The type IV bundle-forming pili (BFP) of enteropathogenic Escherichia coli (EPEC) are required for virulence in orally challenged human volunteers and for the localized adherence and autoaggregation in vitro phenotypes. BFP filament biogenesis and function are encoded by the 14-gene bfp operon. The BFP assembly complex, containing a BfpB-His 6 fusion protein, was chemically cross-linked in situ, and the complex was then purified from BFP-expressing EPEC by a combination of nickel-and BfpB antibody-based affinity chromatography. Characterization of the isolated complex by immunoblotting using BFP protein-specific antibodies showed that at least 10 of the 14 proteins specified by the bfp operon physically interact to form an oligomeric complex. Proteins localized to the outer membrane, inner membrane, and periplasm are within this complex, thus demonstrating that the complex spans the periplasmic space. A combination of immunofluorescence and immuno-gold thin-section transmission electron microscopy studies localized this complex to one pole of the cell.The bundle-forming pili (BFP) of enteropathogenic Escherichia coli (EPEC), a member of the type IV family of pilus proteins, are required for virulence in orally challenged human volunteers and for the localized adherence (LA) and autoaggregation (AA) in vitro phenotypes (2, 16, 24). The 14-gene bfp operon (located on the 69-kb EPEC adherence factor [EAF] plasmid), together with the genes encoding its transcriptional activator PerABC/BfpTVW, specifies the biogenesis of the pilus filament and the AA phenotype in wild-type EPEC strains and when harbored by E. coli strains that normally do not express BFP (5,21,26). Accordingly, the expression of proteins encoded by the bfp operon (in association with certain accessory proteins that are common to both EPEC and lab-adapted E. coli strains) (29) is sufficient for BFP biogenesis and for the BFP-mediated functions that confer the AA phenotype. By contrast, the LA phenotype also requires genes in the chromosomal locus of enterocyte effacement (11,23).In-frame disruption of each of the 14 bfp operon genes (designated bfpA to bfpL) ( Table 1) and analysis of the resulting mutants show that with the exception of bfpH, each of the remaining 13 genes is required for normal BFP filament production and phenotype expression (1, 13). These findings for the BFP system and similar results from the study of