The proton motive force drives the outer membrane transport of cobalamin in Escherichia coli.
Cells of Escherichia coli pump cobalamin (vitamin B12) across their outer membranes into the periplasmic space, and it was concluded previously that this process is potentiated by the proton motive force of the inner membrane. The novelty of such an energy coupling mechanism and its relevance to other outer membrane transport processes have required confirmation of this conclusion by studies with cells in which cobalamin transport is limited to the outer membrane. Accordingly, I have examined the effects of cyanide and of 2,4-dinitrophenol on cobalamin uptake in btuC and atp mutants, which lack inner membrane cobalamin transport and the membrane-bound ATP synthase, respectively. Dinitrophenol eliminated cobalamin transport in all strains, but cyanide inhibited this process only in atp and btuC atp mutant cells, providing conclusive evidence that cobalamin transport across the outer membrane requires specifically the proton motive force of the inner membrane. The coupling of metabolic energy to outer membrane cobalamin transport requires the TonB protein and is stimulated by the ExbB protein. I show here that the tolQ gene product can partly replace the function of the ExbB protein. Cells with mutations in both exbB and tolQ had no measurable cobalamin transport and thus had a phenotype that was essentially the same as TonB-. I conclude that the ExbB protein is a normal component of the energy coupling system for the transport of cobalamin across the outer membrane.
Department of Biochemistry, School of Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.
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Bradbeer
D019297:2,4-Dinitrophenol; D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins / Q000235:genetics; D001693:Biological Transport, Active / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism*; D003486:Cyanides / Q000494:pharmacology; D004140:Dinitrophenols / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000235:genetics; D016296:Mutagenesis; D006180:Proton-Translocating ATPases / Q000235:genetics; D011522:Protons; D014161:Transduction, Genetic; D014805:Vitamin B 12 / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C050021:BtuC peptide, E coli; D003486:Cyanides; D004140:Dinitrophenols; D029968:Escherichia coli Proteins; C052874:ExbB protein, E coli; D008565:Membrane Proteins; D011522:Protons; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; C060792:tolQ protein, E coli; D006180:Proton-Translocating ATPases; D014805:Vitamin B 12; D019297:2,4-Dinitrophenol