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Journal of Bacteriology, July 2007, p. 4662-4670, Vol. 189, No. 13
0021-9193/07/$08.00+0     doi:10.1128/JB.00180-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Deletion and Substitution Analysis of the Escherichia coli TonB Q160 Region

Hema Vakharia-Rao,^1, Kyle A. Kastead,² Marina I. Savenkova,¹ Charles M. Bulathsinghala,² and Kathleen Postle^1,2*

School of Molecular Biosciences, Washington State University, Pullman, Washington 99164,¹ Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802²

Received 3 February 2007/ Accepted 23 April 2007

The active transport of iron siderophores and vitamin B₁₂ across the outer membrane (OM) of Escherichia coli requires OM transporters and the potential energy of the cytoplasmic membrane (CM) proton gradient and CM proteins TonB, ExbB, and ExbD. A region at the amino terminus of the transporter, called the TonB box, directly interacts with TonB Q160 region residues. R158 and R166 in the TonB Q160 region were proposed to play important roles in cocrystal structures of the TonB carboxy terminus with OM transporters BtuB and FhuA. In contrast to predictions based on the crystal structures, none of the single, double, or triple alanyl substitutions at arginyl residues significantly decreased TonB activity. Even the quadruple R154A R158A R166A R171A mutant TonB still retained 30% of wild-type activity. Up to five residues centered on TonB Q160 could be deleted without inactivating TonB or preventing its association with the OM. TonB mutant proteins with nested deletions of 7, 9, or 11 residues centered on TonB Q160 were inactive and appeared never to have associated with the OM. Because the 7-residue-deletion mutant protein (TonB7, lacking residues S157 to Y163) could still form disulfide-linked dimers when combined with W213C or F202C in the TonB carboxy terminus, the TonB7 deletion did not prevent necessary energy-dependent conformational changes that occur in the CM. Thus, it appeared that initial contact with the OM is made through TonB residues S157 to Y163. It is hypothesized that the TonB Q160 region may be part of a large disordered region required to span the periplasm and contact an OM transporter.

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* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-7568. Fax: (814) 863-7024. E-mail: postle{at}psu.edu

Published ahead of print on 4 May 2007.

Present address: Mandel & Adriano Intellectual Property, 55 S. Lake Ave., Suite 710, Pasadena, CA 91101.

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Journal of Bacteriology, July 2007, p. 4662-4670, Vol. 189, No. 13
0021-9193/07/$08.00+0     doi:10.1128/JB.00180-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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