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Journal of Bacteriology, February 2003, p. 1195-1207, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1195-1207.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Analysis of Residues Determining Specificity of Vibrio cholerae TonB1 for Its Receptors

Alexandra R. Mey¹ and Shelley M. Payne^1,2*

Institute for Cellular and Molecular Biology,¹ Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas 78712-1095²

Received 26 July 2002/ Accepted 22 November 2002

In gram-negative organisms, high-affinity transport of iron substrates requires energy transduction to specific outer membrane receptors by the TonB-ExbB-ExbD complex. Vibrio cholerae encodes two TonB proteins, one of which, TonB1, recognizes only a subset of V. cholerae TonB-dependent receptors and does not facilitate transport through Escherichia coli receptors. To investigate the receptor specificity exhibited by V. cholerae TonB1, chimeras were created between V. cholerae TonB1 and E. coli TonB. The activities of the chimeric TonB proteins in iron utilization assays demonstrated that the C-terminal one-third of either TonB confers the receptor specificities associated with the full-length TonB. Single-amino-acid substitutions near the C terminus of V. cholerae TonB1 were identified that allowed TonB1 to recognize E. coli receptors and at least one V. cholerae TonB2-dependent receptor. This indicates that the very C-terminal end of V. cholerae TonB1 determines receptor specificity. The regions of the TonB-dependent receptors involved in specificity for a particular TonB protein were investigated in experiments involving domain switching between V. cholerae and E. coli receptors exhibiting different TonB specificities. Switching the conserved TonB box heptapeptides at the N termini of these receptors did not alter their TonB specificities. However, replacing the amino acid immediately preceding the TonB box in E. coli receptors with an aromatic residue allowed these receptors to use V. cholerae TonB1. Further, site-directed mutagenesis of the TonB box -1 residue in a V. cholerae TonB2-dependent receptor demonstrated that a large hydrophobic amino acid in this position promotes recognition of V. cholerae TonB1. These data suggest that the TonB box -1 position controls productive interactions with V. cholerae TonB1.

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* Corresponding author. Mailing address: The University of Texas, Section of Molecular Genetics and Microbiology, Austin, TX 78712-1095. Phone: (512) 471-9258. Fax: (512) 471-7088. E-mail: payne{at}mail.utexas.edu.

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Journal of Bacteriology, February 2003, p. 1195-1207, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1195-1207.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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