Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter

Authors

  • Kristen M. Skillman,

    1. Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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    • Present address: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

  • Travis J. Barnard,

    1. Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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  • Janine H. Peterson,

    1. Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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  • Rodolfo Ghirlando,

    1. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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  • Harris D. Bernstein

    Corresponding author
    1. Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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E-mail harris_bernstein@nih.gov; Tel. (+1) 301 402 4770; Fax (+1) 301 496 9878.

Summary

Bacterial autotransporters are proteins that contain a small C-terminal ‘β domain’ that facilitates translocation of a large N-terminal ‘passenger domain’ across the outer membrane (OM) by an unknown mechanism. Here we used EspP, an autotransporter produced by Escherichia coli 0157:H7, as a model protein to gain insight into the transport reaction. Initially we found that the passenger domain of a truncated version of EspP (EspPΔ1-851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspPΔ1-851 behaves as a compact monomer and strongly suggest that the channel formed by the β domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N-terminus of EspPΔ1-851 was efficiently translocated across the OM. Further analysis revealed that the passenger domain of wild-type EspP also folds at least partially in the periplasm. To reconcile these data, we propose that the EspP β domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.

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