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Journal of Bacteriology, December 1999, p. 7212-7220, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Genetic Dissection of the Outer Membrane Secretin PulD: Are There Distinct Domains for Multimerization and Secretion Specificity?

Ingrid Guilvout, Kim R. Hardie,dagger Nathalie Sauvonnet, and Anthony P. Pugsley*

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

Received 8 July 1999/Accepted 16 September 1999

Linker and deletion mutagenesis and gene fusions were used to probe the possible domain structure of the dodecameric outer membrane secretin PulD from the pullulanase secretion pathway of Klebsiella oxytoca. Insertions of 24 amino acids close to or within strongly predicted and highly conserved amphipathic beta  strands in the C-terminal half of the polypeptide (the beta  domain) abolished sodium dodecyl sulfate (SDS)-resistant multimer formation that is characteristic of this protein, whereas insertions elsewhere generally had less dramatic effects on multimer formation. However, the beta  domain alone did not form SDS-resistant multimers unless part of the N-terminal region of the protein (the N domain) was produced in trans. All of the insertions except one, close to the C terminus of the protein, abolished function. The N domain alone was highly unstable and did not form SDS-resistant multimers even when the beta  domain was present in trans. We conclude that the beta  domain is a major determinant of multimer stability and that the N domain contributes to multimer formation. The entire or part of the N domain of PulD could be replaced by the corresponding region of the OutD secretin from the pectate lyase secretion pathway of Erwinia chrysanthemi without abolishing pullulanase secretion. This suggests that the N domain of PulD is not involved in substrate recognition, contrary to the role proposed for the N domain of OutD, which binds specifically to pectate lyase secreted by E. chrysanthemi (V. E. Shevchik, J. Robert-Badouy, and G. Condemine, EMBO J. 16:3007-3016, 1997).

* 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 (0) 145688494. Fax: 33 (0) 145688960. E-mail: max{at}pasteur.fr.

dagger Present address: Institute of Infections and Immunity, University of Nottingham, University Hospital, Nottingham NG7 2UH, United Kingdom.

Journal of Bacteriology, December 1999, p. 7212-7220, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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