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Elife. 2014 Sep 2;3:e04234. doi: 10.7554/eLife.04234.

Reconstitution of bacterial autotransporter assembly using purified components.

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Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, United States.


Autotransporters are a superfamily of bacterial virulence factors consisting of an N-terminal extracellular ('passenger') domain and a C-terminal β barrel ('β') domain that resides in the outer membrane (OM). The mechanism by which the passenger domain is secreted is poorly understood. Here we show that a conserved OM protein insertase (the Bam complex) and a molecular chaperone (SurA) are both necessary and sufficient to promote the complete assembly of the Escherichia coli O157:H7 autotransporter EspP in vitro. Our results indicate that the membrane integration of the β domain is the rate-limiting step in autotransporter assembly and that passenger domain translocation does not require the input of external energy. Furthermore, experiments using nanodiscs strongly suggest that autotransporter assembly is catalyzed by a single copy of the Bam complex. Finally, we describe a method to purify a highly active form of the Bam complex that should facilitate the elucidation of its function.


E. coli; biochemistry; infectious disease; membrane proteins; microbiology; protein folding; protein translocation; virulence factors

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