Probing the in vivo dynamics of type I protein secretion complex association through sensitivity to detergents

Unité des Membranes Bactériennes CNRS URA 2172; Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, Institut Pasteur, 75724 Paris Cedex 15 France. Département de Microbiologie, Institut Pasteur, 75724 Paris Cedex 15 France

^* To whom correspondence should be addressed. Email: cwander{at}pasteur.fr.

	Abstract

The Serratia marcescens hemophore is secreted by a type I secretion system consisting of three proteins: a membrane ABC protein, an adaptor protein and the TolC-like outer membrane protein. Assembly of these proteins is induced by substrate binding to the ABC protein. Here we show that a hemophore mutant lacking its last 14 C-terminal amino acids is not secreted, but rather, interacts with the ABC protein and promotes a stable multiprotein complex. Strains expressing the transporter and the mutant protein are sensitive to detergents (SDS). TolC is trapped in the transporter jammed by the truncated substrate and therefore not present at sufficient concentrations to allow the efflux pumps to expell detergents. Using this SDS sensitivity assay, we were able to show that the hemophore interacts with the ABC protein via two non-overlapping sites. We also demonstrate that the C-terminal peptide which, on the overall substrate functions as an intramolecular signal sequence, may also have intermolecular activity and triggers complex dissociation in vivo when provided as a distinct peptide.

The SDS sensitivity test on plates enables the study of type I secretion protein association/dissociation independently of the secretion process itself.