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.
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
-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/
-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.
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