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, 68 (2), 470-7

Expression of the Helicobacter Pylori ureI Gene Is Required for Acidic pH Activation of Cytoplasmic Urease

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Expression of the Helicobacter Pylori ureI Gene Is Required for Acidic pH Activation of Cytoplasmic Urease

D R Scott et al. Infect Immun.

Abstract

ureI encodes an integral cytoplasmic membrane protein. It is present in the urease gene cluster of Helicobacter pylori and is essential for infection and acid survival, but its role is unknown. To determine the function of UreI protein, we produced H. pylori ureI deletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential. We also determined ureI expression, urease activity, and the effect of urea on membrane potential of several gastric and nongastric Helicobacter species. ureI was found to be present in the genome of the gastric Helicobacter species and absent in the nongastric Helicobacter species studied, as determined by PCR. Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric Helicobacter species. When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH. The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited. No acidic pH activation or urea induced membrane potential changes were found in the nongastric Helicobacter species. The ureI gene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.

Figures

FIG. 1
FIG. 1
PCR analysis confirming the replacement of the ureI gene with the kanamycin cassette. Genomic DNA was isolated from the H. pylori wild type and H. pylori ureI mutant strains and used as a template for PCR. PCR primers corresponding to regions of the ureB and ureE gene were used as described in Materials and Methods. A PCR product of 1,281 bp was detected for the H. pylori wild type strains (ATCC 43504, lane 1; ATCC 49503, lane 3). A PCR product of 1,325 bp was detected for the H. pylori ureI mutant strains (ATCC 43504-I, lane 2; ATCC 49503-I, lane 4).
FIG. 2
FIG. 2
pH dependence of urease activity of H. pylori and H. pylori ureI mutant. The urease activity of the intact H. pylori wild type (■) increased rapidly as the medium pH decreased. The urease activity remained high down to a medium pH of 2.5. In contrast, there was no increase of urease activity in the H. pylori ureI mutant (●). The urease activity of the H. pylori ureI mutant lysates displayed a single pH optimum (○). Urease activity was measured radiometrically under strong buffering conditions in the presence of 5 mM urea as described in Materials and Methods (micromoles of CO2 liberated per minute per milligram of protein; n = 3, ± the standard error of the mean [SEM]).
FIG. 3
FIG. 3
The pH dependence of urease activity in intact Helicobacter species. Urease activity was measured radiometrically under strong buffering conditions in the presence of 5 mM urea as described in Materials and Methods. Urease activity of intact H. mustelae (■), H. nemestrinae (●), and H. muridarum (○) (micromoles of CO2 liberated per minute per milligram of protein; n = 3, ±SEM).
FIG. 4
FIG. 4
Effect of detergent permeabilization on the urease activity of intact H. pylori and H. pylori ureI mutant. Addition of 0.01% C12E8 to intact H. pylori and H. pylori ureI mutant increases urease activity at neutral pH. Urease activity was measured radiometrically under strong buffering conditions in the presence of 5 mM urea as described in Materials and Methods (micromoles of CO2 liberated per minute per milligram of protein; n = 3, ±SEM).
FIG. 5
FIG. 5
Effect of urea on the membrane potential of H. pylori wild type and ureI mutants. Membrane potential was measured by using DiSC3(5) at pH 4.5 under pH clamp conditions as described in Materials and Methods. Urea was added as indicated by the arrow and resulted in an increase of membrane potential in H. pylori (A) but not in the H. pylori ureI mutant (B). The horizontal arrow at the point of equilibration of the dye indicates bacterial membrane potential prior to urea addition.
FIG. 6
FIG. 6
Effect of urea on membrane potential of other Helicobacter spp. Membrane potential was measured by using DiSC3(5) at pH 4.5 under pH clamp conditions as described in Materials and Methods. Urea addition resulted in an increase in membrane potential in the gastric Helicobacter species H. pylori, H. mustelae, and H. nemestrinae. Urea addition had no effect on the membrane potential of H. bilis, H. troguntum, H. hepaticus, or H. muridarum (n = 3, ±SEM).
FIG. 7
FIG. 7
Western blot analysis of membrane protein extracts from gastric and nongastric Helicobacter species by using UreI antibodies. Membrane protein extracts were size fractionated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with anti-UreI (UP2/3 and UP4/5 mixture) as described in Materials and Methods. A single band corresponding to a molecular size of about 21 kDa was detected in the lanes containing membrane proteins from H. pylori ATCC 43504 and ATCC 49503 and from H. nemestrinae. No immunoreactivity was detected in the lanes containing membrane proteins from the ureI deletion mutants or the nongastric Helicobacter species.
FIG. 8
FIG. 8
Western blot analysis of membrane protein extracts from H. mustelae with anti-UreI UP2/3 or UP4/5. Membrane protein extracts of H. mustelae were size fractionated on an SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with anti-UreI UP2/3 and UP4/5 as described in Materials and Methods. A band corresponding to a molecular size of about 21 kDa was detected in the lane probed with anti-UreI UP4/5 (lane 2). It was not possible to detect UreI with anti-UreI UP2/3 alone (lane 1).
FIG. 9
FIG. 9
PCR analysis of ureI from genomic DNA of gastric and nongastric Helicobacter species. Genomic DNA was isolated from the different bacterial species and used as template for PCR of ureI as described in Materials and Methods. A single PCR product of about 587 bp was detected in the lanes containing genomic DNA from H. pylori ATCC 43504 and ATCC 49503 and from H. nemestrinae. No PCR product was detected in the lanes containing genomic DNA from the ureI deletion mutants, H. mustelae or the nongastric Helicobacter species.
FIG. 10
FIG. 10
Model of the activation of cytoplasmic urease under acidic conditions. Urea and H+ diffuse into the periplasmic space probably through porins in the outer membrane. Periplasmic acidification results in a conformational change in UreI, permitting the entry of urea into the cytoplasm, where it is rapidly hydrolyzed, producing CO2 and NH3. The ammonia diffuses into the periplasmic space, where it becomes protonated, raising the pH to about 6.5, a level consistent with survival and growth.

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