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Journal of Bacteriology, March 2000, p. 1722-1730, Vol. 182, No. 6
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Membrane Topology of the NixA Nickel Transporter of Helicobacter pylori: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III

John F. Fulkerson Jr. and Harry L. T. Mobley*

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received 5 August 1999/Accepted 21 December 1999

NixA, the high-affinity cytoplasmic membrane nickel transport protein of Helicobacter pylori, imports Ni2+ into the cell for insertion into the active site of the urease metalloenzyme, which is required for gastric colonization. NixA fractionates with the cytoplasmic membrane, and protein cross-linking studies suggest that NixA functions as a monomer. A preliminary topological model of NixA with seven transmembrane domains was previously proposed based on hydropathy, charge dispersion, and homology to other transporters. To test the proposed topology of NixA and relate critical residues to specific structural elements, a series of 21 NixA-LacZ and 21 NixA-PhoA fusions were created along the entire length of the protein. Expression of reporter fusions was confirmed by Western blotting with beta -galactosidase- and alkaline phosphatase-specific antisera. The activities of reporter fusions near to and upstream of the predicted translational initiation demonstrated the presence of an additional amino-terminal transmembrane domain including a membrane localization signal. Activities of fusions immediately adjacent to motifs which have been shown to be requisite for Ni2+ transport localized these motifs entirely within transmembrane domains II and III. Fusion activities localized six additional Asp and Glu residues which reduced Ni2+ transport by >90% when mutated within or immediately adjacent to transmembrane domains II, V, VI, and VII. All fusions strongly support a model of NixA in which the amino and carboxy termini are located in the cytoplasm and the protein possesses eight transmembrane domains.

* Corresponding author. Mailing address: Dept. of Microbiology and Immunology, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201. Phone: (410) 706-0466. Fax: (410) 706-6751. E-mail: hmobley{at}umaryland.edu.

Journal of Bacteriology, March 2000, p. 1722-1730, Vol. 182, No. 6
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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