Site-Directed Mutagenesis of the Campylobacter jejuni Fur Box and the Iron- and Oxygen-Responsive Regulation of fumC

For the foodborne enteric pathogen Campylobacter jejuni, regulation of iron homeostasis is tightly controlled by the ferric uptake regulator Fur. Fur regulates iron-responsive gene expression by binding to the Fur box sequence and a 19 bp Fur box is positioned within the promoter region of the outer membrane haem receptor gene chuA. The fumarase fumC Fur box-like sequence shows three mismatches to the consensus sequence and this variation is predicted to be key to the contrasting iron regulation and Fur-Fur box binding affinity between chuA and fumC. The aims of this study were to determine the functionally important bases in the C. jejuni Fur box that are essential for Fur-Fur box interaction and to assess the interplay of iron and oxygen in the modulation of fumC expression. Site-directed mutagenesis of the 1st, 7th, 10th, 13th and 19th positions was carried out for the chuA and fumC Fur boxes and their interaction with Fur was determined in vitro and in vivo. Two Fur dimers were determined to bind to the Fur box, and although the 1st, 7th, 13th and 19th positions were found to facilitate the interaction of Fur with the Fur box, the architecture of the promoter region is likely to play a more significant role in Fur regulation. fumC encodes the only fumarase in C. jejuni and it is essential for cell growth and for maintaining a functional tricarboxylic acid cycle. Further characterisation of chuA and fumC expression in response to iron and oxygen indicated that both genes are controlled by Fur as well as the peroxide response regulator PerR, and the RacR-RacS two-component system. These observations illustrate the necessity for C. jejuni to cooperatively regulate essential gene expressions using its rather limited set of regulators thus allowing it to adapt to various conditions encountered during transmission and colonisation.