Analysis of Residues Determining Specificity ofVibrio choleraeTonB1 for Its Receptors

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Abstract
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 choleraeencodes two TonB proteins, one of which, TonB1, recognizes only a subset ofV.choleraeTonB-dependent receptors and does not facilitate transport throughEscherichia colireceptors. To investigate the receptor specificity exhibited byV.choleraeTonB1, chimeras were created betweenV.choleraeTonB1 andE.coliTonB. 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 ofV.choleraeTonB1 were identified that allowed TonB1 to recognizeE.colireceptors and at least oneV.choleraeTonB2-dependent receptor. This indicates that the very C-terminal end ofV.choleraeTonB1 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 betweenV.choleraeandE.colireceptors 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 inE.colireceptors with an aromatic residue allowed these receptors to useV.choleraeTonB1. Further, site-directed mutagenesis of the TonB box −1 residue in aV.choleraeTonB2-dependent receptor demonstrated that a large hydrophobic amino acid in this position promotes recognition ofV.choleraeTonB1. These data suggest that the TonB box −1 position controls productive interactions withV.choleraeTonB1.