Characterization of the essential transport function of the AIDA-I autotransporter and evidence supporting structural predictions.
The current model for autodisplay suggests a mechanism that allows a passenger protein to be translocated across the outer membrane by coordinate action of a C-terminal beta-barrel and its preceding linking region. The passenger protein, linker, and beta-barrel are together termed the autotransporter, while the linker and beta-barrel are here referred to as the translocation unit (TU). We characterized the minimal TU necessary for autodisplay with the adhesin-involved-in-diffuse-adherence (AIDA-I) autotransporter. The assumed beta-barrel structure at the C terminus of the AIDA-I autotransporter was studied by constructing a set of seven AIDA-I-cholera toxin B subunit fusion proteins containing various portions of AIDA-I. Surface exposure of the cholera toxin B moiety was assessed by dot blot experiments and trypsin accessibility of the chimeric proteins expressed in Escherichia coli JK321 or UT5600. Export of cholera toxin B strictly depended on a complete predicted beta-barrel region. The absolute necessity for export of a linking region and its influence on expression as an integral part of the TU was also demonstrated. The different electrophoretic mobilities of native and denatured chimeras indicated that the proposed beta-barrel resides within the C-terminal 312 amino acids of AIDA-I. Together these data provide evidence for the predicted beta-barrel structure and support our formerly proposed model of membrane topology of the AIDA-I autotransporter.
Maurer
J
J
Abteilung Infektionsbiologie, Max-Planck-Institut für Biologie, D-72076 Tübingen, Germany.
Jose
J
J
Meyer
T F
TF
D018830:Adhesins, Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002772:Cholera Toxin / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
C075559:AIDA-I protein, E coli; D018830:Adhesins, Escherichia coli; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin