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Journal of Bacteriology, June 2008, p. 4001-4016, Vol. 190, No. 11
0021-9193/08/$08.00+0     doi:10.1128/JB.00135-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane{triangledown}

Wallace A. Kaserer, Xiaoxu Jiang, Qiaobin Xiao, Daniel C. Scott, Matthew Bauler, Daniel Copeland, Salete M. C. Newton, and Phillip E. Klebba*

Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019

Received 25 January 2008/ Accepted 24 March 2008

We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB+ bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepA{Delta}3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, OK 73019. Phone: (405) 325-4969. Fax: (405) 325-6111. E-mail: peklebba{at}ou.edu

{triangledown} Published ahead of print on 4 April 2008.

Journal of Bacteriology, June 2008, p. 4001-4016, Vol. 190, No. 11
0021-9193/08/$08.00+0     doi:10.1128/JB.00135-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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