Structure, localization and function of FanF, a minor component of K99 fibrillae of enterotoxigenic Escherichia coli

Abstract

The DNA sequence of the K99 fanF gene, encoding FanF, was determined. An open reading frame of 999 bp was found. The primary structure of FanF was deduced and analysis revealed the presence of a signal sequence of 22 amino acid residues. The mature protein contains 311 amino acid residues (Mr 33,905 D). The amino acid sequence of FanF showed similarity with the K88ab major subunit FaeG. A specific mouse antiserum against FanF was prepared by constructing and purifying a hybrid Cro-LacZ-FanF protein. Minicell analysis, immunoblotting and immunoelectronmicroscopy revealed a pool of FanF in the periplasm of K99-producing cells and showed, furthermore, that FanF is a minor component of K99 fibrillae, present at the top and in or along the shaft of the K99 fibrillar structures. A fanF mutant plasmid was constructed. Cells harbouring this plasmid produced all K99-specific proteins, except FanF, but produced 0.1% of the K99 fibrillae relative to 'normal' K99-producing cells. Electron microscopic observations showed that cells defective in fanF produce only a few (apparently short) K99 fibrillae. FanF, therefore, was supposed to play a role in initiation and elongation of K99 fibrillae formation. Thin-layer chromatography experiments involving purified receptor material showed that FanF is not required for binding of K99 fibrillae to the ganglioside receptor. Fibrillae produced by an adhesion-negative strain carrying a mutation in the K99 major fibrillar subunit were shown to contain a normal amount of FanF.