Abstract
NixA, the high affinity nickel transport protein of Helicobacter pylori, imports Ni2+ ions across the cytoplasmic membrane for insertion into the active site of the urease metalloenzyme, which is essential for colonization of the gastric mucosa. Twelve conserved aspartate (aspartates 47, 49, 55, 194, 231, and 234), glutamate (glutamates 106, 198, and 274), and histidine (histidines 44, 50, and 79) residues were identified by alignment of NixA with homologous transporters. Polymerase chain reaction-generated site-directed mutants of these residues were expressed in E. coli along with the H. pylori urease gene cluster. Mutations in residues within the predicted periplasmic domains of NixA maintained near wild type levels of Ni2+ uptake and urease activity, as did control mutations of conserved positively charged residues (lysines 140 and 268; arginines 162 and 167). Mutations in highly conserved motifs in predicted helices II and III of NixA abolished Ni2+ uptake and urease activity. Mutations in helices V and VI and the cytoplasmic domains decreased Ni2+ transport rates by >/=90%. Reduction in rates of Ni2+ transport correlated with reduction in urease activities (r = 0.77). Ni2+ transport was inhibited in the presence of Co2+, Cu2+, and Zn2+, indicating that these ions may also be bound or transported by NixA. We conclude that conserved Asp, Glu, and His residues in the transmembrane domains of NixA are critical for the transport of the divalent cations Ni2+, Co2+, Cu2+, and Zn2+ into the cytoplasm of H. pylori.
Highlights
Helicobacter pylori, a Gram-negative, spiral-shaped, microaerophilic bacterium is a well established etiologic agent of gastritis and peptic ulcer disease [1,2,3]
Western Blots Confirm the Presence of Full-length NixA in the Membrane—To show that changes in Ni2ϩ transport and urease activity of NixA containing site-directed mutations were not the result of synthesis of a truncated protein, failure of the protein to be inserted in the membrane, or improper insertion or misfolding of the protein that might render it more susceptible to proteolysis, membrane preparations of E. coli transformed with pBluescript, pUEF204, or each site-directed mutation were assayed by Western blot with affinity-purified polyclonal antiserum raised against an internal polypeptide of NixA (Fig. 3)
H. pylori is a serious human pathogen whose ecological niche is highly restricted to the gastric mucosa of humans, and production of an abundant urease is required for colonization
Summary
Bacterial Strains, Plasmids, and Culture Conditions—E. coli DH5␣ (supE44 ⌬lacU169 (80 lacZ delta M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1), E. coli M15pRep (Qiagen), and E. coli SE5000 (araD139⌬(argF-lac) U169 rpsL150 relA1 flb3501 deoC1 ptsF25 rbsR recA56) were used as recipients of recombinant plasmids [21]. Preparation of a Polyclonal Antiserum to NixA—A 149-base pair PCR-amplified fragment corresponding to gene sequences encoding NixA amino acids 133–180 was subcloned into pBluescript SKϩ (Stratagene), excised at PCR-generated NcoI and BglII sites, and ligated into the His tail vector pQE60 (Qiagen). Western Blotting—Membranes of E. coli SE5000 (pHP808) cotransformed with pUEF204, pBluescript, or site-directed mutants of nixA were isolated from 100-ml overnight cultures. Bacteria from Luria broth cultures (100 ml) of each strain were harvested by centrifugation (5000 ϫ g, 10 min, 4 °C) and resuspended in 25 ml of phosphatebuffered saline containing 1 mM phenylmethylsulfonyl fluoride. Urease Assays—E. coli SE5000 (pHp808) cotransformed individually with pUEF204, pBluescript, or each of the site-directed mutants was grown overnight in 100 ml of Luria broth supplemented with 1 M NiCl2. Protein concentrations were determined by the bicinchoninic acid method using the Pierce BCA assay kit according to the manufacturer’s instructions
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