Summary: Most high-affinity systems for iron uptake in Gram-negative bacteria are thought to employ periplasmic-binding-protein-dependent transport. In , FepB is a periplasmic protein required for uptake of iron complexed to its endogenously-synthesized siderophore enterobactin (Ent). Direct evidence that ferrienterobactin (FeEnt) binds to FepB is lacking because high background binding by FeEnt prevents use of the usual binding protein assays. Here the membrane localization vehicle LppOmpA [Francisco, J. A., Earhart, C. F. Georgiou, G. (1992). 89, 2713-2717] was employed to place FepB in the outer membrane. Plasmid pTX700 was constructed and shown to encode, under operator control, the ‘tribrid‘ protein LppOmpAFepB; the carboxy-terminal FepB portion lacks at most two amino acids of mature FepB. After short induction periods, most of the tribrid was in the outer membrane. A number of LppOmpAFepB species could be detected; some were degradation products and some may be related to the multiplicity of FepB forms previously observed in minicells and maxicells. Outer membrane harbouring the tribrid and lacking FepA, the normal outer membrane receptor for FeEnt, bound approximately four times more FeEnt than outer membrane from uninduced cells, from cells lacking pTX700 and from cells expressing only an LppOmpA ‘dibrid’. Similarly, whole UT5600()/pTX700 cells induced for tribrid synthesis bound FeEnt and this binding was not affected by energy poisons. The results demonstrated that FepB can bind FeEnt, thereby definitively placing FeEnt transport in the periplasmic permease category of transport systems, and that the LppOmpA localization vehicle can be used with periplasmic binding proteins.


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