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Journal of Bacteriology, January 2009, p. 161-168, Vol. 191, No. 1
0021-9193/09/$08.00+0     doi:10.1128/JB.01138-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Type II Secretion System Secretin PulD Localizes in Clusters in the Escherichia coli Outer Membrane{triangledown}

Nienke Buddelmeijer,1 Martin Krehenbrink,1 Frédéric Pecorari,2 and Anthony P. Pugsley1*

Institut Pasteur, Molecular Genetics Unit and CNRS URA2172, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France,1 CNRS UMR 6204, Biotechnology, Biocatalysis and Bioregulation Unit, University of Nantes, 44322 Nantes Cedex 3, France2

Received 13 August 2008/ Accepted 23 October 2008

The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.

* Corresponding author. Mailing address: Institut Pasteur, Molecular Genetics Unit, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: 33-145688494. Fax: 33-145688960. E-mail: max{at}pasteur.fr

{triangledown} Published ahead of print on 31 October 2008.

Journal of Bacteriology, January 2009, p. 161-168, Vol. 191, No. 1
0021-9193/09/$08.00+0     doi:10.1128/JB.01138-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lybarger, S. R., Johnson, T. L., Gray, M. D., Sikora, A. E., Sandkvist, M. (2009). Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae. J. Bacteriol. 191: 3149-3161 [Abstract] [Full Text]  


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