title: Biosynthesis of K88 fimbriae in Escherichia coli: interaction of tip-subunit FaeC with the periplasmic chaperone FaeE and the outer membrane usher FaeD.
abstract: K88 fimbriae are ordered polymeric protein structures at the surface of enterotoxigenic Escherichia coli cells. Their production and assembly requires a molecular chaperone located in the periplasm (FaeE) and a molecular usher located in the outer membrane (FaeD). FaeC is the tip component of the K88 fimbriae. We studied the expression of the subcloned faeC gene, the subcellular localization of FaeC and its interaction with the chaperone and the outer membrane usher. In the absence of the chaperone or the usher, FaeC could not be detected in E. coli cells harbouring the faeC gene and its ribosome binding site under contol of the IPTG inducible lpp/lac promoter/operator. The expression of FaeC was detectable in the presence of chaperone FaeE, but a direct interaction between the chaperone and FaeC was not found. The expression of FaeC was also detectable in cells co-expressing the outer membrane usher FaeD. Overexpression of FaeC after changing the faeC ribosome binding site appeared to induce lethality. Expression of subcloned FaeC in the absence of FaeE or FaeD could be detected when faeC was cloned under the tight control of the ara promoter/operator and when lethality induction was avoided. The direct interaction of FaeC with outer membranes containing the usher FaeD was studied by cell fractionation, isopycnic sucrose density gradient centrifugation, SDS-PAGE and immunoblotting. FaeC was found to bind to outer membranes containing FaeD or a FaeD-PhoA hybrid construct containing 215 amino-terminal residues of FaeD. This binding was not observed when control outer membranes without FaeD were used. No other K88 specific proteins were required for this interaction. The direct interaction between FaeC and FaeD in the outer membranes was shown by affinity blotting experiments. FaeE was not required for this interaction. Together these data indicate that the minor fimbrial subunit FaeC, unlike FaeG, H and F, does not have a strong interaction with the chaperone FaeE in the E. coli periplasm, but directly binds to the outer membrane molecular usher FaeD.
authors.affiliation: Department of Molecular Microbiology, Institute of Molecular Biological Sciences, BioCentrum Amsterdam, Faculty of Biology, Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: O
authors.initials: O
authors.lastname: Mol
authors.forename: W C
authors.initials: WC
authors.lastname: Oudhuis
authors.forename: R P
authors.initials: RP
authors.lastname: Oud
authors.forename: R
authors.initials: R
authors.lastname: Sijbrandi
authors.forename: J
authors.initials: J
authors.lastname: Luirink
authors.forename: N
authors.initials: N
authors.lastname: Harms
authors.forename: B
authors.initials: B
authors.lastname: Oudega
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial; D000954:Antigens, Surface / Q000096:biosynthesis*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D015870:Gene Expression; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D019897:Periplasm
chemical_list: D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C098025:FaeD protein, E coli; C026888:K88 antigen, E coli; D018832:Molecular Chaperones; C070012:faeE protein, E coli; D036781:Fimbriae Proteins