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Journal of Bacteriology, December 2000, p. 6751-6761, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Maturation of IncP Pilin Precursors Resembles the Catalytic Dyad-Like Mechanism of Leader Peptidases

Ralf Eisenbrandt,dagger Markus Kalkum,Dagger Rudi Lurz, and Erich Lanka*

Max-Planck-Institut für Molekulare Genetik, Dahlem, D-14195 Berlin, Germany

Received 12 June 2000/Accepted 6 September 2000

The pilus subunit, the pilin, of conjugative IncP pili is encoded by the trbC gene. IncP pilin is composed of 78 amino acids forming a ring structure (R. Eisenbrandt, M. Kalkum, E.-M. Lai, C. I. Kado, and E. Lanka, J. Biol. Chem. 274:22548-22555, 1999). Three enzymes are involved in maturation of the pilin: LepB of Escherichia coli for signal peptide removal and a yet-unidentified protease for removal of 27 C-terminal residues. Both enzymes are chromosome encoded. Finally, the inner membrane-associated IncP TraF replaces a four-amino-acid C-terminal peptide with the truncated N terminus, yielding the cyclic polypeptide. We refer to the latter process as "prepilin cyclization." We have used site-directed mutagenesis of trbC and traF to unravel the pilin maturation process. Each of the mutants was analyzed for its phenotypes of prepilin cyclization, pilus formation, donor-specific phage adsorption, and conjugative DNA transfer abilities. Effective prepilin cyclization was determined by matrix-assisted laser desorption-ionization-mass spectrometry using an optimized sample preparation technique of whole cells and trans-3-indolyl acrylic acid as a matrix. We found that several amino acid exchanges in the TrbC core sequence allow prepilin cyclization but disable the succeeding pilus assembly. We propose a mechanism explaining how the signal peptidase homologue TraF attacks a C-terminal section of the TrbC core sequence via an activated serine residue. Rather than cleaving and releasing hydrolyzed peptides, TraF presumably reacts as a peptidyl transferase, involving the N terminus of TrbC in the aminolysis of a postulated TraF-acetyl-TrbC intermediate. Under formal loss of a C-terminal tetrapeptide, a new peptide bond is formed in a concerted action, connecting serine 37 with glycine 114 of TrbC.

* Corresponding author. Mailing address: Max-Planck-Institut für Molekulare Genetik, Abteilung Lehrach, Ihnestrasse 73, Dahlem, D-14195 Berlin, Germany. Phone: 49-30-8413-1696. Fax: 49-30-8413-1130. E-mail: lanka{at}molgen.mpg.de.

dagger Present address: Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma, Canto Blanco, E-28049 Madrid, Spain.

Dagger Present address: The Rockefeller University, Mass Spectrometry Laboratory, New York, NY 10021-6399.

Journal of Bacteriology, December 2000, p. 6751-6761, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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