Mey Alexandra R, Payne Shelley M
Institute for Cellular and Molecular Biology. Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas 78712-1095, USA.
J Bacteriol. 2003 Feb;185(4):1195-207. doi: 10.1128/JB.185.4.1195-1207.2003.
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.
在革兰氏阴性菌中，铁底物的高亲和力转运需要通过TonB-ExbB-ExbD复合物将能量传递给特定的外膜受体。霍乱弧菌编码两种TonB蛋白，其中一种TonB1仅识别霍乱弧菌中一部分依赖TonB的受体，且不促进通过大肠杆菌受体的转运。为了研究霍乱弧菌TonB1表现出的受体特异性，构建了霍乱弧菌TonB1与大肠杆菌TonB之间的嵌合体。嵌合TonB蛋白在铁利用测定中的活性表明，两种TonB的C端三分之一赋予了与全长TonB相关的受体特异性。在霍乱弧菌TonB1的C端附近鉴定出单氨基酸取代，使得TonB1能够识别大肠杆菌受体以及至少一种霍乱弧菌TonB2依赖的受体。这表明霍乱弧菌TonB1的最C端决定了受体特异性。在涉及具有不同TonB特异性的霍乱弧菌和大肠杆菌受体之间进行结构域交换的实验中，研究了依赖TonB的受体中参与对特定TonB蛋白特异性识别的区域。在这些受体的N端交换保守的TonB框七肽并未改变它们的TonB特异性。然而，用芳香族残基取代大肠杆菌受体中TonB框之前紧邻的氨基酸，使得这些受体能够利用霍乱弧菌TonB1。此外，对霍乱弧菌TonB2依赖的受体中TonB框-1残基进行定点诱变表明，该位置的一个大的疏水氨基酸促进了对霍乱弧菌TonB1的识别。这些数据表明，TonB框-1位置控制与霍乱弧菌TonB1的有效相互作用。
