title: Initial steps of colicin E1 import across the outer membrane of Escherichia coli.
abstract: Data suggest a two-receptor model for colicin E1 (ColE1) translocation across the outer membrane of Escherichia coli. ColE1 initially binds to the vitamin B(12) receptor BtuB and then translocates through the TolC channel-tunnel, presumably in a mostly unfolded state. Here, we studied the early events in the import of ColE1. Using in vivo approaches, we show that ColE1 is cleaved when added to whole cells. This cleavage requires the presence of the receptor BtuB and the protease OmpT, but not that of TolC. Strains expressing OmpT cleaved ColE1 at K84 and K95 in the N-terminal translocation domain, leading to the removal of the TolQA box, which is essential for ColE1's cytotoxicity. Supported by additional in vivo data, this suggests that a function of OmpT is to degrade colicin at the cell surface and thus protect sensitive E. coli cells from infection by E colicins. A genetic strategy for isolating tolC mutations that confer resistance to ColE1, without affecting other TolC functions, is also described. We provide further in vivo evidence of the multistep interaction between TolC and ColE1 by using cross-linking followed by copurification via histidine-tagged TolC. First, secondary binding of ColE1 to TolC is dependent on primary binding to BtuB. Second, alterations to a residue in the TolC channel interfere with the translocation of ColE1 across the TolC pore rather than with the binding of ColE1 to TolC. In contrast, a substitution at a residue exposed on the cell surface abolishes both binding and translocation of ColE1.
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA.
authors.forename: Muriel
authors.initials: M
authors.lastname: Masi
authors.forename: Phu
authors.initials: P
authors.lastname: Vuong
authors.forename: Matthew
authors.initials: M
authors.lastname: Humbard
authors.forename: Karen
authors.initials: K
authors.lastname: Malone
authors.forename: Rajeev
authors.initials: R
authors.lastname: Misra
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism*; D003087:Colicins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D006460:Hemolysin Proteins / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D018272:Porins / Q000378:metabolism; D011487:Protein Conformation; D021381:Protein Transport
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C044592:BtuB protein, E coli; D003087:Colicins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D018272:Porins; C405564:ompT protein, bacteria