Reconstitution of bacterial autotransporter assembly using purified components

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Abstract

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.

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Giselle Roman-Hernandez

National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, United States

Competing interests
The authors declare that no competing interests exist.
Janine H Peterson

National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, United States

Competing interests
The authors declare that no competing interests exist.
Harris D Bernstein

National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, United States

For correspondence
harris_bernstein@nih.gov

Competing interests
The authors declare that no competing interests exist.

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This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.