Interaction of TonB with the Outer Membrane Receptor FpvA ofPseudomonas aeruginosa

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Abstract
Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane ofPseudomonas aeruginosais dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonBpp) was constructed. This protein was, after expression inEscherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathioneS-transferase (GST) tag. Purified GST-TonBppwas able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm ofP. aeruginosainhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonBppinteractions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.