Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL.
Erwinia chrysanthemi secretes, by the type II secretory pathway, a large number of enzymes, including cellulases and pectinases. This process requires the products of the out genes, which are widely conserved in Gram-negative bacteria. The Out proteins are thought to form a membrane-associated multiprotein complex. Here, we investigated interaction between OutE, the putative ATP binding component, and OutL, an inner membrane protein. We showed, by limited proteolysis, genetic suppression and the yeast two-hybrid system, that OutE and OutL interact directly. Analysis of truncated forms of OutE demonstrated that the N terminus of OutE (residues 1-97) is important for the OutE/OutL interaction. Moreover, results from the yeast two-hybrid system suggested that OutE and OutL are each able to form homomultimers. The region required for homomultimerisation of OutE is located in its C terminus. Limited proteolysis assay indicated that OutE induces a conformational change in OutL, in both its cytoplasmic and periplasmic domains. Moreover, the secretion process requires a conformational change in OutE which depends on both the interaction with OutL and on the presence of an intact Walker A motif in OutE. Our results support the view that interaction occurring on the cytoplasmic side influences the events occurring in the outer membrane. We discuss a model in which OutE uses ATP to control the assembly of the type II secretion machinery.
Py
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B
Laboratoire de Chimie Bactérienne, IBSM, CNRS, 31 chemin Joseph Aiguier, Marseille Cedex 20, 13402, France. py@ibsm.cnrs-mrs.fr
Loiseau
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L
Barras
F
F
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D016974:Dickeya chrysanthemi / Q000378:metabolism*; D006822:Hybrid Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D028044:Phosphate-Binding Proteins; D015003:Yeasts / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; C120189:OutE protein, Erwinia chrysanthemi; D028044:Phosphate-Binding Proteins