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Journal of Bacteriology, April 2000, p. 2142-2152, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Multiple Interactions between Pullulanase Secreton Components Involved in Stabilization and Cytoplasmic Membrane Association of PulE

Odile M. Possot, Guillaume Vignon, Natalia Bomchil,dagger Frank Ebel, and Anthony P. Pugsley*

Unité de Génétique Moléculaire, CNRS, URA 1773, Institut Pasteur, 75724 Paris, Cedex 15, France

Received 2 September 1999/Accepted 14 January 2000

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.

* Corresponding author. Mailing address: Unité de Génétique Moléculaire, CNRS, URA 1773, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, Cedex 15, France. Phone: 33-1-45688494. Fax: 33-1-45688960. E-mail: max{at}pasteur.fr.

dagger Present address: Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.

Journal of Bacteriology, April 2000, p. 2142-2152, Vol. 182, No. 8
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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