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Journal of Bacteriology, October 2003, p. 5747-5754, Vol. 185, No. 19
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.19.5747-5754.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

In Vivo Evidence for TonB Dimerization

Annette Sauter,1 S. Peter Howard,1,2 and Volkmar Braun1*

Mikrobiologie/Membranphysiologie, Universität Tübingen, D-72076 Tübingen, Germany,1 Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E52

Received 21 April 2003/ Accepted 30 June 2003

TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and {phi}80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (Pctx::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated Pctx::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

* Corresponding author. Mailing address: Mikrobiologie/Membranphysiologie, Universität Tübingen, Auf der Morgenstelle, 28 D-72076 Tübingen, Tübingen, Germany. Phone: (49) 7071-2972096. Fax: (49) 7071-295843. E-mail: volkmar.braun{at}mikrobio.uni-tuebingen.de.

Journal of Bacteriology, October 2003, p. 5747-5754, Vol. 185, No. 19
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.19.5747-5754.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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