PERMISSIVE LINKER INSERTION SITES IN THE OUTER-MEMBRANE PROTEIN OF 987P FIMBRIAE OF ESCHERICHIA-COLI

The FasD protein is essential for the biogenesis of 987P fimbriae of E scherichia coli. In this study, subcellular fractionation was used to demonstrate that FasD is an outer membrane protein. In addition, the a ccessibility of FasD to proteases established the presence of surface- exposed FasD domains on both sides of the outer membrane. The fasD gen e was sequenced, and the deduced amino acid sequence was shown to shar e homologous domains with a family of outer membrane proteins from var ious fimbrial systems. Similar to porins, fimbrial outer membrane prot eins are relatively polar, lack typical hydrophobic membrane-spanning domains, and possess secondary structures predicted to be rich in turn s and amphipathic beta-sheets. On the basis of the experimental data a nd structural predictions, FasD is postulated to consist essentially o f surface-exposed turns and loops and membrane-spanning interacting am phipathic beta-strands. In an attempt to test this prediction, the fas D gene was submitted to random in-frame linker insertion mutagenesis. Preliminary experiments demonstrated that it was possible to produce f asD mutants, whose products remain functional for fimbrial export and assembly. Subsequently, 11 fasD alleles, containing linker inserts enc oding beta-turn-inducing residues, were shown to express functional pr oteins. The insertion sites were designated permissive sites. The inse rts used are expected to be least detrimental to the function of FasD when they are inserted into surface-exposed domains not directly invol ved in fimbrial export. In contrast, FasD is not expected to accommoda te such residues in its amphipathic P-strands without being destabiliz ed in the membrane and losing function. All permissive sites were sequ enced and shown to be located in or one residue away from predicted tu rns. In contrast, 5 of 10 sequenced nonpermissive sites were mapped to predicted amphipathic beta-strands. These results are consistent with the structural predictions for FasD.