A cis-Encoded Antisense Small RNA Regulated by the HP0165-HP0166 Two-Component System Controls Expression of ureB in Helicobacter pylori

Colonization of healthy human and animal stomachs is a property of gastric Helicobacter species, including the human pathogen, Helicobacter pylori. H. pylori maintains a relatively neutral periplasmic pH in the face of a highly intragastric acidic environment, preserving cytoplasmic pH homeostasis and its proton motive force. This acid acclimation is distinct from the acid tolerance/resistance of other neutralophiles that transit but do not colonize the stomach (22, 47, 48). The most important component of acid acclimation is the nickel metaloenzyme urease, which generates the buffers NH3 and HCO3− from the metabolism of ambient urea, maintaining both cytoplasmic and periplasmic pHs to enable the organism to survive and grow in the stomach. The organism expresses very high levels of the urease A and B subunits, more than any other known ureolytic microbe, accounting for as much as 8% of the total bacterial protein (34). The expression of active urease requires six of the seven genes of the urease gene cluster, ureA and ureB for the catalytic subunits and ureE, ureF, ureG, and ureH, necessary for nickel insertion into the apourease, UreA/UreB. The third gene of the urease cluster, ureI, encodes a pH-gated urea channel that increases urea access to intrabacterial urease in acid (51, 71). Because of the importance of this enzyme for survival in an acidic environment, most attention has been focused on its upregulation under acidic conditions. There is increased urease gene transcription by the activation of the two-component system (TCS) HP0165-HP0166 (ArsRS) (39, 41) and the cytoplasmic histidine kinase HP0244 (74). Although increased urease activity in the presence of acid is beneficial, this activity in the absence of acid and in the presence of urea is lethal to the organism in vitro (14, 33). Gastric pH can climb to as high as pH 6.0 after a meal due to the strong buffering effect of food, and urea is always present in gastric juice. Hence, urease may act as a double-edged sword, being essential for survival and colonization on the highly acidic gastric surface but lethal at relatively neutral intragastric pH. Therefore, H. pylori may downregulate urease activity at a relatively neutral pH to ensure its survival. The transcriptional induction of urease genes (ureA, ureB, and ureI) in response to low pH (pH 5.0) is mediated mainly by the HP0165-HP0166 TCS (39-41). The phosphorylated response regulator HP0166 binds to extended regions overlapping the PureA and PureI promoters (41). In addition to the TCS, transcription of the ureA and ureB genes is positively regulated by NikR in response to increasing concentrations of Ni2+ in the surrounding medium (63, 64). While transcriptional control of gene expression is clearly of primary importance in prokaryotes, these organisms also employ regulatory mechanisms to control translation, initiation, and mRNA stability. To achieve this, many bacteria rely on the expression of small, noncoding RNAs (70). In bacteria, noncoding regulatory RNAs are usually between 50 and 300 nucleotides (nt) in length and are thus known as small RNAs (sRNAs) (67). Most sRNAs that have been characterized act as posttranscriptional regulators by interacting with specific mRNA targets, modulating message stability and/or altering mRNA accessibility to the translational machinery (30). sRNAs are involved in a number of cellular processes, including translational quality control, by blocking or freeing ribosome binding sites (4, 31), acid resistance in Escherichia coli (38, 59), and iron homeostasis (11, 19), and have recently been implicated in regulating the virulence of several pathogens (23, 46). It is now widely accepted that bacterial sRNAs play central roles in gene expression regulation in response to environmental changes. Many sRNAs act by posttranscriptional regulation of mRNAs via base-pairing interactions (57). Two classes of bacterial base-pairing sRNAs can be distinguished (70). There are cis-encoded sRNAs that are located in the same noncoding DNA region and that are therefore fully complementary to their targets over a long sequence stretch, whereas trans-encoded sRNAs are located in another chromosomal location and are only partially complementary to their target mRNAs. The known regulatory mechanisms employed by cis-encoded antisense sRNAs include transcription attenuation, translation inhibition, inhibition of primer maturation, and promotion or inhibition of mRNA degradation (7). The majority of regulation by known trans-encoded sRNAs is negative (1, 25). Base pairing between the sRNA and its target mRNA usually leads to repression of protein levels through translational inhibition, mRNA degradation, or both. In many cases, the RNA chaperone Hfq is required for trans-encoded sRNA-mediated regulation, presumably to facilitate RNA-RNA interactions due to limited complementarity between the sRNA and target mRNA (1). Although the majority of these sRNAs were discovered in E. coli, small RNAs appear to be ubiquitous in bacteria (5, 28, 66, 69, 75). A recent primary transcriptome study in H. pylori using a novel dRNA-seq approach (54) identified an unexpected wealth of sRNAs from H. pylori, with ∼60 of them being validated, despite the lack of a conserved Hfq protein. These results show that many sRNA-mediated regulations are yet to be discovered in H. pylori. Many of the small RNAs are transcribed under the control of TCSs (15, 24, 62), providing additional control of expression of genes that are primarily regulated at the transcriptional level via these TCSs (12). In the current study, we have identified a novel urease regulatory mechanism by an sRNA-mediated downregulation of ureB expression, which is controlled primarily by the HP0165-HP0166 TCS. Shown here is the presence of an antisense sRNA (5′ureB-sRNA) that is cis encoded by the noncoding strand of the 5′ ureB gene, which regulates ureAB expression at a posttranscriptional level by targeted degradation of the sRNA-mRNA duplexes, resulting in a truncated ureAB mRNA leading to reduced translation of functional UreB protein. This 5′ureB-sRNA is downregulated by HP0165 in response to acidic pH; hence, UreB expression increases in acid media. Electrophoretic mobility shift assay (EMSA) shows that a nonphosphorylatable HP0166 response regulator binds to the promoter region of 5′ureB-sRNA, whereas phosphorylated HP0166 does not. Overexpression of 5′ureB-sRNA greatly increases the 1.4-kb transcript level and decreases urease activity. This novel regulatory mechanism maintains a high urease level at acidic pH but permits a relatively rapid decrease of urease activity when the organism is transiting to neutral pH.