PORINS OF VIBRIO-CHOLERAE - PURIFICATION AND CHARACTERIZATION OF OMPU

Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been p urified to homogeneity, The synthesis of all the three proteins is reg ulated by the osmolarity of the growth medium. The pore-forming abilit y of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded , The 27-kDa protein, OmpX, though osmoregulated, is not a porin. OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl. OmpU is an acidic protein and is a homotrimer of 38-kDa monomeri c units, Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit, R emoval of Ca2+ irreversibly disrupts the structure and pore-forming ab ility of the protein, The pore size of OmpU is 1.6 nm, and the specifi c activity of the OmpU channel is two- to threefold higher than that o f Escherichia coli porin OmpF, synthesis of which resembles that of Om pU with respect to the osmolarity of the growth medium, The pore size of OmpT, which is analogous to OmpC of E. coli, is smaller than that o f OmpU, Southern blot hybridization of V. cholerae genomic DNA digeste d with several restriction endonucleases with nick-translated. coli om pF as the probe revealed no nucleotide sequence homolog between the om pU and ompF genes, OmpU is also not antigenically related to OmpF, Ant i-OmpF antiserum, however, cross-reacted with the 45-kDa V, cholerae o uter membrane protein, OmpS, the synthesis of which is regulated by th e presence of maltose in the growth medium, OmpU hemagglutinated with rabbit and human blood. This toxR-regulated protein is one of the poss ible virulence determinants in V. cholerae (V. L. Miller and J. J. Mek alanos, J. Bacteriol, 170:2575-2583, 1988).