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Journal of Bacteriology, September 2004, p. 6220-6229, Vol. 186, No. 18
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.18.6220-6229.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The hFbpABC Transporter from Haemophilus influenzae Functions as a Binding-Protein-Dependent ABC Transporter with High Specificity and Affinity for Ferric Iron

Damon S. Anderson,¹ Pratima Adhikari,¹ Andrew J. Nowalk,² Cheng Y. Chen,³ and Timothy A. Mietzner^1*

Department of Molecular Genetics and Biochemistry,¹ Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania,² Sexually Transmitted Diseases and Tuberculosis Laboratory Research, Division of AIDS, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia³

Received 4 June 2004/ Accepted 9 June 2004

Pathogenic Haemophilus influenzae, Neisseria spp. (Neisseria gonorrhoeae and N. meningitidis), Serratia marcescens, and other gram-negative bacteria utilize a periplasm-to-cytosol FbpABC iron transporter. In this study, we investigated the H. influenzae FbpABC transporter in a siderophore-deficient Escherichia coli background to assess biochemical aspects of FbpABC transporter function. Using a radiolabeled Fe³⁺ transport assay, we established an apparent K_m = 0.9 µM and V_max = 1.8 pmol/10⁷cells/min for FbpABC-mediated transport. Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide m-chlorophenyl hydrazone had no effect. Metal competition experiments demonstrated that the transporter has high specificity for Fe³⁺ and selectivity for trivalent metals, including Ga³⁺ and Al³⁺, over divalent metals. Metal sensitivity experiments showed that several divalent metals, including copper, nickel, and zinc, exhibited general toxicity towards E. coli. Significantly, gallium-induced toxicity was specific only to E. coli expressing FbpABC. A single-amino-acid mutation in the gene encoding the periplasmic binding protein, FbpA(Y196I), resulted in a greatly diminished iron binding affinity K_d = 5.2 x 10^–4 M^–1, 14 orders of magnitude weaker than that of the wild-type protein. Surprisingly, the mutant transporter [FbpA(Y196I)BC] exhibited substantial transport activity, 35% of wild-type transport, with K_m = 1.2 µM and V_max = 0.5 pmol/10⁷cells/min. We conclude that the FbpABC complexes possess basic characteristics representative of the family of bacterial binding protein-dependent ABC transporters. However, the specificity and high-affinity binding characteristics suggest that the FbpABC transporters function as specialized transporters satisfying the strict chemical requirements of ferric iron (Fe³⁺) binding and membrane transport.

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* Corresponding author. Mailing address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Room E1240 Biomedical Science Tower, Lothrop St., Pittsburgh, PA 15261. Phone: (412) 648-9244. Fax: (412) 624-1401. E-mail: mietzner{at}pitt.edu.

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Journal of Bacteriology, September 2004, p. 6220-6229, Vol. 186, No. 18
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.18.6220-6229.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Anderson, D. S., Adhikari, P., Weaver, K. D., Crumbliss, A. L., Mietzner, T. A. (2007). The Haemophilus influenzae hFbpABC Fe3+ Transporter: Analysis of the Membrane Permease and Development of a Gallium-Based Screen for Mutants. J. Bacteriol. 189: 5130-5141 [Abstract] [Full Text]  
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