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Journal of Bacteriology, October 2009, p. 6300-6311, Vol. 191, No. 20
0021-9193/09/$08.00+0     doi:10.1128/JB.00833-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Genome-Scale Proteomic Screen Identifies a Role for DnaK in Chaperoning of Polar Autotransporters in Shigella{triangledown}

Anuradha Janakiraman,1,2 Kathryn R. Fixen,3 Andrew N. Gray,3 Hironori Niki,4,5 and Marcia B. Goldberg1,3*

Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts 02139,1 Department of Biology, City College and Graduate Center of the City University of New York, New York, New York 10031,2 Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115,3 Radioisotope Center,4 Microbial Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka, Japan5

Received 25 June 2009/ Accepted 7 August 2009

Autotransporters are outer membrane proteins that are widely distributed among gram-negative bacteria. Like other autotransporters, the Shigella autotransporter IcsA, which is required for actin assembly during infection, is secreted at the bacterial pole. In the bacterial cytoplasm, IcsA localizes to poles and potential cell division sites independent of the cell division protein FtsZ. To identify bacterial proteins involved in the targeting of IcsA to the pole in the bacterial cytoplasm, we screened a genome-scale library of Escherichia coli proteins tagged with green fluorescent protein (GFP) for those that displayed a localization pattern similar to that of IcsA-GFP in cells that lack functional FtsZ using a strain carrying a temperature-sensitive ftsZ allele. For each protein that mimicked the localization of IcsA-GFP, we tested whether IcsA localization was dependent on the presence of the protein. Although these approaches did not identify a polar receptor for IcsA, the cytoplasmic chaperone DnaK both mimicked IcsA localization at elevated temperatures as a GFP fusion and was required for the localization of IcsA to the pole in the cytoplasm of E. coli. DnaK was also required for IcsA secretion at the pole in Shigella flexneri. The localization of DnaK-GFP to poles and potential cell division sites was dependent on elevated growth temperature and independent of the presence of IcsA or functional FtsZ; native DnaK was found to be enhanced at midcell and the poles. A second Shigella autotransporter, SepA, also required DnaK for secretion, consistent with a role of DnaK more generally in the chaperoning of autotransporter proteins in the bacterial cytoplasm.

* Corresponding author. Mailing address: Bacterial Pathogenesis, Massachusetts General Hospital, 65 Landsdowne Street, Cambridge, MA 02139. Phone: (617) 768-8740. Fax: (617) 768-8738. E-mail: mgoldberg1{at}partners.org

{triangledown} Published ahead of print on 14 August 2009.

Journal of Bacteriology, October 2009, p. 6300-6311, Vol. 191, No. 20
0021-9193/09/$08.00+0     doi:10.1128/JB.00833-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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