OUTER-MEMBRANE TRANSLOCATION ARREST OF THE TCPA PILIN SUBUNIT IN RFB MUTANTS OF VIBRIO-CHOLERAE O1 STRAIN 569B

The toxin-coregulated pilus (TCP) of Vibrio cholerae is a type 4-relat ed fimbrial adhesin and a useful model for the study of type 4 pilus b iogenesis and related bacterial macromolecular transport pathways, Tra nsposon mutagenesis of the putative perosamine biosynthesis genes in t he rfb operon of V. cholerae 569B eliminates lipopolysaccharide (LPS) O-antigen biosynthesis but also leads to a specific defect in TCP expo rt. Localization of TcpA is made difficult by the hydrophobic nature o f this bundle-forming pilin, which floats anomalously in sucrose densi ty gradients, but the processed form of TcpA can be found in membrane and periplasmic fractions prepared from these strains. While TcpA cann ot be detected by surface immunogold labelling in transmission electro n microscope preparations, EDTA pretreatment facilitates immunofluores cent antibody labelling of whole cells, and ultrathin cryosectioning t echniques confirm membrane and periplasmic accumulation of TcpA, Salt and detergent extraction, protease accessibility, and chemical cross-l inking experiments suggest that although TcpA has not been assembled o n the cell surface, subunit interactions are otherwise identical to th ose within TCP, In addition, TcpA-mediated fucose-resistant hemaggluti nation of murine erythrocytes is preserved in whole-cell lysates, sugg esting that TcpA has obtained its mature conformation. These data loca lize a stage of type 4 pilin translocation to the outer membrane, at w hich stage export failure leads to the accumulation of pilin subunits in a configuration similar to that within the mature fiber. Possible c andidates for the outer membrane defect are discussed.