Postweaning diarrhoea (PWD) and oedema disease (OD) are the leading cause of serious illness in early-weaned pigs. These diseases, which give rise to slowed growth and death in piglets, impose an enormous economic burden on the pig industry worldwide, including Australia. The causative agents of PWD and OD are strains of enterotoxigenic Escherichia coli (ETEC). The key virulence factors produced by pig ETEC include Shiga toxin, heat-labile (LT) and/or heat-stable enterotoxin (STa or STb) as well as fimbrial adhesins (F4 or F18) which are required by ETEC to attach to the pig gut mucosal surface to deliver toxins into the epithelium.
A previous study from our lab showed that FedR, a member of the AraC family of transcriptional regulators, can activate the expression of two targets in pig ETEC, the F18 operon which contains five genes fedABCDF, each encoding a different component of the F18 fimbriae and the fedR operon which encodes its own protein. To understand the molecular mechanism of FedR-mediated activation, we carried out a series of experiments to identify the promoter and operator sequences of the fedABCDF and the fedR operons. Our results indicated that the transcription of each operon is driven by a single sigma-70 promoter. Furthermore, an 8-bp palindromic sequence (named FedR box) related to the consensus TGTGCACA and located upstream of the promoters of the two target operons were shown to be responsible for FedR-mediated activation.
In addition, we performed random and site-directed mutagenesis to map the various functional domains of the FedR protein. Further biochemical and genetic assays will be used to determine whether these mutations are defective in DNA binding, dimer formation or the interaction with RNA polymerase.