TY - JOUR
AU - Bereswill, Stefan
AB - Abstract In this review, we summarize how genomic approaches contributed to the understanding of the biology of the recently discovered pathogen Helicobacter pylori. Comparative genomics provided new insights into H. pylori's spectacular genetic diversity and generated exiting hypotheses on its evolutionary history. Transcriptomic studies provided original information on the mechanisms of H. pylori gastric adaptation that are central to its virulence. DNA-arrays, gastric pathologies, evolution, transcriptome, diversity Introduction Landmarks in the ‘short’ history of Helicobacter pylori research Reports on spiral bacteria in the stomach of humans and mammals (Bizzozero, 1893) date back to the 19th century (Konjetzny, 1923). However, diseases of the upper intestinal tract remained mostly incurable before the discovery of H. pylori. Scientific proof for an infectious origin of gastric diseases was not provided until 1982, when Robin Warren and Barry Marshall isolated H. pylori from gastric biopsy samples and established associations between infection and mucosal inflammation (Marshall & Warren, 1984). Subsequently, several researchers performed self-infection experiments, which resulted in H. pylori-induced gastritis. To fulfill Koch's postulates, the intentionally absorbed bacteria were reisolated from gastric biopsies, and inflammation was cured by subsequent antibiotic treatment (Marshall et al., 1985). Because the H. pylori-related diseases represent a common problem that many individuals encounter during their lifetime, the discovery of H. pylori led to a major breakthrough in gastroenterology, for which Marshall and Warren were awarded the Nobel Prize in Physiology or Medicine in 2005 (http://nobelprize.org). Their discoveries offered the possibility to treat gastric diseases by antimicrobial therapy and provided a novel understanding of mechanisms underlying gastritis, ulcerations, lymphomas and gastric cancer (Suerbaum & Michetti, 2002). About 10 years ago, the scientific investigation of H. pylori and infection-related diseases was greatly promoted by the release of the genome sequences of two strains, 26695 (Tomb et al., 1997) and J99 (Alm et al., 1999). The genome era opened the possibility to study gene contents of individual strains, the overall genetic instability and micro-evolution within individual hosts and the global gene expression of a strain in several conditions. In this review, we highlight recent discoveries made by comparative and functional genomics. The results demonstrate how a consequent application of genome-based research tools contributes to a better understanding of bacterial evolution and adaptation. To display novel insights (reviewed in Kusters et al., 2006) adequately, we discuss possible mechanisms involved in colonization of the restricted and hostile natural reservoir including interspecies transmission, genetic diversity and regulatory pathways. Comparative genomics Genetic variability and gastric adaptation Helicobacter pylori belongs to the epsilon subdivision of Proteobacteria, and is classified in the Helicobacteraceae family of the Campylobacterales order (Eppinger et al., 2004). Infection with the highly motile Gram-negative rods is specific to the stomach of humans and primates. No other natural reservoir has been convincingly demonstrated. Transfer is inter-human, mainly familial. After initial infection, it is thought that H. pylori adapts rapidly to its unique gastric niche. The bacteria colonize the gastric mucus layer and persist livelong in close contact with epithelial cells. In the gastric mucosa, H. pylori is continuously faced with harsh physiological conditions and a vigorous immune response. The need for adaptation to the extremely changing micro-environment and to individual hosts (van Vliet et al., 2001) is probably the cause of a high degree of interstrain genetic variation observed in the H. pylori population worldwide. Point mutations, insertions and deletions of genes (Blaser, 1994) and intergenic regions (Bereswill et al., 2000a) present in all isolates are thought to provide specific mechanisms to subvert host immunity (Cooke et al., 2005) and to adapt to unfavourable conditions (van Vliet et al., 2001). In addition, the gene order varies among strains (Jiang et al., 1996). The interstrain diversity of H. pylori is extended by plasmids (Hofreuter & Haas, 2002) and there is evidence that active import of DNA via specific uptake machineries (Karnholz et al., 2006) promotes genetic variability in the gastric pathogen. Helicobacter pylori diversity at the genome level The release of the genome sequences of two H. pylori strains (Tomb et al., 1997; Alm et al., 1999) offered the possibility to study genetic instability and micro-evolution in more detail. At that time, about one-third of the c. 1600 genes predicted in the 1.6 Mbp genome were considered to be H. pylori-specific due to the absence of homologues in other organisms (Marais et al., 1999; Boneca et al., 2003). The fact that even these two genomes display a high degree of diversity in terms of insertions, deletions, overall genome structure and plasticity, led to the proposal that the H. pylori population represents a ‘quasi-species’ (Covacci & Rappuoli, 1998) with a panmictic structure due to free recombination (Suerbaum et al., 1998; Suerbaum & Achtman, 2004). The repertoire of H. pylori to generate genetic variability is completed by sequence changes affecting phase-variable genes in which mutations can shut gene expression on or off (De Vries et al., 2001). Comprehensive analysis of homopolymeric tracts and dinucleotide repeats in the phase-variable gene subsets deduced from strains 26695 and J99 revealed nucleotide variations that may be present or absent among the strains (Salaun et al., 2004). Definition of the core genome and the variable gene pool The fact that 6–7% of genes in the two H. pylori genomes are strain-specific (Alm et al., 1999) provided the first evidence of the presence of a variable gene pool, which is indicative of horizontal gene transfer between H. pylori strains. Because these strain-specific genes could be involved in gastric adaptation during coevolution, this flexible gene pool was extensively investigated in many strains worldwide. In silico analysis of the two H. pylori genomes (Garcia-Vallve et al., 2002; Saunders et al., 2005) revealed that both housekeeping genes and virulence genes are transferred among H. pylori strains. For 69 genes, divergent codon-usage and GC-content provided evidence that these genes were obtained from other species. Genetic variability and DNA exchange among H. pylori strains was further studied by micro-array analysis. The results demonstrate that gene contents of H. pylori isolates from the same (Israel et al., 2001) and different (Salama et al., 2000) individuals display between 3% and 22% variability, respectively. Based on the global gene distribution in 15 unrelated H. pylori strains from different geographical origins (Salama et al., 2000), the core genome was estimated to consist of 1280 genes, including those coding for central (house-keeping), conserved and essential functions (Salama et al., 2000). More than 300 genes are not homogeneously distributed and many of the dispensable genes are located in the so-called ‘plasticity zones’ and in the cag pathogenicity island. Other variable genes are involved in the synthesis of surface structures and in DNA modification or transposition. It is striking that many genes in the flexible pool encode H. pylori-specific proteins or conserved proteins of so far unknown functions. Identification of species- and genus-specific genes The taxonomical classification and assignment of H. pylori to the epsilon subdivision of Proteobacteria opened the way to define species-specific genes as well as genes exchanged and shared by members of this bacterial group. Thus, the availability of whole genomes of Helicobacter hepaticus (Suerbaum et al., 2004), Campylobacter jejuni (Parkhill et al., 2000), and Wolinella succinogenes (Baar et al., 2003) offered the opportunity to extend our knowledge of H. pylori evolution (Eppinger et al., 2004). Whole genome clustering of H. pylori and C. jejuni demonstrated that 648 H. pylori genes are species-specific (Janssen et al., 2001). The fact that 95% of the 162 H. pylori genes displaying interstrain variability are species-specific supports the assumption that genes of the flexible gene pool are exchanged among Helicobacter species but are not transferred to bacteria of other genus. Finally, a comprehensive analysis of the genomes available from the four different Campylobacterales species revealed that H. pylori and the other pathogens have lost many regulatory circuits and sensing systems still conserved in an epsilon Proteobacteria such as W. succinogenes, presenting the capacity to be free living (Eppinger et al., 2004). Thus, this bacterial group fulfills the paradigm of reductive evolution (related to host adaptation), as Wolinella contains complete metabolic pathways and a high number of sensing systems. Based on recent comparisons of the H. pylori genomes with that of Helicobacter acinonychis, which colonizes felines, it was postulated that there was a host jump about 200 000 years ago at which H. pylori was transferred from humans to cats (Eppinger et al., 2006). Extended micro-array analysis of the gene distribution and Multi Locus Sequence typing (MLST) on seven core genes performed with 56 H. pylori strains (Gressmann et al., 2005) revealed that 25% of genes that are common to both 26685 and J99 are missing in at least one isolate and that 21% of genes were absent or variable in H. acinonychis. In addition, this study concludes that there is a core genome of only 1111 genes and predicts that the cag pathogenicity island genes were acquired en bloc after speciation. Variable genes are small, possess unusual GC contents and encode mostly proteins of unknown function or outer membrane proteins (OMPs). Genes for proteins of unknown function and transposases were predicted to have been acquired prior to speciation (Gressmann et al., 2005). Finally, comparison of the Campylobacterales genomes to the genetic information of the sequence databases allowed the identification of genes and molecular signatures that are unique to the members of the epsilon Proteobacteria (Gupta, 2006). Worldwide coevolution and spreading with the human host The rapid progress in sequencing technologies and related in silico software tools for sequence analysis has paved the way for investigations focussed on the gene content and genetic diversity of H. pylori populations worldwide. It became clear that H. pylori colonized humans more than 100 000 years ago (Covacci et al., 1999) and that a significant correlation between H. pylori genotypes and human entities worldwide can be unraveled (reviewed in Suerbaum & Achtman, 2004). Using multi-locus sequence analysis of selected core genes in H. pylori isolates from ethnical subpopulations of humans worldwide, it was established that the global H. pylori population can be subdivided into seven genetically distinct subpopulations that derived their gene pools from ancestral populations arising in Africa, Central Asia, and East Asia (Falush et al., 2003a). The optimization of the mathematical basis for the use of multilocus genotype data (Falush et al., 2003b) revealed close associations between H. pylori subtypes and human subpopulations in one continent or even ethnic subgroups within small geographic regions (Wirth et al., 2004). Thus, H. pylori subtypes can be used to trace human migrations during history and the spreading of distinct H. pylori subtypes could be attributed to prehistoric and modern migratory fluxes (Suerbaum & Achtman, 2004; Linz et al., 2007). Microevolution and genetic variability within single human hosts Because the occurrence of gene loss or gain in single hosts may play an important role in gastric adaptation, the resulting micro-evolution is a driving force for genetic diversity of H. pylori. The gene contents of H. pylori strains isolated sequentially from single patients during persistent infection confirmed that gene loss and acquisition of exogenous DNA occurs (Israel et al., 2001). Furthermore, multilocus sequence analysis of 10 genes in paired isolates from 26 different individuals (Falush et al., 2001) showed that point mutations occur in the stomach of a single host, and that mostly small mosaic DNA segments with a median size of 417 bp are exchanged. Calculations of mutation and recombination frequencies with respect to insert sizes revealed that genetic diversity displayed by the panmictic population structure is a result of continuous DNA exchange between parental strains (without mutations) and daughter strains, which have accumulated mutations. This was supported by gene content analysis of isolates taken from single patients at different time points, which demonstrated that the great majority of genetic changes were caused by homologous recombination, indicating that adaptation of H. pylori to the host individual is more frequently mediated by sequence changes acquired by recombination events rather than loss or gain of genes (Kraft et al., 2006). Helicobacter pylori evolution during early infection and disease progression The identification of genes associated with severe pathologies represents a major challenge in H. pylori research. Molecular analysis during the pregenomic era established that the sequences of virulence-associated genes coding for the vacuolating cytotoxin VacA (Atherton et al., 1995) and the CagA protein (Blaser, 1994) vary considerably among strains. In addition, specific genotypes of VacA are associated with ulcer development (Atherton et al., 1995) and the presence of the cag pathogenicity island promoting injection of the CagA protein into host cells correlates with pronounced inflammation and more severe pathologies. Although H. pylori causes acute superficial gastritis in nearly all of its human hosts, some infected individuals develop chronic atrophic gastritis (ChAG). This pathology is characterized by diminished numbers of acid-producing parietal cells and increased risk for the development of gastric adenocarcinoma. The complete genome analysis of the first H. pylori isolate from a patient with ChAG gave further insights in gene sets that could be involved in causing this specific disease outcome (Oh et al., 2006). Whole-genome analysis of additional H. pylori isolates from an individual who progressed from ChAG to gastric adenocarcinoma revealed a gene signature shared among ChAG strains, as well as genes that may have been lost or gained during progression to adenocarcinoma. Many of these genes encode components of metal uptake and utilization pathways, outer membrane proteins, and virulence factors indicating that the bacteria adapt effectively to environmental changes during ChAG disease progression. The rhesus macaque model has also contributed greatly to the examination of genomic changes in H. pylori that occur early during experimental infection (Solnick et al., 2004). Micro-array analysis demonstrated that H. pylori recovered from infected macaques carried deletions in the locus coding for BabA, an adhesin that mediates attachment of H. pylori to gastric epithelia. In some isolates the babA gene was not expressed or was replaced by babB, which encodes a related protein. Absence of babA and duplication of babB was also seen in H. pylori isolates derived from human clinical samples, suggesting that this gene conversion is of relevance to the human host and might reflect diverging selective pressures for adhesion either across hosts or within an individual (Colbeck et al., 2006). The conclusion that changes in babA and babB expression represent a dynamic response in the H. pylori outer membrane that facilitates adherence to the gastric epithelium and promotes chronic infection was further supported by extensive genotypic diversity displayed by human H. pylori isolates as well as within a strain colonizing an individual patient. Functional genomics Transcriptomics to study H. pylori host adaptation and pathogenesis In this part of the review we will focus on genomic studies designed to explore dynamic aspects of the lifestyle of H. pylori that are relevant to the long-term interaction of this pathogen with its host and its potential ‘adaptive evolution’ after transmission or during disease progression. The strategies and factors of H. pylori that allow it persistently to colonize the gastric niche are particularly interesting to study as they are on the one hand related to its pathogenicity (see below) and on the other are unique because of its original lifestyle. Helicobacter pylori colonizes a single niche that no other microbe is able to inhabit permanently and therefore it does not have to struggle with competitors. However, this pathogen has to deal with adverse and unstable environmental conditions such as mild to strong acidity, fluctuating nutrient availability, changes in oxygen tension and an intense immune response of the host. Despite these parameters, H. pylori is a particularly successfully pathogen proliferating in its host stomach over decades. In the following paragraphs, we will only focus on the numerous transcriptomic studies of H. pylori over the last 5 years, which are listed in Table 1. These studies have revealed that H. pylori has an important adaptation potential through modulation of gene expression. Whole genome transcriptional profiling studies are designed to define the changes in the transcriptome of an organism submitted to a modification of its environment/growth conditions or carrying a mutation. In the case of H. pylori a major effort was concentrated on examining its response to conditions mimicking those encountered during colonization of the host. We will mainly discuss the known functions regulated under these conditions, however a large amount of unknown genes are also regulated. Table 1 Studies using whole genome transcriptional profiling of Helicobacter pylori Condition or regulator protein tested  Experimental procedure  Strains used  Total number of regulated genes  Reference  Helicobacter pyloritranscriptome in conditions mimicking those encountered in the host  In response to acidity            Growth on plates (48 h) at pH 5.5 vs. 7  26695  84  Ang (2001)    Exposure to pH 4 vs. 7 during 30 min  26695  11  Allan et al. (2001)    Exposure to pH 5 of a liquid culture grown at pH 7, time course from 0.5 to 2 h  G27  118  Merrell et al., (2003a)    Exposure to pH 4.5, 5.5, 6.2, 7.4 with and without 5 mM urea during 30 min  26695  300  Wen et al. (2003)    Exponential growth in liquid medium at pH 5 and 7  26695  111  Bury-Moné. (2004)  As a function of growth phase  Growth in liquid medium from exponential to stationary phase, time course from 0 to 50 h  SS1  325  Thompson et al. (2003)  In response to iron starvation  Iron chelation or add-back of exponential and stationary phase, time course from 0 to 100 min  SS1  183  Merrell et al., (2003b)  In response to AGS cell attachment  Bacteria attached to AGS cells during 4 h vs. in the same conditions without cells  69a  43  Kim et al. (2004)  Helicobacter pyloritranscriptome in mutants deficient in transcriptional regulators  NikR  WT vs. ΔnikR mutant, in the presence of nickel  SS1  42  Contreras et al. (2003)  Fur  WT vs. Δfur mutant in iron-restricted or iron-replete conditions, grown for 20 h  26695  97 iron-responsive regulation/43 Fur-dependent regulation  Ernst et al., (2005a)  Fur  WT vs. Δfur mutant, exponential and stationary growth phase  G27  29  Gancz et al. (2006)  Fur  WT vs. Δfur mutant, exponential and stationary growth phase  G27  26 in exponential phase, 90 in stationary phase  Danielli et al. (2006)  ArsRS (HP165)  WT vs. a HP165 mutant  26695 and B128  7  Forsyth et al. (2002)  σ28, FlgM  WT vs. mutants  N6  NA  Josenhans et al. (2002)  σ54, FleRS, FlhF, FlhA  WT vs. mutants and mutants vs. mutants  N6 and 88–3887 (a motile variant of 26695)  NA  Niehaus et al. (2004)  Helicobacter pyloritranscriptome in mutants deficient in transcriptional regulators in response to acidity  Fur  WT vs. Δfur mutant, exposure to pH 5 of a liquid culture grown at pH 7, time course 0.5–1.5 h  G27  95  Gancz et al. (2006)  NikR and Fur  ΔnikR-Δfur mutant during growth at pH 5 vs. 7  26695  36  Bury-Moné. (2004)  ArsR-S (HP166-165)  WT vs. ΔarsS mutant, exposure to pH 5 for 1 h  G27  109  Pflock et al., (2006a)  ArsR-S (HP166-165)  WT vs. arsS mutant, exposure to pH 5 for 75 min  J99  68 on 101 considered genes  Loh & Cover (2006)  CrdRS (HP1365-HP1364)  WT vs. a crdS mutant, exposure to pH 5 for 75 min  J99  63 on 101 considered genes  Loh & Cover (2006)  Condition or regulator protein tested  Experimental procedure  Strains used  Total number of regulated genes  Reference  Helicobacter pyloritranscriptome in conditions mimicking those encountered in the host  In response to acidity            Growth on plates (48 h) at pH 5.5 vs. 7  26695  84  Ang (2001)    Exposure to pH 4 vs. 7 during 30 min  26695  11  Allan et al. (2001)    Exposure to pH 5 of a liquid culture grown at pH 7, time course from 0.5 to 2 h  G27  118  Merrell et al., (2003a)    Exposure to pH 4.5, 5.5, 6.2, 7.4 with and without 5 mM urea during 30 min  26695  300  Wen et al. (2003)    Exponential growth in liquid medium at pH 5 and 7  26695  111  Bury-Moné. (2004)  As a function of growth phase  Growth in liquid medium from exponential to stationary phase, time course from 0 to 50 h  SS1  325  Thompson et al. (2003)  In response to iron starvation  Iron chelation or add-back of exponential and stationary phase, time course from 0 to 100 min  SS1  183  Merrell et al., (2003b)  In response to AGS cell attachment  Bacteria attached to AGS cells during 4 h vs. in the same conditions without cells  69a  43  Kim et al. (2004)  Helicobacter pyloritranscriptome in mutants deficient in transcriptional regulators  NikR  WT vs. ΔnikR mutant, in the presence of nickel  SS1  42  Contreras et al. (2003)  Fur  WT vs. Δfur mutant in iron-restricted or iron-replete conditions, grown for 20 h  26695  97 iron-responsive regulation/43 Fur-dependent regulation  Ernst et al., (2005a)  Fur  WT vs. Δfur mutant, exponential and stationary growth phase  G27  29  Gancz et al. (2006)  Fur  WT vs. Δfur mutant, exponential and stationary growth phase  G27  26 in exponential phase, 90 in stationary phase  Danielli et al. (2006)  ArsRS (HP165)  WT vs. a HP165 mutant  26695 and B128  7  Forsyth et al. (2002)  σ28, FlgM  WT vs. mutants  N6  NA  Josenhans et al. (2002)  σ54, FleRS, FlhF, FlhA  WT vs. mutants and mutants vs. mutants  N6 and 88–3887 (a motile variant of 26695)  NA  Niehaus et al. (2004)  Helicobacter pyloritranscriptome in mutants deficient in transcriptional regulators in response to acidity  Fur  WT vs. Δfur mutant, exposure to pH 5 of a liquid culture grown at pH 7, time course 0.5–1.5 h  G27  95  Gancz et al. (2006)  NikR and Fur  ΔnikR-Δfur mutant during growth at pH 5 vs. 7  26695  36  Bury-Moné. (2004)  ArsR-S (HP166-165)  WT vs. ΔarsS mutant, exposure to pH 5 for 1 h  G27  109  Pflock et al., (2006a)  ArsR-S (HP166-165)  WT vs. arsS mutant, exposure to pH 5 for 75 min  J99  68 on 101 considered genes  Loh & Cover (2006)  CrdRS (HP1365-HP1364)  WT vs. a crdS mutant, exposure to pH 5 for 75 min  J99  63 on 101 considered genes  Loh & Cover (2006)  NikR, nickel-responsive regulator. Fur, iron-responsive regulator. ArsR-S, two-component system involved in Acid Responsive Signaling. ArsR, response regulator; ArsS, sensor histidine kinase. σ28 (FliA), FlgM (anti-σ28), σ54 (RpoN), FleRS, FlhF, FlhA: regulators of the flagellar genes expression. CrdRS: two-component system involved in regulation of Copper Resistance Determinant. CrdR, response regulator; CrdS, sensor histidine kinase. NA, not applicable. View Large Table 1 Studies using whole genome transcriptional profiling of Helicobacter pylori Condition or regulator protein tested  Experimental procedure  Strains used  Total number of regulated genes  Reference  Helicobacter pyloritranscriptome in conditions mimicking those encountered in the host  In response to acidity            Growth on plates (48 h) at pH 5.5 vs. 7  26695  84  Ang (2001)    Exposure to pH 4 vs. 7 during 30 min  26695  11  Allan et al. (2001)    Exposure to pH 5 of a liquid culture grown at pH 7, time course from 0.5 to 2 h  G27  118  Merrell et al., (2003a)    Exposure to pH 4.5, 5.5, 6.2, 7.4 with and without 5 mM urea during 30 min  26695  300  Wen et al. (2003)    Exponential growth in liquid medium at pH 5 and 7  26695  111  Bury-Moné. (2004)  As a function of growth phase  Growth in liquid medium from exponential to stationary phase, time course from 0 to 50 h  SS1  325  Thompson et al. (2003)  In response to iron starvation  Iron chelation or add-back of exponential and stationary phase, time course from 0 to 100 min  SS1  183  Merrell et al., (2003b)  In response to AGS cell attachment  Bacteria attached to AGS cells during 4 h vs. in the same conditions without cells  69a  43  Kim et al. (2004)  Helicobacter pyloritranscriptome in mutants deficient in transcriptional regulators  NikR  WT vs. ΔnikR mutant, in the presence of nickel  SS1  42  Contreras et al. (2003)  Fur  WT vs. Δfur mutant in iron-restricted or iron-replete conditions, grown for 20 h  26695  97 iron-responsive regulation/43 Fur-dependent regulation  Ernst et al., (2005a)  Fur  WT vs. Δfur mutant, exponential and stationary growth phase  G27  29  Gancz et al. (2006)  Fur  WT vs. Δfur mutant, exponential and stationary growth phase  G27  26 in exponential phase, 90 in stationary phase  Danielli et al. (2006)  ArsRS (HP165)  WT vs. a HP165 mutant  26695 and B128  7  Forsyth et al. (2002)  σ28, FlgM  WT vs. mutants  N6  NA  Josenhans et al. (2002)  σ54, FleRS, FlhF, FlhA  WT vs. mutants and mutants vs. mutants  N6 and 88–3887 (a motile variant of 26695)  NA  Niehaus et al. (2004)  Helicobacter pyloritranscriptome in mutants deficient in transcriptional regulators in response to acidity  Fur  WT vs. Δfur mutant, exposure to pH 5 of a liquid culture grown at pH 7, time course 0.5–1.5 h  G27  95  Gancz et al. (2006)  NikR and Fur  ΔnikR-Δfur mutant during growth at pH 5 vs. 7  26695  36  Bury-Moné. (2004)  ArsR-S (HP166-165)  WT vs. ΔarsS mutant, exposure to pH 5 for 1 h  G27  109  Pflock et al., (2006a)  ArsR-S (HP166-165)  WT vs. arsS mutant, exposure to pH 5 for 75 min  J99  68 on 101 considered genes  Loh & Cover (2006)  CrdRS (HP1365-HP1364)  WT vs. a crdS mutant, exposure to pH 5 for 75 min  J99  63 on 101 considered genes  Loh & Cover (2006)  Condition or regulator protein tested  Experimental procedure  Strains used  Total number of regulated genes  Reference  Helicobacter pyloritranscriptome in conditions mimicking those encountered in the host  In response to acidity            Growth on plates (48 h) at pH 5.5 vs. 7  26695  84  Ang (2001)    Exposure to pH 4 vs. 7 during 30 min  26695  11  Allan et al. (2001)    Exposure to pH 5 of a liquid culture grown at pH 7, time course from 0.5 to 2 h  G27  118  Merrell et al., (2003a)    Exposure to pH 4.5, 5.5, 6.2, 7.4 with and without 5 mM urea during 30 min  26695  300  Wen et al. (2003)    Exponential growth in liquid medium at pH 5 and 7  26695  111  Bury-Moné. (2004)  As a function of growth phase  Growth in liquid medium from exponential to stationary phase, time course from 0 to 50 h  SS1  325  Thompson et al. (2003)  In response to iron starvation  Iron chelation or add-back of exponential and stationary phase, time course from 0 to 100 min  SS1  183  Merrell et al., (2003b)  In response to AGS cell attachment  Bacteria attached to AGS cells during 4 h vs. in the same conditions without cells  69a  43  Kim et al. (2004)  Helicobacter pyloritranscriptome in mutants deficient in transcriptional regulators  NikR  WT vs. ΔnikR mutant, in the presence of nickel  SS1  42  Contreras et al. (2003)  Fur  WT vs. Δfur mutant in iron-restricted or iron-replete conditions, grown for 20 h  26695  97 iron-responsive regulation/43 Fur-dependent regulation  Ernst et al., (2005a)  Fur  WT vs. Δfur mutant, exponential and stationary growth phase  G27  29  Gancz et al. (2006)  Fur  WT vs. Δfur mutant, exponential and stationary growth phase  G27  26 in exponential phase, 90 in stationary phase  Danielli et al. (2006)  ArsRS (HP165)  WT vs. a HP165 mutant  26695 and B128  7  Forsyth et al. (2002)  σ28, FlgM  WT vs. mutants  N6  NA  Josenhans et al. (2002)  σ54, FleRS, FlhF, FlhA  WT vs. mutants and mutants vs. mutants  N6 and 88–3887 (a motile variant of 26695)  NA  Niehaus et al. (2004)  Helicobacter pyloritranscriptome in mutants deficient in transcriptional regulators in response to acidity  Fur  WT vs. Δfur mutant, exposure to pH 5 of a liquid culture grown at pH 7, time course 0.5–1.5 h  G27  95  Gancz et al. (2006)  NikR and Fur  ΔnikR-Δfur mutant during growth at pH 5 vs. 7  26695  36  Bury-Moné. (2004)  ArsR-S (HP166-165)  WT vs. ΔarsS mutant, exposure to pH 5 for 1 h  G27  109  Pflock et al., (2006a)  ArsR-S (HP166-165)  WT vs. arsS mutant, exposure to pH 5 for 75 min  J99  68 on 101 considered genes  Loh & Cover (2006)  CrdRS (HP1365-HP1364)  WT vs. a crdS mutant, exposure to pH 5 for 75 min  J99  63 on 101 considered genes  Loh & Cover (2006)  NikR, nickel-responsive regulator. Fur, iron-responsive regulator. ArsR-S, two-component system involved in Acid Responsive Signaling. ArsR, response regulator; ArsS, sensor histidine kinase. σ28 (FliA), FlgM (anti-σ28), σ54 (RpoN), FleRS, FlhF, FlhA: regulators of the flagellar genes expression. CrdRS: two-component system involved in regulation of Copper Resistance Determinant. CrdR, response regulator; CrdS, sensor histidine kinase. NA, not applicable. View Large Transcriptome analysis in conditions mimicking those encountered in the host Acidity One of the most remarkable properties of H. pylori is its capacity to persistently colonize an acidic niche. This organism is able to survive extreme acidity in the gastric lumen upon the initial phase of infection (median pH of 1.4), but colonization is mainly established within the gastric mucus layer, where a moderately acidic pH prevails (Schreiber et al., 2004). Adhesion to the epithelial cells at a pH close to neutrality only concerns about 20% of the H. pylori population and this process is thought to be dynamic, with bacteria attaching and detaching from the cells. Thus, within its host H. pylori is submitted to fluctuating pH to which it needs to respond rapidly and in an appropriate and co-ordinated way. Therefore, the influence of pH on global gene expression was the first environmental condition to be extensively investigated by transcriptional profiling (Allan et al., 2001; Ang et al., 2001; Merrell et al., 2003a; Wen et al., 2003; Bury-Moné et al., 2004; see Table 1). As central mechanisms in acid resistance are related to a tight regulation of urease activity and expression (summarized in Stingl & De Reuse, 2005), the complexity of the in vivo response of H. pylori to pH is difficult to transpose into in vitro experiments. Accordingly, several conditions were tested, which, together with different analysis methods, probably accounts for the poor overlap between the five transcriptomic studies both in the nature and the total number of regulated genes (Table 1 and discussed in Bury-Moné et al., 2004). Strikingly, two studies (Merrell et al., 2003a; Bury-Moné et al., 2004), although performed in different conditions, reported a similar number of acid-regulated genes (118 and 111), of which 22% were in common. We will briefly summarize some converging conclusions emerging from these studies. It was found that low pH modifies gene expression to induce mechanisms of protection against protons. Ammonia production by H. pylori is the major strategy for acid resistance and this compound is also described to be cytotoxic either alone or in conjunction with neutrophil metabolites (Sommi et al., 1996). Expression of urease genes is induced by acidity as well as those of other ammonia-producing enzymes, the AmiE amidase (Skouloubris et al., 1997) and the AmiF formamidase (Skouloubris et al., 2001). Another protection strategy is suggested by the reiterated observation of down-regulation in the expression of membrane proteins including transporters, permeases and OMPs. A common theme was also the enhanced expression of genes related to motility, including structural proteins of the flagellar apparatus and motor-related proteins. Stimulation of H. pylori motility by acid was indeed visualized by video-microscopy (Merrell et al., 2003a) and might be indicative of a strategy to escape acidity or of a pH-driven response directing the bacteria through the mucus pH gradient to a suitable site for multiplication or adhesion, as suggested by the work of Schreiber et al., (2004). The two studies also indicated enhanced expression of the VacA cytotoxin and the SabA adhesin at neutral pH as compared to pH 5 (Merrell et al., 2003a; Bury-Moné et al., 2004). This regulation, confirmed at the protein level for VacA by comparative two-dimensional gels (Jungblut et al., 2000), might optimize the production of virulence factors in the neutral vicinity of the gastric epithelial cells. Finally, our analysis of the response of H. pylori to exponential growth at pH 5 uncovered a novel category of acid-regulated genes encoding proteins related to metal metabolism or regulated by metal availability (Bury-Moné et al., 2004). Solubility of metal ions is known to be enhanced by acidity, accordingly as a protection mechanism against their toxic effects, we found enhanced expression of a metal storage protein (Hpn-like) and diminished expression of nickel and iron transport systems such as NixA or FecA, respectively. Acid down-regulation of the expression of two transcriptional regulators, the iron-responsive regulator Fur (van Vliet et al., 2003; Bury-Moné et al., 2004) and the HP165-HP166 genes encoding a two-component regulatory system (recently designated ArsRS), was also observed (Wen et al., 2003, 2006; Bury-Moné et al., 2004), leading to the early proposition confirmed by subsequent studies that these may be involved in the adaptive response to low pH (see below). Growth phase, iron starvation and attachment to gastric cells A careful time course transcriptome study was performed to identify the growth-phase-dependent genes of H. pylori (Thompson et al., 2003) A major switch in gene expression was observed during the transition between late logarithmic and stationary phase with the expression of several genes related to virulence being modified. During this switch, genes encoding the neutrophil activating protein and bacterioferritin (NapA) and the FlaA flagellin were induced, whereas genes encoding proteins involved in iron homeostasis were regulated as if the bacteria were submitted to iron overload. The regulatory mechanisms governing the entry into stationary phase are still a black box in the case of H. pylori, in particular there is no homologue of the alternative sigma factor RpoS central to this switch in Escherichia coli. HrcA, a heat-shock regulator found to be up-regulated during the Log-Stat switch, might be a first candidate to play a role in the growth phase switch (Thompson et al., 2003). During stomach colonization, H. pylori undoubtedly experiences periods of iron overload and starvation as the human body sequesters this ion to prevent it causing oxidative stress and to restrict bacterial proliferation. Iron homeostasis consists in a fine-tuned balance of its uptake, efflux, utilization and storage. Indeed, this ion is essential for several bacterial functions but is toxic when in excess. A time course transcriptomic study was performed in response to iron starvation in both exponential and stationary phase (Merrell et al., 2003b). Of a total of 183 differentially regulated genes, there were only 30 in common between the two growth phases. The recent observation of a phase-dependent expression of the Fur protein i.e et al., enhanced production in the stationary phase (Danielli et al., 2006) might partially explain this observation. Most interestingly, in contrast to Fur from other bacteria, the H. pylori Fur protein repressed iron storage factors such as the Pfr ferritin (Bereswill et al., 1998) in the absence of iron (Bereswill et al., 2000b). This unusual function of Fur was independently confirmed and the binding site of iron-free Fur on the pfr gene promoter was subsequently identified (Delany et al., 2002). In addition, some virulence factors were affected as a function of iron. VacA and NapA were induced by iron starvation and motility was affected in a complex growth phase-dependent manner. The two amidase genes were induced by iron starvation in both growth phases, and a weaker induction of the ureAB genes was measured only in stationary phase (Merrell et al., 2003b). To gain an insight into the H. pylori response to adhesion to gastric epithelial cells, Kim et al., (2004) compared the global gene expression after attachment to AGS gastric cancer cells with that of bacteria incubated in the cell culture medium. Only 43 genes presented differential expression, among which down-regulation of genes related to motility was in agreement with close adhesion. Transcriptome analysis in mutants deficient in regulators When results from gene expression profiling lead to hypotheses on processes or mechanisms that are switched on or off in response to a change of environment, these obviously need to be directly experimentally validated. To understand how an organism responds to an environmental change, one should try to identify the extracellular signal sensed, the transcriptional regulator(s) transmitting this signal and whether more than one regulator is involved to define regulatory cascades and networks of coregulated genes. Helicobacter pylori is recognized as presenting a reduced number of sigma factors (the house-keeping σ80, and two alternative sigma factors σ54 and σ28) and few transcriptional regulators: NikR and Fur metalloregulators responding to nickel and iron, respectively, the carbon storage regulator CsrA, and two heat-shock regulators, HspR and HrcA. The amount of two-component regulatory systems is dramatically reduced in H. pylori as compared to the 36 such systems found in E. coli. These signal transduction systems are specialized in the transformation of environmental stimuli into transcriptional regulation and usually comprise two cognate proteins, a histidine kinase sensor and a response regulator. Apart from two orphan response regulators, HP1043 and HP1021, three complete two-component systems have been well characterized in H. pylori HP166-HP165 (AsrRS, Pflock et al., 2004, 2005, 2006a), HP1365-HP1364 (CrdRS, Waidner et al., 2005) and HP703-HP244 (FleRS, Niehaus et al., 2004), involved in the response to acidity, to copper ions and in flagellar gene expression, respectively. A few other annotated transcriptional regulators have not yet been characterized. The low number of regulators found in H. pylori is proposed to be related to its unique environmental niche and small genome size. A genome-wide regulon (all the genes regulated by a given system or regulator) can be defined by comparison of the transcriptome of a wild-type strain and that of an isogenic mutant deficient in the regulator of interest and/or with that of a strain submitted to a defined stimulus. Such an approach was used to unravel the complex transcriptional hierarchy and feedback regulation of flagellar system (Niehaus et al., 2004), in particular the role of the two sigma factors recruited for the expression of flagellar genes, σ54 and σ28 (FliA) with its cognate anti-sigma FlgM (Colland et al., 2001; Josenhans et al., 2002). These studies emphasized the complexity of this system and revealed unique features of the H. pylori system, such as the σ54- and σ28-independent expression of chemotaxis and flagellar motor genes and the absence of a true master regulator. The regulon of the nickel-responsive regulator NikR was investigated in conditions of nickel excess by comparing the response of a wild-type strain to that of a ΔnikR mutant (Contreras et al., 2003). This study revealed that NikR is a pleiotropic regulator, in contrast to its homologue of E. coli, which only represses the NikABC nickel uptake system under conditions of nickel excess (de Pina et al., 1999). In H. pylori, NikR associated to nickel becomes active as an autoregulator controlling nickel uptake (NixA permease), metabolism (structural subunits of urease, a nickel metallo-enzyme) and storage (Hpn, Hpn-like), thereby contributing to nickel homeostasis (Contreras et al., 2003). In addition, NikR also controlled the expression of stress response and flagellar genes (Contreras et al., 2003). Although NikR was thought to be a repressor, its regulon comprised both negatively and positively regulated genes. Direct binding of NikR to promoter regions of activated target genes such as ureAB confirmed its role as a nickel-responsive activator (Delany et al., 2005; Ernst et al., 2005b). Finally, an original overlap between the nickel and iron metabolism was observed as NikR not only represses the expression of the iron-responsive regulator Fur but also that of different proteins involved in iron acquisition (Contreras et al., 2003; Delany et al., 2005). The role of Fur, the iron-responsive regulator, was assessed by transcriptional profiling of both a wild-type strain 26695 and a Δfur mutant in iron-restricted and iron-replete conditions (Ernst et al., 2005a). Pairwise comparisons of these four conditions resulted in complex regulatory patterns with both genes positively or negatively regulated by Fur bound to iron or in apo-Fur form and also in a significant number of Fur-independent iron-regulated genes. Fur+Iron repressed genes comprised those involved in iron uptake and cofactor metabolism, as well as the amidases. Similar results were found with a Δfur mutant in another genetic background (strain G27; Gancz et al., 2006). The iron-free Fur-repressed genes comprised the oxidative stress response protein SodB and hydrogenase subunits. To clarify these complex iron and Fur regulatory patterns and to distinguish between direct and indirect effects, it is essential to combine both individual gene expression measurements (as for SodB; Ernst et al., 2005b or for amiE, van Vliet et al., 2003) and global Fur-DNA binding assays [the recently published Fur chromatin immunoprecipitation (Danielli et al., 2006) will be discussed below]. Among the few two-component systems of H. pylori, the HP165-HP166 attracted more attention as it was found to play a central role in the H. pylori adaptive response, in particular to acidity, and it has therefore been renamed Ars for Acid Responsive Signaling (Pflock et al., 2005). Indeed, a mutant of HP165, the ArsS histidine kinase, is unable to colonize a mouse animal model (Panthel et al., 2003) and the ArsR response regulator (HP166) of the OmpR family is essential for in vitro growth of H. pylori (Beier & Frank, 2000). Although the essential target of this system is still unknown, this suggests unusual distinct functions for ArsR in its phosphorylated and unphosphorylated forms, the latter regulating the expression of an essential gene. Two global studies were performed to identify targets of the ArsRS system, one using a DNA magnetocapture assay with bound recombinant ArsR (Dietz et al., 2002) and the other using DNA arrays with a strain deficient in ArsS (Forsyth et al., 2002). The absence of overlap between these studies emphasizes the difficulties and possible side effects of these approaches. However, recent analysis of ArsRS-mediated acid regulation by these two groups (Loh & Cover, 2006; Pflock et al., 2006a) confirmed some of the data, such as the negative autoregulation of ArsR (Dietz et al., 2002) and the regulation of the Hpn (HP1432) and arginase (HP1399) encoding genes (Forsyth et al., 2002). Role of NikR, Fur, ArsRS and CrdRS in the acid response Analysis and comparison of the numerous transcriptional profiling studies presented above revealed striking overlaps between the subset of genes differentially regulated under several environmental conditions or in mutant strains. Complex regulatory networks became apparent, in particular for the responses to acidity and metal ions. At least three transcriptional regulators were found to be involved in the response of H. pylori to acidity. The precursor work of Bijlsma et al., (2002) showed that Fur, the iron-responsive regulator was, in H. pylori, as in other bacteria (e.g. Salmonella enterica serovar Typhimurium) required for acid resistance. The involvement of NikR and ArsR (HP166) in addition to Fur as effectors of the global response of H. pylori to acidity was revealed by the comparison of our transcriptome data with those of mutants deficient in these regulators (Bury-Moné et al., 2004). For the first time, a nickel-responsive regulator, NikR, was involved in global acid response; this was discussed in a review (van Vliet et al., 2004b). To assess the role of these effectors in the acid response, several gene expression profiling experiments were performed in mutants exposed to low pH (Table 1). We found that during growth at pH 5, the amount of acid-responding genes dropped from 101 in a wild-type strain to only 36 genes in an isogenic Δfur-ΔnikR double mutant (Bury-Moné et al., 2004). In a recent study, Merrell's group used time course transcriptome analysis to dissect iron and pH regulation in H. pylori and compared acid-regulated genes in a wild type and in a Δfur mutant (Gancz et al., 2006). They obtained a list of 95 genes which were aberrantly regulated at acidic pH in the absence of Fur with a strong overlap with the previously identified acid-regulated genes. Fur-dependent acid-regulated genes comprised genes involved in ammonia production (amidase, asparaginase), detoxification (SodB and the KatA catalase), pathogenicity (proteins of the cag PAI and NapA) and transcriptional regulation (ArsR). In addition, 89% of the Fur/acid-regulated genes had been identified previously as being regulated by Fur, iron or acid. ArsR (HP166) is an OmpR-like response regulator similar to its orthologue of S. enterica known to be involved in stationary phase-dependent pH-induced acid tolerance via positive autoregulation (Bang et al., 2002). Low pH was shown to be a signal triggering the autophosphorylation of the H. pylori ArsS histidine kinase and the subsequent phosphorylation of its cognate response regulator ArsR (Pflock et al., 2004). Comparison of the transcriptome of a ΔarsS mutant vs. wild-type strain exposed to pH 5 revealed differential expression of as many as 109 genes, among which were several already identified acid-regulated genes such as those encoding urease subunits and the AmiE and AmiF amidases (Pflock et al., 2005, 2006a). For the three corresponding promoter regions, direct binding of phosphorylated ArsR protein was demonstrated. Another study on the role of two-component systems in the H. pylori acid response was published at the same time (Loh & Cover, 2006). In that work, global gene regulation in response to acidity of mutants deficient in ArsS (HP165), CrdS (HP1364) and FleS (HP244) was compared to that of the wild-type strain. No difference was observed with the FleS mutant. In contrast, of 101 analyzed acid-regulated genes, 68 and 63 were no longer responding to acidity in the HP165 and HP1364 mutants, respectively, with about 70% common genes (Loh & Cover, 2006). To make the picture more complete, one should mention negative auto-regulation of each of the three best characterized regulators, Fur, NikR and ArsRS, autorepression being stimulated by acidity for Fur and ArsRS. An additional level of complexity is given by cross-regulation; NikR negatively controls the expression of fur (Bury-Moné et al., 2004; van Vliet et al., 2004a) and vice versa (Delany et al., 2005). Furthermore, ArsR does not regulate the expression of NikR and Fur (Pflock et al., 2006a,b) but fur represses the expression of arsR in response to iron (Gancz et al., 2006; Merrell et al., 2003b), suggesting that the ArsR acid response is part of a complex regulatory network including acid- and metal-dependent regulation. Conclusions Understanding how H. pylori is able to persistently colonize its unique gastric environment has been a fascinating issue for the research community almost since its discovery. Classical genetic screens are difficult to apply to the study of H. pylori mainly because of the lack of a direct random mutagenesis system. This limitation and the young ‘age’ of this pathogen probably explain why genomics rapidly became an approach of choice to study its life cycle. A plethora of gene expression profiling studies has been published in the last 5 years (Table 1) generating a considerable amount of data, which are often difficult to merge. Although new regulators and adaptive mechanisms have started to be identified and transcriptional networks unraveled, a closer look at the data gives a rather confusing image, with the expression of a significant number of genes varying in every condition or mutant strain tested. Despite that, a common theme of these transcriptome analyses is the convincing connection of metal metabolism, acid response and virulence as testified by overlapping regulons. Accordingly, H. pylori strains deficient in each of the four transcriptional regulators involved in these responses (Fur, NikR, ArsRS, CrdRS) presented attenuated or abolished colonization capacities in mice- or gerbil-infection models (Panthel et al., 2003; Bury-Moné et al., 2004; Gancz et al., 2006). Three metal-responsive regulators are involved in the acid regulation; as metal ions are more soluble at low pH, a tempting hypothesis is that higher metal bioavailability is a signal sensed by H. pylori during acid stress (Bury-Moné et al., 2004; van Vliet et al., 2004a,b). Whether the ArsS sensor directly senses protons remains to be examined. We previously proposed a model (Bury-Moné et al., 2004) in which acidity is a ‘spatial-temporal’ signal for H. pylori, indicating its location in the stomach either in the acidic mucus or close to the neutral epithelial cells and accordingly regulating several virulence factors. The study of Schreiber. (2004) on the spatial orientation of H. pylori in the gastric mucus is nicely compatible with such a model. With the data available at present, a hierarchy in the regulatory networks of H. pylori cannot be established. Overlapping regulatory pathways are evocative of the existence of a master regulator like ComK controlling more than 100 genes involved in competence in Bacillus subtilis (Maamar & Dubnau, 2005). However, such a ‘conductor’ has not yet been identified in H. pylori. In addition, a major concern with transcriptomic studies is that they highlight both directly regulated genes and genes differentially expressed as a consequence of indirect effects. Growth phase strongly influences global gene expression in H. pylori (Merrell et al., 2003b; Thompson et al., 2003), including Fur production itself (Danielli et al., 2006) and it might well be that the different conditions tested have influenced this parameter. Direct transcriptional regulation was tested to validate the genomic data. Using electrophoresis mobility shift assays (EMSA) or DNAseI protection assays, direct binding of Fur, NikR, phosphorylated-ArsR and CrdR to the promoter region of a number of genes has been demonstrated (Delany et al., 2002, 2005; Contreras et al., 2003; van Vliet et al., 2003, 2004; Pflock et al., 2004, 2005, 2006; Ernst et al., 2005c; Waidner et al., 2005; Wen et al., 2006). Although this should constitute final proof of direct regulation, in some cases there are reservations. First, some of these experiments were performed with µM amounts of purified regulators, whereas protein–DNA binding is usually achieved at nm range concentrations. Second, despite numerous attempts, no clear Fur, NikR or ArsR binding site consensus (box) could be established which might suggest that additional parameters or cofactors are still to be identified. An interesting genome-wide location analysis of Fur binding has been published recently (Danielli et al., 2006). To globally identify the Fur-binding target, Fur chromatin immunoprecipitation (ChIP) was applied; Fur-IP-DNA pools were hybridized to H. pylori DNA-arrays. Two hundred candidate targets were first identified. Among these regions, only those also found by transcriptome analysis of wild type vs. Δfur mutant were retained. This resulted in a Fur regulon consisting of 59 genes directly regulated, 25 of which are positively regulated (Danielli et al., 2006). Global gene expression in H. pylori: what is still to be done? No study has yet reported the transcriptome of a single NikR mutant in response to acidity. In addition, ChIP strategies will be interesting to apply to the study NikR and phosphorylated ArsR, for instance, in H. pylori cells incubated under different conditions. The difficulty to define consensus binding boxes for the H. pylori regulators is intriguing. Bio-informatic approaches might help define such a binding sequence; alternatively, this might suggest an additional adaptor molecule still to be identified. Analysis of the H. pylori transcriptome during in vivo infection has never been reported and remains challenging because of small amounts of bacterial mRNA that can be extracted from human or animal biopsies. Recent advances in cDNA amplification techniques will permit this in the future. As far as adaptation is concerned, it is interesting to observe that H. pylori possessed both a short-term strategy involving co-ordinated control of gene expression, which is an immediate and reversible response affecting the entire bacterial population, as well as a long-term strategy implying genome plasticity not homogeneous among the population but that can be stabilized under external selective pressure. Finally, these studies have revealed a fascinating link between the high adaptation capacity of H. pylori and its virulence and opened a broad investigation field. Acknowledgements We would like to thank Kristine Schauer for critical reading. References Allan E. Clayton C.L. McLaren A. et al.   . ( 2001) Characterization of low-pH responses of Helicobacter pylori using genomic DNA arrays. Microbiology  147: 2285– 2292. Google Scholar CrossRef Search ADS PubMed  Alm R.A. Ling LSL Moir D.T. et al.   . ( 1999) Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature  397: 176– 180. Google Scholar CrossRef Search ADS PubMed  Ang S. Lee C.Z. Peck K. et al.   . ( 2001) Acid-induced expression in Helicobacter pylori: study in genomic scale by microarray. Infect Immun  69: 1679– 1686. Google Scholar CrossRef Search ADS PubMed  Atherton J.C. Cao P. Peek R.M. et al.   . ( 1995) Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. J Biol Chem  270: 17771– 17777. Google Scholar CrossRef Search ADS PubMed  Baar C. Eppinger M. Raddatz G. et al.   . ( 2003) Complete genome sequence and analysis of Wolinella succinogenes. Proc Natl Acad Sci USA  100: 11690– 11695. Google Scholar CrossRef Search ADS PubMed  Bang I.S. Audia J.P. Park Y.K. Foster J.W. ( 2002) Autoregulation of the ompR response regulator by acid shock and control of the Salmonella enterica acid tolerance response. Mol Microbiol  44: 1235– 1250. Google Scholar CrossRef Search ADS PubMed  Beier D. Frank R. ( 2000) Molecular characterization of two-component systems of Helicobacter pylori. J Bacteriol  182: 2068– 2076. Google Scholar CrossRef Search ADS PubMed  Bereswill S. Waidner U. Odenbreit S. et al.   . ( 1998) Structural, functional and mutational analysis of the pfr gene encoding a ferritin from Helicobacter pylori. Microbiology  144: 2505– 2516. Google Scholar CrossRef Search ADS PubMed  Bereswill S. Schönenberger R. Thies C. et al.   . ( 2000a) New approaches for genotyping of Helicobacter pylori based on amplification of polymorphisms in intergenic DNA regions and at the insertion site of the cag pathogenicity island. Med Microbiol Immunol  189: 105– 113. Google Scholar CrossRef Search ADS   Bereswill S. Greiner S. Van Vliet AHM et al.   . ( 2000b) Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori. J Bacteriol  182: 5948– 5953. Google Scholar CrossRef Search ADS   Bijlsma JJE Waidner B. Van Vliet AHM et al.   . ( 2002) The Helicobacter pylori homologue of the ferric uptake regulator is involved in acid resistance. Infect Immun  70: 606– 611. Google Scholar CrossRef Search ADS PubMed  Bizzozero G. ( 1893) Ueber die schlauchfoermigen Druesen des Magendarmkanals und die Beziehungen ihres Epithels zu dem Oberflaechenepithel der Schleimhaut. Arch Mikr Anat  42: 82. Google Scholar CrossRef Search ADS   Blaser M.J. ( 1994) Helicobacter pylori phenotypes associated with peptic ulceration. Scand J Gastroenterol  205: 1– 5. Google Scholar CrossRef Search ADS   Boneca I.G. De Reuse H. Epinat J.C. et al.   . ( 2003) A revised annotation and comparative analysis of Helicobacter pylori genomes. Nucleic Acids Res  31: 1704– 1714. Google Scholar CrossRef Search ADS PubMed  Bury-Moné S. Thiberge J.M. Contreras M. et al.   . ( 2004) Responsiveness to acidity via metal ion regulators mediates virulence in the gastric pathogen Helicobacter pylori. Mol Microbiol  53: 623– 638. Google Scholar CrossRef Search ADS PubMed  Colbeck J.C. Hansen L.M. Fong J.M. Solnick J.V. ( 2006) Genotypic profile of the outer membrane proteins BabA and BabB in clinical isolates of Helicobacter pylori. Infect Immun  74: 4375– 4378. Google Scholar CrossRef Search ADS PubMed  Colland F. Rain J.C. Gounon P. et al.   . ( 2001) Identification of the Helicobacter pylori anti-σ28 factor. Mol Microbiol  41: 477– 487. Google Scholar CrossRef Search ADS PubMed  Contreras M. Thiberge J.M. Mandrand-Berthelot M.A. Labigne A. ( 2003) Characterization of the roles of NikR, a nickel-responsive pleiotropic autoregulator of Helicobacter pylori. Mol Microbiol  49: 947– 963. Google Scholar CrossRef Search ADS PubMed  Cooke C.L. Huff J.L. Solnick J.V. ( 2005) The role of genome diversity and immune evasion in persistent infection with Helicobacter pylori. FEMS Immunol Med Microbiol  45: 11– 23. Google Scholar CrossRef Search ADS PubMed  Covacci A. Rappuoli R. ( 1998) Helicobacter pylori molecular evolution of a bacterial quasi-species. Curr Opin Microbiol  1: 96– 102. Google Scholar CrossRef Search ADS PubMed  Covacci A. Telford J.L. Del Giudice G. et al.   . ( 1999) Helicobacter pylori virulence and genetic geography. Science  284: 1328– 1333. Google Scholar CrossRef Search ADS PubMed  Danielli A. Roncarati D. Delany I. et al.   . ( 2006) In vivo dissection of the Helicobacter pylori Fur regulatory circuit by genome-wide location analysis. J Bacteriol  188: 4654– 4662. Google Scholar CrossRef Search ADS PubMed  Delany I. Spohn G. Pacheco ABF et al.   . ( 2002) Autoregulation of Helicobacter pylori Fur revealed by functional analysis of the iron-binding site. Mol Microbiol  46: 1107– 1122. Google Scholar CrossRef Search ADS PubMed  Delany I. Ieva R. Soragni A. et al.   . ( 2005) In vitro analysis of protein-operator interactions of the NikR and Fur metal-responsive regulators of coregulated genes in Helicobacter pylori. J Bacteriol  187: 7703– 7715. Google Scholar CrossRef Search ADS PubMed  De Pina K. Desjardin V. Mandrand-Berthelot M.A. et al.   . ( 1999) Isolation and characterization of the nikR gene encoding a nickel-responsive regulator in Escherichia coli. J Bacteriol  181: 670– 674. Google Scholar PubMed  De Vries N. Van Vliet AHM Kusters J.G. ( 2001) Gene regulation. Helicobacter pylori: Physiology and Genetics  ( Mobley HLT Mendz G.L. Hazell S.L., eds), pp. 321– 334. ASM Press, Washington, DC. Google Scholar CrossRef Search ADS   Dietz P. Gerlach G. Beier D. ( 2002) Identification of target genes regulated by the two-component system HP166-HP165 of Helicobacter pylori. J Bacteriol  184: 350– 362. Google Scholar CrossRef Search ADS PubMed  Eppinger M. Baar C. Raddatz G. et al.   . ( 2004) Comparative analysis of four Campylobacterales. Nat Rev Microbiol  2: 872– 885. Google Scholar CrossRef Search ADS PubMed  Eppinger M. Baar C. Linz B. et al.   . ( 2006) Who ate whom? Adaptive Helicobacter genomic changes that accompanied a host jump from early humans to large felines. PLoS Genet  2: e120. Google Scholar CrossRef Search ADS PubMed  Ernst F.D. Bereswill S. Waidner B. et al.   . ( 2005a) Transcriptional profiling of Helicobacter pylori Fur- and iron-regulated gene expression. Microbiology  151: 533– 546. Google Scholar CrossRef Search ADS   Ernst F.D. Homuth G. Stoof J. et al.   . ( 2005b) Iron-responsive regulation of the Helicobacter pylori iron-cofactored superoxide dismutase SodB is mediated by Fur. J Bacteriol  187: 3687– 3692. Google Scholar CrossRef Search ADS   Ernst F.D. Kuipers E.J. Heijens A. et al.   . ( 2005c) The nickel-responsive regulator NikR controls activation and repression of gene transcription in Helicobacter pylori. Infect Immun  73: 7252– 7258. Google Scholar CrossRef Search ADS   Falush D. Kraft C. Taylor N.S. et al.   . ( 2001) Recombination and mutation during long-term gastric colonization by Helicobacter pylori: estimates of clock rates, recombination size, and minimal age. Proc Natl Acad Sci USA  98: 15056– 15061. Google Scholar CrossRef Search ADS PubMed  Falush D. Wirth T. Linz B. et al.   . ( 2003a) Traces of human migrations in Helicobacter pylori populations. Science  299: 1582– 1585. Google Scholar CrossRef Search ADS   Falush D. Stephens M. Pritchard J.K. ( 2003b) Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics  164: 1567– 1587. Forsyth M.H. Cao P. Garcia P.P. et al.   . ( 2002) Genome-wide transcriptional profiling in a histidine kinase mutant of Helicobacter pylori identifies members of a regulon. J Bacteriol  184: 4630– 4635. Google Scholar CrossRef Search ADS PubMed  Gancz H. Censini S. Merrell D.S. ( 2006) Iron and pH homeostasis intersect at the level of Fur regulation in the gastric pathogen Helicobacter pylori. Infect Immun  74: 602– 614. Google Scholar CrossRef Search ADS PubMed  Garcia-Vallve S. Janssen P.J. Ouzounis C.A. ( 2002) Genetic variation between Helicobacter pylori strains: gene acquisition or loss? Trends Microbiol  10: 445– 447. Google Scholar CrossRef Search ADS PubMed  Gressmann H. Linz B. Ghai R. Pleissner K.P. et al.   . ( 2005) Gain and loss of multiple genes during the evolution of Helicobacter pylori. PLoS Genet  1: e43. Google Scholar CrossRef Search ADS PubMed  Gupta R.S. ( 2006) Molecular signatures (unique proteins and conserved indels) that are specific for the epsilon proteobacteria (Campylobacterales). BMC Genomics  7: 167. Google Scholar CrossRef Search ADS PubMed  Hofreuter D. Haas R. ( 2002) Characterization of two cryptic Helicobacter pylori plasmids: a putative source for horizontal gene transfer and gene shuffling. J Bacteriol  184: 2755– 2766. Google Scholar CrossRef Search ADS PubMed  Israel D.A. Salama N. Krishna U. et al.   . ( 2001) Helicobacter pylori genetic diversity within the gastric niche of a single human host. Proc Natl Acad Sci USA  98: 14625– 14630. Google Scholar CrossRef Search ADS PubMed  Janssen P.J. Audit B. Ouzounis C.A. ( 2001) Strain-specific genes of Helicobacter pylori: distribution, function and dynamics. Nucleic Acids Res  29: 4395– 4404. Google Scholar CrossRef Search ADS PubMed  Jiang Q. Hiratsuka K. Taylor D.E. ( 1996) Variability of gene order in different Helicobacter pylori strains contributes to genome diversity. Mol Microbiol  20: 833– 842. Google Scholar CrossRef Search ADS PubMed  Josenhans C. Niehus E. Amersbach S. et al.   . ( 2002) Functional characterization of the antagonist flagellar late regulators FliA and FlgM of Helicobacter pylori and their effects on the H. pylori transcriptome. Mol Microbiol  43: 307– 322. Google Scholar CrossRef Search ADS PubMed  Jungblut P.R. Bumann D. Haas G. et al.   . ( 2000) Comparative proteome analysis of Helicobacter pylori. Mol Microbiol  36: 710– 725. Google Scholar CrossRef Search ADS PubMed  Karnholz A. Hoefler C. Odenbreit S. et al.   . ( 2006) Functional and topological characterization of novel components of the comB DNA transformation competence system in Helicobacter pylori. J Bacteriol  188: 882– 893. Google Scholar CrossRef Search ADS PubMed  Kim N. Marcus E.A. Wen Y. et al.   . ( 2004) Genes of Helicobacter pylori regulated by attachment to AGS cells. Infect Immun  72: 2358– 2368. Google Scholar CrossRef Search ADS PubMed  Konjetzny G.E. ( 1923) Chronische Gastritis und Duodenitis als Ursache des Magenduodenalgeschwuers. Beitraege zur pathologischen Anatomie und allgemeinen Pathologie  71: 595– 618. Kraft C. Stack A. Josenhans C. et al.   . ( 2006) Genomic changes during chronic Helicobacter pylori infection. J Bacteriol  188: 249– 254. Google Scholar CrossRef Search ADS PubMed  Kusters J.G. Van Vliet A.H. Kuipers E.J. ( 2006) Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev  19: 449– 490. Google Scholar CrossRef Search ADS PubMed  Linz B. Balloux F. Moodley Y. et al.   . ( 2007) An African origin for the intimate association between humans and Helicobacter pylori. Nature  445: 915– 918. Google Scholar CrossRef Search ADS PubMed  Loh J.T. Cover T.L. ( 2006) Requirement of histidine kinases HP0165 and HP1364 for acid resistance in Helicobacter pylori. Infect Immun  74: 3052– 3059. Google Scholar CrossRef Search ADS PubMed  Maamar H. Dubnau D. ( 2005) Bistability in the Bacillus subtilis K-state (competence) system requires a positive feedback loop. Mol Microbiol  56: 615– 624. Google Scholar CrossRef Search ADS PubMed  Marais A. Mendz G.L. Hazell S.L. Megraud F. ( 1999) Metabolism and genetics of Helicobacter pylori: the genome era. Microbiol Mol Biol Rev  63: 642– 674. Google Scholar PubMed  Marshall B.J. Warren J.R. ( 1984) Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet  1: 1311– 1315. Google Scholar CrossRef Search ADS PubMed  Marshall B.J. Armstrong J.A. McGechie D.B. et al.   . ( 1985) Attempt to fulfil Koch's postulates for pyloric Campylobacter. Med J Aust  142: 436– 439. Google Scholar PubMed  Merrell D.S. Goodrich M.L. Otto G. et al.   . ( 2003a) pH-regulated gene expression of the gastric pathogen Helicobacter pylori. Infect Immun  71: 3529– 3539. Google Scholar CrossRef Search ADS   Merrell D.S. Thompson L.J. Kim C.C. et al.   . ( 2003b) Growth phase-dependent response of Helicobacter pylori to iron starvation. Infect Immun  71: 6510– 6525. Google Scholar CrossRef Search ADS   Niehaus E. Gressmann H. Ye F. et al.   . ( 2004) Genome-wide analysis of transcriptional hierarchy and feedback regulation in the flagellar system of Helicobacter pylori. Mol Microbiol  52: 947– 961. Google Scholar CrossRef Search ADS PubMed  Oh J.D. Kling-Backhed H. Giannakis M. et al.   . ( 2006) The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain: evolution during disease progression. Proc Natl Acad Sci USA  103: 9999– 10004. Google Scholar CrossRef Search ADS PubMed  Panthel K. Dietz P. Haas R. Beier D. ( 2003) Two-component systems of Helicobacter pylori contribute to virulence in a mouse model infection model. Infect Immun  71: 5381– 5385. Google Scholar CrossRef Search ADS PubMed  Parkhill J. Wren B.W. Mungall K. et al.   . ( 2000) The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature  403: 665– 668. Google Scholar CrossRef Search ADS PubMed  Pflock M. Dietz P. Shaer J. Beier D. ( 2004) Genetic evidence for histidine kinase HP165 being an acid sensor of Helicobacter pylori. FEMS Microbiol Lett  234: 51– 61. Google Scholar CrossRef Search ADS PubMed  Pflock M. Kennard S. Delany I. et al.   . ( 2005) Acid-induced activation of the urease promoters is mediated directly by the ArsRS two-component system of Helicobacter pylori. Infect Immun  73: 6437– 6445. Google Scholar CrossRef Search ADS PubMed  Pflock M. Finsterer N. Joseph B. et al.   . ( 2006a) Characterization of the ArsRS regulon of Helicobacter pylori, involved in acid adaptation. J Bacteriol  188: 3449– 3462. Google Scholar CrossRef Search ADS   Pflock M. Kennard S. Finsterer N. Beier D. ( 2006b) Acid-responsive gene regulation in the human pathogen Helicobacter pylori. J Biotechnol  126: 52– 60. Google Scholar CrossRef Search ADS   Salama N. Guillemin K. McDaniel T.K. et al.   . ( 2000) A whole-genome microarray reveals genetic diversity among Helicobacter pylori strains. Proc Natl Acad Sci USA  97: 14668– 14673. Google Scholar CrossRef Search ADS PubMed  Salaun L. Linz B. Suerbaum S. Saunders N.J. ( 2004) The diversity within an expanded and redefined repertoire of phase-variable genes in Helicobacter pylori. Microbiology  150: 817– 830. Google Scholar CrossRef Search ADS PubMed  Saunders N.J. Boonmee P. Peden J.F. Jarvis S.A. ( 2005) Inter-species horizontal transfer resulting in core-genome and niche-adaptive variation within Helicobacter pylori. BMC Genomics  6: 9. Google Scholar CrossRef Search ADS PubMed  Schreiber S. Konradt M. Groll C. et al.   . ( 2004) The spatial orientation of Helicobacter pylori in the gastric mucus. Proc Natl Acad Sci USA  101: 5024– 5029. Google Scholar CrossRef Search ADS PubMed  Skouloubris S. Labigne A. De Reuse H. ( 1997) Identification and characterization of an aliphatic amidase in Helicobacter pylori. Mol Microbiol  25: 989– 998. Google Scholar CrossRef Search ADS PubMed  Skouloubris S. Labigne A. De Reuse H. ( 2001) The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogs. Mol Microbiol  40: 596– 609. Google Scholar CrossRef Search ADS PubMed  Solnick J.V. Hansen L.M. Salama N.R. et al.   . ( 2004) Modification of Helicobacter pylori outer membrane protein expression during experimental infection of rhesus macaques. Proc Natl Acad Sci USA  101: 2106– 2111. Google Scholar CrossRef Search ADS PubMed  Sommi P. Ricci V. Fiocca R. et al.   . ( 1996) Significance of ammonia in the genesis of gastric epithelial lesions induced by Helicobacter pylori: an in vitro study with different bacterial strains and urea concentrations. Digestion  57: 299– 304. Google Scholar CrossRef Search ADS PubMed  Stingl K. De Reuse H. ( 2005) Staying alive overdosed: how does Helicobacter pylori control urease activity? Int J Med Microbiol  295: 307– 315. Google Scholar CrossRef Search ADS PubMed  Suerbaum S. Achtman M. ( 2004) Helicobacter pylori recombination, population structure and human migrations. Int J Med Microbiol  294: 133– 139. Google Scholar CrossRef Search ADS PubMed  Suerbaum S. Michetti P. ( 2002) Helicobacter pylori infection. N Engl J Med  347: 1175– 1186. Google Scholar CrossRef Search ADS PubMed  Suerbaum S. Smith J.M. Bapumia K. et al.   . ( 1998) Free recombination within Helicobacter pylori. Proc Natl Acad Sci USA  95: 12619– 12624. Google Scholar CrossRef Search ADS PubMed  Suerbaum S. Josenhans C. Sterzenbach T. et al.   . ( 2004) The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus. Proc Natl Acad Sci USA  100: 7901– 7906. Google Scholar CrossRef Search ADS   Thompson L. Merrell D.S. Neilan B.A. et al.   . ( 2003) Gene expression profiling of Helicobacter pylori reveals a growth-phase-dependent switch in virulence gene expression. Infect Immun  71: 2643– 2655. Google Scholar CrossRef Search ADS PubMed  Tomb J.F. White O. Kerlavage A.R. Clayton R.A. et al.   . ( 1997) The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature  388: 539– 547. Google Scholar CrossRef Search ADS PubMed  Van Vliet A.H. Kuipers E.J. Stoof J. et al.   . ( 2004a) Acid-responsive gene induction of ammonia-producing enzymes in Helicobacter pylori is mediated via a metal-responsive repressor cascade. Infect Immun  72: 766– 773. Google Scholar CrossRef Search ADS   Van Vliet AHM Bereswill S. Kusters J.G. ( 2001) Ion metabolism and transport. Helicobacter pylori: Physiology and genetics  ( Mobley H.L. Mendz G.L. Hazell S.L., ed), pp. 193– 206. ASM Press, Washington, DC. Google Scholar CrossRef Search ADS   Van Vliet AHM Stoof J. Poppelaars S.W. et al.   . ( 2003) Differential regulation of amidase- and formamidase-mediated ammonia production by the Helicobacter pylori Fur repressor. J Biol Chem  278: 9052– 9057. Google Scholar CrossRef Search ADS PubMed  Van Vliet AHM Ernst F.D. Kusters J.G. ( 2004b) NikR-mediated regulation of Helicobacter pylori acid adaptation. Trends Microbiol  12: 489– 494. Google Scholar CrossRef Search ADS   Waidner B. Melchers K. Stähler F.N. et al.   . ( 2005) The Helicobacter pylori CrdRS two-component regulation system (HP1364/HP1365) is required for copper-mediated induction of the copper resistance determinant CrdA. J Bacteriol  187: 4683– 4688. Google Scholar CrossRef Search ADS PubMed  Wen Y. Marcus E.A. Matrubutham U. et al.   . ( 2003) Acid-adaptive genes of Helicobacter pylori. Infect Immun  71: 5921– 5939. Google Scholar CrossRef Search ADS PubMed  Wen Y. Feng J. Scott D.R. et al.   . ( 2006) Involvement of the HP0165-HP0166 two-component system in expression of some acidic-pH-upregulated genes of Helicobacter pylori. J Bacteriol  188: 1750– 1761. Google Scholar CrossRef Search ADS PubMed  Wirth T. Wang X. Linz B. et al.   . ( 2004) Distinguishing human ethnic groups by means of sequences from Helicobacter pylori: lessons from Ladakh. Proc Natl Acad Sci USA  101: 4746– 4751. Google Scholar CrossRef Search ADS PubMed  © 2007 Federation of European Microbiological Societies
TI - Ten years after the first Helicobacter pylori genome: comparative and functional genomics provide new insights in the variability and adaptability of a persistent pathogen
JF - Journal of the Endocrine Society
DO - 10.1111/j.1574-695X.2007.00244.x
DA - 2007-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/ten-years-after-the-first-helicobacter-pylori-genome-comparative-and-8fXBnBY0gC
SP - 165
EP - 176
VL - 50
IS - 2
DP - DeepDyve
ER -