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PMID: 15342592 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

The hFbpABC transporter from Haemophilus influenzae functions as a binding-protein-dependent ABC transporter with high specificity and affinity for ferric iron.

Journal of bacteriology ·Vol. 186 ·No. 18 ·2004-09-00 ·Pages 6220-9

Anderson DS, Adhikari P, Nowalk AJ, Chen CY, Mietzner TA

Abstract

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 Km=0.9 microM and Vmax=1.8 pmol/10(7)cells/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 Kd=5.2 x 10(-4) M(-1), approximately 14 orders of magnitude weaker than that of the wild-type protein. Surprisingly, the mutant transporter [FbpA(Y196I)BC] exhibited substantial transport activity, approximately 35% of wild-type transport, with Km=1.2 microM and Vmax=0.5 pmol/10(7)cells/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.

MeSH Terms
ATP-Binding Cassette Transporters/genetics,physiology Amino Acid Substitution Bacterial Proteins/genetics,metabolism Biological Transport, Active Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology Cations/metabolism Copper/toxicity Enzyme Inhibitors/pharmacology Escherichia coli/genetics,metabolism Ferric Compounds/metabolism Gallium/toxicity Genetic Complementation Test Haemophilus influenzae/drug effects,genetics,metabolism Iron/analysis,metabolism Mutation, Missense Nickel/toxicity Periplasmic Binding Proteins/genetics,metabolism Substrate Specificity Uncoupling Agents/pharmacology Vanadates/pharmacology Zinc/toxicity
Chemicals
ATP-Binding Cassette Transporters Bacterial Proteins Cations Enzyme Inhibitors Ferric Compounds Periplasmic Binding Proteins Uncoupling Agents Vanadates Carbonyl Cyanide m-Chlorophenyl Hydrazone Copper Nickel Gallium Iron Zinc
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Anderson Damon S
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Room E1240 Biomedical Science Tower, Lothrop St., Pittsburgh, PA 15261, USA.
Adhikari Pratima
Nowalk Andrew J
Chen Cheng Y
Mietzner Timothy A
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/JB.186.18.6220-6229.2004
Published
2004-09-00
Pages
6220-9
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC515168
Subset
IM
Grants
NIAID NIH HHS · R29 AI3226 · United States
External Links
PubMed source DOI: 10.1128/JB.186.18.6220-6229.2004 PMC Full Text View journal details
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