Epitope tagging analysis of the outer membrane folding of the molecular usher FaeD involved in K88 fimbriae biosynthesis in Escherichia coli.

To analyse the outer membrane folding of the molecular usher FaeD, tagged derivatives were prepared and their expression, tag-localisation and functioning in K88 fimbriae biosynthesis was studied. A semi-random insertion mutagenesis approach with factor Xa cleavage sites yielded six tagged FaeD derivatives. A site-directed mutagenesis approach in which c-myc epitopes were inserted yielded twenty-one different derivatives. Four tagged FaeD constructs were not expressed in the outer membrane as full-sized proteins to levels that could be detected by using immunoblotting analyses. Two of these had an insertion in the amino-terminal part of FaeD, whereas the other two had a tag inserted in the carboxyl-terminal part. The latter ones yielded stable carboxyl-terminally shortened truncates of about 70 kDa, as did other mutations in this region. Six tagged derivatives were expressed but the location of the tag with respect to the outer membrane could not be determined, possibly due to shielding. Functional analysis showed that insertion of a tag in two regions of FaeD, a central region of approximately 200 amino acid residues (a.a. 200-400) and the carboxyl-terminal region (a.a. 600-end), resulted in a defective K88 fimbriae biosynthesis. In-frame deletions in the amino-terminal region of FaeD abolished fimbriae production. The integrity of these regions is obviously essential for fimbriae biosynthesis. Based on the results and with the aid of a computer analysis programme for the prediction of outer membrane beta-strands, a folding model with 22 membrane spanning beta-strands and two periplasmioc domains has been developed.