Helicobacter pylori is the only neutralophile that has been able to colonize the human stomach by using a variety of acid-adaptive mechanisms. One of the adaptive mechanisms is increased buffering due to expression of an acid-activated inner membrane urea channel, UreI, and a neutral pH-optimum intrabacterial urease. To delineate other possible adaptive mechanisms, changes in gene expression in response to acid exposure were examined using genomic microarrays of H. pylori exposed to different levels of external pH (7.4, 6.2, 5.5, and 4.5) for 30 min in the absence and presence of 5 mM urea. Gene expression was correlated with intrabacterial pH measured using 2,7-bis-(2-carboxyethyl)-5-carboxyfluorescein and compared to that observed with exposure to 42°C for 30 min. Microarrays containing the 1,534 open reading frames of H. pylori strain 26695 were hybridized with cDNAs from control (pH 7.4; labeled with Cy3) and acidic (labeled with Cy5) conditions. The intrabacterial pH was 8.1 at pH 7.4, fell to 5.3 at pH 4.5, and rose to 6.2 with urea. About 200 genes were up-regulated and ϳ100 genes were down-regulated at pH 4.5 in the absence of urea, and about half that number changed in the presence of urea. These genes included pH-homeostatic, transcriptional regulatory, motility, cell envelope, and pathogenicity genes. The up-regulation of some pH-homeostatic genes was confirmed by real-time PCR. There was little overlap with the genes induced by temperature stress. These results suggest that H. pylori has evolved multifaceted acid-adaptive mechanisms enabling it to colonize the stomach that may be novel targets for eliminating infection.

The role of the periplasmic ␣-carbonic anhydrase (␣-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid survival in vitro. Intrabacterial urease generation of NH 3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate bioenergetics for survival and growth. Since ␣-CA catalyzes the conversion of CO 2 to HCO 3 ؊ , the role of CO 2 in periplasmic buffering was studied using an ␣-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed that ␣-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the ␣-CA knockout. In the mutant or in the presence of acetazolamide, there was an ϳ3 log 10 decrease in acid survival. In acid, absence of ␣-CA activity decreased membrane integrity, as observed using membrane-permeant and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition to wild-type organisms in acid was absent in the ␣-CA knockout mutant and in the presence of acetazolamide, although UreI and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in the absence of ␣-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH 3 efflux from the cytoplasm but also on the conversion of CO 2 , produced by urease, to HCO 3 ؊ by the periplasmic ␣-CA.

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.

Helicobacter pylori is a gastric-dwelling pathogen responsible, with acid secretion, for peptic ulcer and a 20-fold increase in the risk of gastric cancer. Several transcriptomes have been described after short-term exposure to acidity in vitro, but there are no data identifying the effects of chronic gastric exposure on bacterial gene expression. Comparison of the in vivo to the in vitro transcriptome at pH 7.4 identified several groups of genes of known function that increased expression >2-fold, and three of these respond both to acidity in vitro and to gastric infection. Almost all known acid acclimation genes are highly up-regulated. These include ureA, ureB, and rocF and the pH-gated urea channel, ureI. There is also up-regulation of two groups of motility and chemotaxis genes and for pathogenicity island genes, especially cagA, a predictor for pathogenicity. Most of these genes interact with HP0166, the response element of the pH-sensing two-component histidine kinase, HP0165/HP0166, ArsRS. Based on the pH profile of survival of ureI deletion mutants in vitro and their inability to survive in gastric acidity, the habitat of the organism at the gastric surface is acidic with a pH < 4.0. Hence, the pH of the habitat of H. pylori on the surface of the stomach largely determines the regulation of these specific groups of genes.acid acclimation ͉ gastric pH ͉ global transcriptome ͉ microarray