Dependence of Helicobacter pylori urease activity on the nickel-sequestering ability of the UreE accessory protein

Abstract

The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in E. coli and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type H. pylori and in hypA and hypB mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between H. pylori urease activity and the nickel-sequestering ability of the UreE accessory protein.

FIG. 1.

Protein sequences of H. pylori UreE, UreE variants constructed in this study, and other UreE proteins. The carboxyl-terminal region (residues 80 through 170) of H. pylori 43504 (Hp 43504), as deduced from the DNA sequence ([this study], and shown to be identical to H. pylori 85P [7]) is compared to the homologous UreE regions from H. pylori 26695 (Hp 26695 [32]), Bacillus pasteurii (Bp [5]), Proteus mirabilis (Pm [14]), and Klebsiella aerogenes (Ka [24]). Also shown are the sequences of the UreE variants engineered in this study. Histidine residues shown to be involved in nickel binding in K. aerogenes are indicated in boldface type (30). One of these residues also shown to be involved in nickel binding in B. pasteurii (29) and conserved in H. pylori is shaded. The histidine-rich carboxyl-terminal tail of K. aerogenes (used in this study) is underlined. The hexahistidine tag and additional amino acids added by the construction are shown in italics.

FIG. 2.

Western blot analysis of UreE expression in H. pylori. Whole-cell lysate (25 μg) of H. pylori and purified UreE (500 ng) were subjected to SDS-PAGE, transferred to nitrocellulose, and blotted with antibodies raised against purified UreE. Lane 1, hypB[Cm]; lane 2, hypB[HPE]; lane 3, ureE mutant; lane 4, purified UreE; lane 5, hypB[HPE6]; lane 6, hypB[HPE10]; lane 7, hypB[HPE10SF]. The position of a molecular mass standard is indicated on the left.

FIG. 3.

Urease activities of H. pylori 43504 and hypA and hypB mutants harboring an additional copy of ureE or one of its variants in the chromosome. Constructs introduced by homologous recombination within the hp0405 gene region of the chromosome of either strain are indicated. Results shown are the averages of three to four independent growth experiments; error bars denote standard deviations. TPI, triose phosphate isomerase.

FIG. 4.

Western blot analysis of UreB expression in H. pylori. Whole-cell lysate (6 μg) of H. pylori was subjected to SDS-PAGE, transferred to nitrocellulose, and blotted with antibodies directed to the urease large subunit UreB. Lane 1, 43504[Cm]; lane 2, hypA[HPE10SF]; lane 3, hypA[HPE10]; lane 4, hypB[HPE6]; lane 5, hypB[HPE]; lane 6, hypB[Cm]; lane 7, hypB. The positions of molecular mass standards are indicated on the left.

FIG. 5.

SDS-PAGE of purified UreE variants. Lanes 1, 4, 7, and 10 contain noninduced cell extracts of strain Rosetta harboring pET-HP (for expression of UreE), pET-HP6 (UreE-His₆), pET-HP10 (UreE-His₁₀), and pET-HP10SF (UreE-His₁₀SF), respectively. Lanes 2, 5, 8, and 11 contain cell extracts after 3 h of IPTG induction of UreE, UreE-His₆, UreE-His₁₀, and UreE-His₁₀SF, respectively. UreE was purified by ammonium sulfate precipitation and cation exchange chromatography (lane 3). UreE-His₆, UreE-His₁₀, and UreE-His₁₀SF were purified through an Ni-NTA column (lanes 6, 9, and 12, respectively). The positions of molecular mass standards are indicated on the left.

FIG. 6.

Nickel ion binding by UreE, UreE-His₆, UreE-His₁₀, and UreE-His₁₀SF proteins based on equilibrium dialysis. Each purified protein (at 6 to 7 μM monomer concentration) in 50 mM NaCl (pH 8.25) was equilibrated with the indicated concentrations of NiCl₂, and the number of nickel ions bound per monomer was determined by graphite furnace atomic absorption spectrophotometry. Standard deviations were ≤10% for each set of measurements.