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Helicobacter pylori encounters a wide range of pH within the human stomach. In a comparison of H. pylori cultured in vitro under neutral or acidic conditions, about 15% of genes are differentially expressed, and corresponding changes are detectable for many of the encoded proteins. The ArsRS two-component system (TCS), comprised of the sensor kinase ArsS and its cognate response regulator ArsR, has an important role in mediating pH-responsive changes in H. pylori gene expression. In this study, we sought to delineate the pH-responsive ArsRS regulon and further define the role of ArsR in pH-responsive gene expression. We compared H. pylori strains containing an intact ArsRS system with an arsS null mutant or strains containing site-specific mutations of a conserved aspartate residue (D52) in ArsR, which is phosphorylated in response to signals relayed by the cognate sensor kinase ArsS. We identified 178 genes that were pH-responsive in strains containing an intact ArsRS system but not in ΔarsS or arsR mutants. These constituents of the pH-responsive ArsRS regulon include genes involved in acid acclimatization (ureAB, amidases), oxidative stress responses (katA, sodB), transcriptional regulation related to iron or nickel homeostasis (fur, nikR), and genes encoding outer membrane proteins [including sabA, alpA, alpB, hopD (labA), and horA]. When comparing H. pylori strains containing an intact ArsRS TCS with arsRS mutants, each cultured at neutral pH, relatively few genes are differentially expressed. Collectively, these data suggest that ArsRS-mediated gene regulation has an important role in H. pylori adaptation to changing pH conditions.

Purpose. We investigated the transcription of adhesin-encoding genes sabA, hopZ and labA in Helicobacter pylori strain J99. Each possesses a repeating homopolymeric nucleotide tract within their promoter regions, and sabA and hopZ possess repeats within their 5′ coding regions. Methodology. We altered the repeat lengths associated with the adhesin genes and quantified mRNA levels by real-time quantitative PCR. Using adherence to AGS cells and IL-8 assays, we examined the effects of altered transcript levels. We assessed the role of ArsRS in transcription using an arsS null mutant and by examining ArsR binding to promoter regions via electrophoretic mobility shift assays. Results. Extensions or truncations of promoter region repeats in hopZ and labA increased transcript levels, mirroring results shown by our lab and others for mutations in the sabA promoter. Altered lengths of the poly-cytosine thymine tract within the 5′ coding region of sabA demonstrated that switching from phase-off to phase-on significantly increased mRNA levels. However, mutations in the poly-thymine tract of sabA, which increased mRNA levels, do not behave synergistically with phase-on mutations. Phase-on mutations of sabA resulted in increased H. pylori adherence to AGS cells, but only a modest effect on IL-8. hopZ and labA, and sabA paralogue sabB, transcript levels were increased in an arsS mutant and ArsR bound the promoter regions for each of these genes in vitro. Conclusion. This work highlights the complex nature of adhesin regulation, its impact on H. pylori attachment and the pervasive role of ArsRS in adhesin expression. Such regulation may help facilitate the decades-long persistence of infection.

The pathogenesis of diseases elicited by the gastric pathogen Helicobacter pylori is partially determined by the effectiveness of adaptation to the variably acidic environment of the host stomach. Adaptation includes appropriate adherence to the gastric epithelium via outer membrane protein adhesins such as SabA. The expression of sabA is subject to regulation via phase variation in the promoter and coding regions as well as repression by the two-component system ArsRS. In this study, we investigated the role of a homopolymeric thymine [poly(T)] tract −50 to −33 relative to the sabA transcriptional start site in H. pylori strain J99. We quantified sabA expression in H. pylori J99 by quantitative reverse transcription-PCR (RT-PCR), demonstrating significant changes in sabA expression associated with experimental manipulations of poly(T) tract length. Mimicking the length increase of this tract by adding adenines instead of thymines had similar effects, while the addition of other nucleotides failed to affect sabA expression in the same manner. We hypothesize that modification of the poly(T) tract changes DNA topology, affecting regulatory protein interaction(s) or RNA polymerase binding efficiency. Additionally, we characterized the interaction between the sabA promoter region and ArsR, a response regulator affecting sabA expression. Using recombinant ArsR in electrophoretic mobility shift assays (EMSA), we localized binding to a sequence with partial dyad symmetry −20 and +38 relative to the sabA +1 site. The control of sabA expression by both ArsRS and phase variation at two distinct repeat regions suggests the control of sabA expression is both complex and vital to H. pylori infection.

Helicobacter pylori is a genetically diverse bacterial species that colonizes the stomach in about half of the human population. Most persons colonized by H. pylori remain asymptomatic, but the presence of this organism is a risk factor for gastric cancer. Multiple populations and subpopulations of H. pylori with distinct geographic distributions are recognized. Genetic differences among these populations might be a factor underlying geographic variation in gastric cancer incidence. Relatively little is known about the genomic features of African H. pylori strains compared to other populations of strains. In this study, we first analyzed the genomes of H. pylori strains from seven globally distributed populations or subpopulations and identified encoded proteins that exhibited the highest levels of sequence divergence. These included secreted proteins, an LPS glycosyltransferase, fucosyltransferases, proteins involved in molybdopterin biosynthesis, and Clp protease adaptor (ClpS). Among proteins encoded by the cag pathogenicity island, CagA and CagQ exhibited the highest levels of sequence diversity. We then identified proteins in strains of Western African origin (classified as hspWAfrica by MLST analysis) with sequences that were highly divergent compared to those in other populations of strains. These included ATP-dependent Clp protease, ClpS, and proteins of unknown function. Three of the divergent proteins sequences identified in West African strains were characterized by distinct insertions or deletions up to 8 amino acids in length. These polymorphisms in rapidly evolving proteins represent robust genetic signatures for H. pylori strains of West African origin.

Phase variation is frequently utilized by bacterial species to affect gene expression such that phenotypic variants are maintained within populations, ensuring survival as environmental or host conditions change. Unusual among Helicobacter pylori phase variable or contingency genes is arsS, encoding a sensory histidine kinase involved in the acid acclimation of the organism. The presence of a 3′ homopolymeric cytosine tract of variable length in arsS among Helicobacter pylori strains allows for the expression of various functional ArsS isoforms, differing in carboxy-terminal protein domains. In this study, we analyzed this 3′ arsS region via amplified fragment length polymorphism (AFLP) and sequencing analyses for H. pylori populations from 3 different gastric sites of 12 patients. Our data indicate the presence of multiple arsS alleles within each population of H. pylori derived from the gastric antrum, cardia, or corpus of these patients. We also show that H. pylori, derived from the same anatomical site and patient, are predicted to express multiple ArsS isoforms in each population investigated. Furthermore, we identify a polymorphic deletion within arsS that generates another alternate ArsS C-terminal end. These findings suggest that four C-terminal variations of ArsS adds to the complexity of the ArsRS acid adaptation mechanism as a whole and may influence the ability of H. pylori to persist in the gastric niche for decades.

Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA ) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct "cross-talk" between HpArsRS and HpNikR at neutral pH has been demonstrated.

Helicobacter pylori is a genetically diverse bacterial species that chronically infects human stomachs and sometimes causes severe gastroduodenal disease. Studies of polymorphic DNA sequences can suggest geographic origins of individual strains. Here, we describe a 180-bp insertion (ins180), which is just after the translation stop of a gene of unknown function, near the promoter of jhp0152- jhp0151 two-component signal transduction genes in strain J99, and absent from this site in strain 26695. This ins180 insertion was found in 9 of 9 Gambian (West African), 9 of 20 (45%) South African, and 9 of 40 (23%) Spanish strains but in only 2 of 20 (10%) North American strains and none of 20 Lithuanian, 20 Indian, and 20 Japanese strains. Four South African isolates that lacked ins180 and that belonged to an unusual outlier group contained a 480-bp insertion at this site (ins480), whereas none of 181 other strains screened contained ins480. In further tests 56% (10 of 18) of strains from African Americans but only 17% (3 of 18) of strains from Caucasian Americans carried ins180 ( P < 0.05). Thus, the H. pylori strains of modern African Americans seem to retain traces of African roots, despite the multiple generations since their ancestors were taken from West Africa. Fragmentary ins180-like sequences were found at numerous sites in H. pylori genomes, always between genes. Such sequences might affect regulation of transcription and could facilitate genome rearrangement by homologous recombination. Apparent differences between African-American and Caucasian-American H. pylori gene pools may bear on our understanding of H. pylori transmission and disease outcome.

Summary To identify Helicobacter pylori genes with expression that is enhanced under low pH conditions, we used subtractive hybridization methodology. We identified 28 acid-induced genes, of which 18 have known or putative functions. Six pairs of genes were co-transcribed. Primer extension analysis identified single or multiple transcriptional start points (tsp) for 14 of the 22 loci. Sequence analysis of the −10 regions upstream of the tsps revealed consensus motifs for multiple RNA polymerase sigma factors present in H. pylori (σ80, σ54 and σ28). No sequences resembling the −35 Escherichia coli consensus sequence (TTGACA) were present upstream of any of the genes. Both increased gene transcription and decreased mRNA decay contribute to the observed increase in H. pylori transcript abundance at acid pH. These studies document the complex response of H. pylori to environmental pH changes, and provide insight into mechanisms used for intragastric survival.

Broth culture supernatants from Tox + Helicobacter pylori strains induce vacuolation of HeLa cells in vitro and contain VacA in concentrations that are higher than those found in supernatants from Tox − H. pylori strains. To investigate the basis for this phenomenon, we analyzed the transcription of the vacuolating cytotoxin gene ( vacA ) in eight Tox + strains (each with a type s1/m1 vacA genotype) and nine Tox − strains (each with a type s2/m2 vacA genotype). Most of the Tox + and Tox − strains tested used the same vacA transcriptional start point, but Tox + strains yielded significantly stronger primer extension signal intensities than did Tox − strains (mean densitometry values of 15.8 ± 1.9 versus 8.9 ± 1.7, P = 0.0016). Correspondingly, when we introduced vacA :: xylE transcriptional fusions into the chromosomes of a Tox + strain (60190) and a Tox − strain (86-313), the level of XylE activity in 60190 vacA :: xylE was about 30-fold higher than that in 86-313 vacA :: xylE . Sequence analysis and promoter exchange experiments indicated that the different levels of vacA transcription in these two strains cannot be explained solely by a difference in promoter strength. These data indicate that Tox + and Tox − H. pylori strains typically differ not only in the VacA amino acid sequence but also in the level of vacA transcription.

In an analysis of Helicobacter pylori genomic DNA by macroarray methodology, genomic DNA from a panel of cag pathogenicity island (PAI)-negative H. pylori clinical isolates failed to hybridize with 27 genes located outside the cag PAI in a cag PAI-positive reference strain. PCR analyses confirmed that HP0217 (encoding a lipopolysaccharide biosynthetic protein) and HP1079 (encoding a protein of unknown function) were present significantly more frequently in cagA -positive strains than in cagA -negative strains. A low G+C content of these two genes suggests they were acquired by horizontal transfer events.

To identify putative members of a regulon controlled by the H. pylori sensory histidine kinase HP0164 (HK0164), we constructed HK0164 null mutant H. pylori strains and analyzed bacterial gene transcription using DNA arrays. Seven genes were differentially expressed in multiple HK0164 mutant strains compared to their expression in control strains. Strain-dependent variations in differential expression were also detected. These results indicate that the signal transduction circuit utilizing HK0164 controls the transcription of at least seven genes in H. pylori .

Analysis of 12 Helicobacter pylori promoters indicates the existence of a consensus −10 hexamer (TAtaaT) but little conservation of −35 sequences. In this study, mutations in either the H. pylori vacA −10 region or the −35 region resulted in decreased vacA transcription and suggested that an extended −10 motif is utilized. Thus, despite the lack of a −35 consensus sequence for H. pylori promoters, the −35 region plays a functional role in vacA transcription.

The gyrAB genes encoding the entire B and a portion of the A subunit of DNA gyrase (E.C. 5.99.1.3) from Mycoplasma gallisepticum (Mg), strain S6, were cloned and sequenced. These gyrAB genes are co-transcribed as a single, polycistronic mRNA transcript. The Mg gyrB appears unique among prokaryotic gyrB in its use of GUG as a start codon.