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Journal of Bacteriology, August 2001, p. 4435-4450, Vol. 183, No. 15
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.15.4435-4450.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Novel Topology of BfpE, a Cytoplasmic Membrane Protein Required for Type IV Fimbrial Biogenesis in Enteropathogenic Escherichia coli

T. Eric Blank and Michael S. Donnenberg*

Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received 21 August 2000/Accepted 7 May 2001

Enteropathogenic Escherichia coli (EPEC) produces the bundle-forming pilus (BFP), a type IV fimbria that has been implicated in virulence, autoaggregation, and localized adherence to epithelial cells. The bfpE gene is one of a cluster of bfp genes previously shown to encode functions that direct BFP biosynthesis. Here, we show that an EPEC strain carrying a nonpolar mutation in bfpE fails to autoaggregate, adhere to HEp-2 cells, or form BFP, thereby demonstrating that BfpE is required for BFP biogenesis. BfpE is a cytoplasmic membrane protein of the GspF family. To determine the membrane topology of BfpE, we fused bfpE derivatives containing 3' truncations and/or internal deletions to alkaline phosphatase and/or beta -galactosidase reporter genes, whose products are active only when localized to the periplasm or cytoplasm, respectively. In addition, we constructed BfpE sandwich fusions using a dual alkaline phosphatase/beta -galactosidase reporter cassette and analyzed BfpE deletion derivatives by sucrose density flotation gradient fractionation. The data from these analyses support a topology in which BfpE contains four hydrophobic transmembrane (TM) segments, a large cytoplasmic segment at its N terminus, and a large periplasmic segment near its C terminus. This topology is dramatically different from that of OutF, another member of the GspF family, which has three TM segments and is predominantly cytoplasmic. These findings provide a structural basis for predicting protein-protein interactions required for assembly of the BFP biogenesis machinery.

* Corresponding Author. Mailing Address: Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine, 10 South Pine Street, Medical School Teaching Facility 9-00, Baltimore, MD 21201-1192. Phone: (410) 706-7560. Fax: (410) 706-8700. E-mail: mdonnenb{at}umaryland.edu.

Journal of Bacteriology, August 2001, p. 4435-4450, Vol. 183, No. 15
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.15.4435-4450.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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