Demonstration of ferric L-parabactin-binding activity in the outer membrane of Paracoccus denitrificans.
Under low-iron conditions, Paracoccus denitrificans excretes a catecholamine siderophore, L-parabactin, to sequester and utilize iron. In this report, we demonstrate the presence of stereospecific high-affinity ferric L-parabactin-binding activity associated with P. denitrificans membranes grown in low-iron medium. Isolated outer membrane components were shown to be three to four times higher in specific activity for ferric L-parabactin. The same amount of binding activity existed whether or not the radiolabel was present in the metal (55Fe) or the ligand (3H) portion of ferric parabactin chelate, suggesting that binding was to the intact complex. Ion-exchange chromatography of a Triton X-100-solubilized outer membrane mixture on DEAE-cellulose resulted in a 10-fold increase in binding activity relative to that present in whole membranes. Polypeptide profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of each stage of the purification showed that binding activity copurified with one or more of the low-iron-induced outer membrane proteins in the 80-kilodalton (kDa) region. Membrane proteins and [55Fe]ferric L-parabactin electrophoresed in nondenaturing gels demonstrated the presence of membrane component(s) which stereo-specifically bound ferric L-parabactin, thus providing independent confirmation of the binding assay results. Moreover, when the band labeled by [55Fe]ferric L-parabactin was excised and profiled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 80-kDa polypeptides were the major components present. These results demonstrate the presence of a high-affinity ferric L-parabactin receptor in P. denitrificans membranes and suggest that one or more of the 80-kDa low-iron-induced polypeptides are components of the ferric L-parabactin receptor.
Bergeron
R J
RJ
Department of Medicinal Chemistry, J. Hillis Miller Health Center, University of Florida, Gainesville 32610-0485.
Weimar
W R
WR
Dionis
J B
JB
D001345:Autoradiography; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007502:Iron Chelating Agents / Q000302:isolation & purification* / Q000378:metabolism*; D010080:Oxazoles / Q000302:isolation & purification / Q000378:metabolism*; D010231:Paracoccus denitrificans / Q000378:metabolism* / Q000648:ultrastructure
D001425:Bacterial Outer Membrane Proteins; D007502:Iron Chelating Agents; D010080:Oxazoles; C042991:parabactin