantly reduced in Δfur, and expression of 140 orfs was significantly increased in Δfur. Then, the upstream regions of those orfs were subjected to in silico analysis for the presence of the novel Fur-binding site, and 39 orfs (out of 148 Fur-activated orfs) and 29 orfs (out of 140 Fur-repressed orfs) were found to contain highly homologous sequences. To verify the role of this sequence, 4 genes were chosen for further study: from the Fur-activated group, ftr (formate transporter) and pts (phosphotransferase system); and from the Fur-repressed group, irp (iron regulated protein) and vuuA (vulnibactin outermembrane receptor). The primer extension assays were performed to identify the promoter regions of the selected genes. Then, transcription fusions including the putative regulatory region and the promoter(s) for each gene were constructed. In addition, the putative novel Fur-binding sites were mutagenized and then used for construction of transcriptional fusions. Fusion assays showed that expression of each fusion was consistent with the data derived DNA microarray, and that expression of the mutagenized fusions was no longer regulated by Fur. Therefore, the direct repeats of 5'-AAATTGT-3' is another consensus Fur-binding site, although it is not widely distributed as classical Fur box, and it is involved in both activation and repression by Fur in V. vulnificus.