Structural and topographical studies of the type IV bundle-forming pilus assembly complex of enteropathogenic Escherichia coli.
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-His6 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.
Department of Medicine (Infectious Diseases and Geographic Medicine), Stanford Medical School, Stanford, California 94305, USA.
Jaiweon
J
Hwang
David
D
Bieber
Sandra W
SW
Ramer
Cheng-Yen
CY
Wu
Gary K
GK
Schoolnik
D016764:Cell Polarity; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000378:metabolism*; D015151:Immunoblotting; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009876:Operon; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D036781:Fimbriae Proteins