title: Release of immunity protein requires functional endonuclease colicin import machinery.
abstract: Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.
authors.affiliation: Laboratoire d'Ingéniérie des Systèmes Macromoléculaires, Institut de Biologie Structurale et Microbiologie, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. duche@ibsm.cnrs-mrs.fr
authors.forename: Denis
authors.initials: D
authors.lastname: Duché
authors.forename: Aurélie
authors.initials: A
authors.lastname: Frenkian
authors.forename: Valérie
authors.initials: V
authors.lastname: Prima
authors.forename: Roland
authors.initials: R
authors.lastname: Lloubès
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003087:Colicins / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D018272:Porins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C044592:BtuB protein, E coli; D003087:Colicins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C109237:OmpF protein; D018272:Porins; D011993:Recombinant Fusion Proteins; C007104:colicin immunity proteins; D049452:Green Fluorescent Proteins; D004720:Endonucleases