Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier

ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The i rreversible inhibition of urease when the organism was exposed to a protono phore (3,3',4',5-tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants o f several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gen e (kan(R)). Mutants carrying the modified ureAB-kan(R)-EFGH operon all show ed wild-type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild-type bacteria showed high levels of urease activity below pH 4.0, this ability was not re tained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid-derived H. pylori ureI restored wild-type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy-ethylene-8-laurylether; C12E8), suggested a membrane-limited acce ss of urea to internal urease at neutral pH. Measurement of C-14-urea uptak e into Xenopus oocytes injected with ureI cRNA showed acid activation of up take only in injected oocytes. Acceleration of urea uptake by UreI therefor e mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori s urvival in environments below pH 4.0. ureI-independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.