The major surface-located protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin, molecular mass, 38 kDa, 341 amino acids. To define precisely the molecular reactivities of nine mouse monoclonal antibodies (MAbs) against Hib porin, overlapping hexapeptides corresponding to the entire sequence of porin were synthesized. The epitopes recognized by the MAbs were mapped by enzyme-linked immunosorbent assay to stretches of 6 to 11 amino acids. Antigenic sites between amino acids 112 and 126, 148 and 153, 162 and 172, and 318 and 325 were identified. The antigenic sites between amino acids 162 and 172 and between amino acids 318 and 325 were determined by flow cytometry to be on the bacterial cell surface. Four MAbs, POR.2, POR.3, POR.4, and POR.5, that react with amino acids 162 to 172 were able to discriminate among porins from the three major outer membrane protein subtypes of Hib, i.e., 1H, 2L, and 6U. A model for the topological organization of Hib porin was created by calculating the hydrophobicity, amphiphilicity, and turn propensity in its amino acid sequence. Determination of the molecular reactivities of the anti-Hib porin MAbs provided substantive evidence for the orientation of selected regions of porin in the outer membrane of Hib.

The ferrichrome-iron receptor of Escherichia coli K-12 is FhuA (Me, 78,992), the first component of an energy-dependent, high-affinity iron uptake pathway. FhuA is also the cognate receptor for bacteriophages T5, Ti, +80, and UC-1, for colicin M and microcin 25, and for albomycin. To probe the topological organization of FhuA which enables recognition of these different ligands, we generated a library of 16 Under conditions of iron deprivation, many gram-negative bacteria derepress the expression of a class of surface receptors called iron-regulated outer membrane proteins. The function of these proteins is to bind specific siderophores and to initiate their internalization, thereby enabling the cell to satisfy its iron requirement (5). Siderophores have widely differing structures, but all share a high affinity for the ferric ion (24). Assimilation of the ferric siderophore requires a specific receptor as well as other periplasmic and cytoplasmic membrane proteins, including TonB (28). A cytoplasmic membrane protein complex including TonB, ExbB, and ExbD is thought to couple the electrochemical potential of the cytoplasmic membrane to the outer membrane siderophore receptor, a step which allows subsequent translocation of the bound ligand into the periplasm (37,38 proteins, including FepA (26), FhuA (23), FoxA (1), and LamB (9). These models incorporate some structural elements in common: amphiphilic sequences constitute antiparallel 3 sheets which traverse the outer membrane; intervening sequences are thought to constitute loops that are exposed at the cell surface or in the periplasm.Predictions of outer membrane protein topology can be evaluated with a number of tools, including monoclonal antibodies (MAbs) (26,34,39), insertion and deletion mutagenesis (4, 6, 7, 23), and reporter protein fusions (27)

The major surface-located, channel-forming protein in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; MA, 37,782). In order to generate Hib porin that is devoid of lipooligosaccharides and capsular polysaccharide, the Hib porin gene ompP2 was subcloned into a plasmid vector and recombinant 3334