FepA with globular domain deletions lacks activity.
TonB-gated transporters have beta-barrels containing an amino-terminal globular domain that occludes the interior of the barrel. Mutations in the globular domain prevent transport of ligands across the outer membrane. Surprisingly, FepA with deletions of the globular domain (amino acids 3 to 150 and 17 to 150) was previously reported to retain significant sensitivity to colicins B and D and to use ferric enterochelin, all in a TonB-dependent fashion. To further understand TonB interaction with the beta-barrel, in the present study, proteins with deletions of amino acids 1 to 152, 7 to 152, 20 to 152, and 17 to 150 in fepA were constructed and expressed in a deltafepA strain. In contrast to previous studies of fepA globular domain deletions, constructs in this study did not retain sensitivity to colicin B and conferred only marginal sensitivity to colicin D. Consistent with these observations, they failed to bind colicin B and detectably cross-link to TonB in vivo. To address this discrepancy, constructs were tested in other strains, one of which (RWB18-60) did support activity of the FepA globular domain deletion proteins constructed in this study. The characteristics of that strain, as well as the strain in which the deltaFhuA globular domain mutants were seen to be active, suggests the hypothesis that interprotein complementation by two individually nonfunctional proteins restores TonB-dependent activity.
Vakharia
Hema L
HL
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234, USA.
Postle
Kathleen
K
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003087:Colicins / Q000378:metabolism / Q000494:pharmacology; D004758:Enterobactin / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D008565:Membrane Proteins / Q000378:metabolism; D011956:Receptors, Cell Surface
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C029489:enterobactin receptor; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D004758:Enterobactin; C014554:ferrienterochelin