Transcriptional regulatory networks (TRNs) transduce environmental signals into coordinated output expression of the genome. Accordingly, they are central for the adaptation of bacteria to their living environments and in host–pathogen interactions. Few attempts have been made to describe a TRN for a human pathogen, because even in model organisms, such as Escherichia coli, the analysis is hindered by the large number of transcription factors involved. In light of the paucity of regulators, the gastric human pathogen Helicobacter pylori represents a very appealing system for understanding how bacterial TRNs are wired up to support infection in the host. Herein, we review and analyze the available molecular and “-omic” data in a coherent ensemble, including protein–DNA and protein–protein interactions relevant for transcriptional control of pathogenic responses. The analysis covers ∼80% of the annotated H. pylori regulators, and provides to our knowledge the first in-depth description of a TRN for an important pathogen. The emerging picture indicates a shallow TRN, made of four main modules (origons) that process the physiological responses needed to colonize the gastric niche. Specific network motifs confer distinct transcriptional response dynamics to the TRN, while long regulatory cascades are absent. Rather than having a plethora of specialized regulators, the TRN of H. pylori appears to transduce separate environmental inputs by using different combinations of a small set of regulators.
Citation: Danielli A, Amore G, Scarlato V (2010) Built Shallow to Maintain Homeostasis and Persistent Infection: Insight into the Transcriptional Regulatory Network of the Gastric Human Pathogen Helicobacter pylori. PLoS Pathog 6(6): e1000938. doi:10.1371/journal.ppat.1000938
Editor: Marianne Manchester, University of California San Diego, United States of America
Published: June 10, 2010
Copyright: © 2010 Danielli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was partially supported by funding from the Italian Ministry for Research (PRIN) to AD, and from the University of Bologna (ex60% and Strategic Project) to VS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Helicobacter pylori is a Gram-negative bacterium, recognized as the principal causative agent of chronic inflammation of the mucous membranes of the human stomach and of peptic ulcer worldwide , . It is one of the major risk factors for gastric neoplasia, including lymphomas and gastric cancers . Survival in the gastric niche depends on the concerted expression of virulence factors and housekeeping genes, which enables H. pylori to withstand the stresses imposed by the harsh acidic environment and to counteract the host's responses , . The restricted gastric habitat of H. pylori has been associated with reduced functional redundancy of its small genome (∼1.6 Mb), characterized by a constrained number of transcriptional regulators , . In analogy to other bacteria, the environmental signals are transduced into coordinated output expression by a transcriptional regulatory network (TRN) that can be reconstructed by describing how transcriptional regulators and target genes are interconnected in local network structures –.
TRNs commonly exhibit multilayered hierarchical structures, composed of regulatory modules (origons) that include the concerted activity of transcription factors (TFs) regulating related physiological functions . Origons therefore embody regulatory sub-networks that originate at a distinct class of sensor TFs, rooting a signal input node . Each origon comprises a master regulator, sensing a specific signal, followed by intermediate TFs and regulatory interactions that ultimately feed into output gene targets. Within or between origons, TFs and target genes are wired in specific local patterns of interconnection termed motifs (Figure 1). They enable the system to respond with specific dynamics, according to the biological nature of the triggering signal . Interestingly, it has been shown that extensive tinkering of transcriptional interactions at the local level has contributed to the evolution of prokaryotic TRNs by wiring orthologous genes in different types of motifs . In fact, these local network structures do not evolve as rigid units with fixed patterns, but tend to be conserved in organisms sharing similar living environments, regardless of their phylogenetic distance . Accordingly, identification of definite motifs and TRN organization in H. pylori may represent important findings for understanding the regulatory circuits of other human microbial pathogens.
A. Single input motif (SIM); a single transcription factor A regulates a set of operons (X, Y, Z); frequently A is autoregulated. B. multicomponent loop (MCL); transcription factor A regulates another regulator B, which in turn can regulate the transcription of A; each regulator can also control transcription of a separate set of target operons. C. Feed forward loops (FFLs) are three node motifs, occurring with high frequency in prokaryotic TRNs. A top regulator, A, which controls a second regulator, B, and a target gene, X, which is regulated by both A and B. The regulators in this motif (A, B) have an asymmetric position and hierarchy, as A regulates two targets, while B only one. According to the net signs feeding into X, FFL can be coherent (same sign) or incoherent (opposite sign), negative or positive, conferring different response kinetics  D. Bifan motif (BM); a set of operons (X, Y) is each regulated by the combination of two (A, B; BM) or multiple (A, B, C, etc.; multi input motif, not shown) transcription factors.
The H. pylori TRN
For the analysis, we have modeled the H. pylori TRN using BioTapestry software , by integrating the regulatory connections gathered from molecular, transcriptome, and ChIP-chip data available in the literature. To filter out indirect connections and pleiotropic effects, regulatory interactions were considered only for targets subjected to direct transcriptional control, defined by ascertained TF binding to regulatory elements of the target gene. We examined 685 non-redundant transcriptional regulations and identified 224 direct interactions (Dataset S1), separated according to the sensor TF serving as rooting node of the origon. The model, supplied as an interactive Java plug-in file (Model S1, Text S1), covers approximately 80% of the annotated regulators in H. pylori, a value that exceeds that reported for the most studied model organism Escherichia coli (50% coverage) , where hundreds of regulators make the dissection of the TRN difficult.
In fact, H. pylori appears to enroll only 17 bona fide TFs (Table 1), encompassed in four main origons, which process the key physiological responses needed to colonize the gastric niche: 1) heat and stress response, 2) motility and chemotaxis, 3) acid acclimation, and 4) metal ion homeostasis.
The Heat Shock and Stress Response Origon
The heat shock origon is amongst the best-understood regulatory modules in H. pylori. Whereas most Gram-negatives employ specialized sigma factors (σ32) to positively regulate the transcriptional responses to heat shock, H. pylori has evolved an opposite strategy, commonly found in Gram-positive bacteria, that implements two repressors with homology to Bacillus subtilis HrcA ,  and Streptomyces spp. HspR . Specifically, the heat shock origon of H. pylori is composed of two TFs (HspR and HrcA) directly repressing three main target operons, including the groESL chaperone genes (Figure 2A). All three operons are responsive to heat shock and are activated by the presence of misfolded proteins or stress signals , . HspR alone represses transcription of the cbpA operon, thereby negatively autoregulating its own synthesis . On the contrary, both HspR and HrcA are required for dual repression of the groESL and hrcA operons . The DNA-binding activity of both repressors is enhanced by the product of the groESL target gene , suggesting that HrcA and HspR are involved with the GroE chaperonin system in a feedback regulatory loop, complying with the B. subtilis “titration model” . This model postulates that upon heat shock, GroESL is titrated away by misfolded polypeptides, thereby relieving HspR/HrcA repression, and triggering the stress response.
A. The heat shock origon is composed of two TFs (HspR and HrcA) repressing directly three main target operons: I) the cbp operon, encoding, respectively, a DnaJ-homologue CbpA, the HspR regulator itself, and a gene product of unknown function with homology to a helicase (HP1026); II) the hrcA operon, coding for the HrcA regulator, DnaK, and the GrpE co-chaperone; and III) the groESL operon, encoding conserved GroES and GroEL chaperones. Block arrows: open reading frames. Green boxes: HrcA operators; blue boxes, HspR operators. −10 and −35 boxes are depicted by black and white squares, respectively. TF–DNA interactions and direct transcriptional control are depicted by black lines; red dotted lines represent protein–protein interactions important for feedback control of the circuit; arrowheads denote positive regulation; hammerheads indicate negative regulation. B. Incoherent type-2 FFL wiring of HspR, HrcA, and the groESL operon, responsible for the prompt derepression kinetics of Pgro upon heat shock. Red line: derepression kinetic of the intact FFL motif; blue dotted line: derepression kinetic of a mutated Pgro promoter in which the HspR operator has been deleted. The membrane association of HrcA in H. pylori  may confer additional sensory potential to the circuit, e.g., through sensing of periplasmic misfolded peptides, or need to release HrcA from the inner membrane by a stress-inducible co-factor.
Interestingly, although both TFs are required for full regulation, binding in vitro to their respective adjacent operators occurs in an independent, non-cooperative manner. A coherent explanation for two biochemically independent but functionally interconnected regulators can be evinced by the logical scheme of the heat shock response circuit (Figure 2B). In fact, the heat shock module is wired in what appears to be an exquisite example of an incoherent feed forward loop (FFL, Figure 1C). Incoherent FFLs greatly accelerate the response kinetics of a regulation cascade at the same steady state levels , . Accordingly, in wild-type H. pylori strains the Pgro promoter is promptly derepressed (<120 seconds) upon temperature shift to 42°C . In a mutant in which the binding site of HspR has been deleted, and HrcA is still functional, the derepression of the Pgro promoter is observed only after 60 minutes of temperature up-shift . These data strongly argue in favor of a bona fide incoherent type-2 FFL motif governing GroESL chaperone synthesis. This has important implications, as the altered homeostasis of chaperones appears to indirectly influence virulence gene expression, as well as the assembly/expression of the flagellar apparatus (see below). Interestingly, the incoherent type-2 FFL is absent in E. coli (0 out of 42 described FFLs) and rare in Saccharomyces cerevisiae (3/56) . Thus, the HspR/HrcA FFL may reflect evolutionary adaptation to the host environment that H. pylori encounters as a pathogen. In this light, it is interesting to recall that a similar heat shock regulation mode has been reported in Staphylococcus aureus (CstR/HrcA) .
The Flagellar Biosynthesis Module
Flagellar, chemotaxis, and motor protein encoding genes are key virulence factors in H. pylori. Their deletion results in attenuated infections in animal models –, possibly because of a failure to move in response to favorable or noxious gradients. In contrast to other well-characterized systems, the ∼40 motility genes of H. pylori are unclustered, frequently scattered within multicistronic gene operons , , . Along with other pleiotropic regulatory effects, such as polarity and DNA supercoiling , their transcription is hierarchically regulated, employing a remarkable fraction of dedicated TFs (7 out of 17, Table 1).
As for other bacteria, flagellar genes are typically positively regulated and hierarchically organized in three main classes according to their activating sigma factor , : class I encompasses gene targets transcribed by the vegetative σ80-containing RNA polymerase (RNAP), and comprises mostly flagellar regulatory genes (flgR, flgS, rpoN, flhA); class II includes specific targets of the alternative σ54 factor (RpoN), and encode components of the flagellar basal body and hook; finally, class III genes encode late flagellar structures, transcribed by σ28-(FliA)-containing RNAP. Other regulators include the FlgRS two-component system constituted by a NtrB-like cytoplasmic sensory histidine kinase (FlgS) and a NtrC-like response regulator (FlgR) –, and the CheA/CheY/CheY2 system regulating chemotactic responses , .
Notably, a master regulator similar to the enterobacterial FlhDC is absent, possibly because motility and chemotaxis are essential in vivo and need to be constitutively expressed in H. pylori. Conversely, the flagellar regulatory module adopts a short σ regulatory cascade (σ80>σ54>σ28) initiated by the housekeeping σ80 factor (Figure 3), where each σ factor activates a dedicated target gene class with single input motif (SIM) circuitry (Figure 1A). SIMs frequently occur in systems expressing gene products that form assemblies with controlled stoichiometry , and exhibit time-shifted dynamics , . Thus, the flagellar regulatory chain of H. pylori is appropriate to guarantee the correct sequential expression of early, middle, and late flagellar components. In analogy to E. coli , a putative type-1 coherent FFL with OR logic found at the bottom of the σ cascade may contribute to regulate an intermediate class of flagellar genes , and to sustain flagellar gene expression in the presence of discontinuous triggering signals. However, formal demonstration of the dynamic responses governed by this FFL has yet to be provided. In fact, despite the presence of a σ54 promoter consensus sequence upstream of the fliA gene, a direct regulation of RpoN on fliA is elusive to date , .
RNAP, RNA polymerase; phosphorylation events are indicated by a light blue circle. CheAY, two component system involved in chemotaxis; FlgS, NtrB-like cytoplasmic histidine kinase; FlgR, NtrC-like response regulator. FlgM, anti-σ28 factor. FlhA, FlhF, HP0958: accessory factors. Note sequential activation of class I, II, and III due to σ regulatory cascade. A pink triangle depicts a putative coherent FFL with OR logic proposed to control expression of intermediate class flagellar genes .
Besides TF–promoter (protein–DNA) interactions, several accessory proteins involved in post-transcriptional feedback mechanisms have been described: the anti-σ28 factor FlgM, itself transcribed as a class II gene, is involved in FliA inhibition of class III gene transcription , . In addition, components of the basal body, FlhA and FlhF, appear to modulate rpoN- and fliA-dependent transcription of middle and late flagellar genes . Moreover, the HP0958 protein described as an RpoN chaperone  also acts as an mRNA decay-promoting factor for the flaA transcript . Thus, although the exact molecular mechanisms are not fully understood, it is clear that, in analogy to the heat shock origon controlled by GroESL levels, protein–protein interactions of accessory factors with TFs are central for feedback modulation of the flagellar regulatory circuit. Ectopic regulatory inputs, deriving from external origons, also feed into the flagellar circuit (see below). In addition, the output of the HspR-HrcA module indirectly affects motility, likely through altered flagellar biosynthesis occurring in strains where the intracellular levels of chaperones are deregulated . This links the flagellar circuit with the sensing of stresses.
The Acid Acclimation Origon
The capacity to grow under the harsh acidic conditions encountered in the stomach is a distinctive feature of H. pylori and is associated with virulence , . Accordingly, the regulated expression of a dedicated set of so-called acid acclimation genes (the ure urease operon, aliphatic amidases amiE and amiF, arginase roc, etc.) allows H. pylori to keep acidity of the bacterial periplasm close to neutrality, and to maintain physiologic pH levels in the cytoplasm in the presence of urea and urease activity –. Transcription of acid acclimation operons is under control of the housekeeping σ80 factor and regulated principally by the essential acid response regulator ArsR . ArsR is autoregulated and is encoded by an operon that also encompasses the cognate transmembrane ArsS histidine kinase . It has been proposed that the signal sensed by ArsS is acidification of the periplasm, transduced through changes in protonation of several histidine residues (pKa ∼6.0) encompassed in the extracytosolic sensory domain . This stimulus triggers phosphorylation of ArsR, thereby promoting its DNA-binding activity towards a specific set of promoters , . However, whilst arsR appears to be an essential gene, strains carrying a point mutation that disrupts the phosphoacceptor site in the ArsR receiver domain, as well as arsS deletion mutants, are viable , . Despite some discrepancies in the experimental datasets of several systematic studies, possibly arising from the use of diverse strains , –, , , the emerging evidence points to the existence of distinct regulatory targets, which are controlled according to the phosphorylation status of ArsR:
- A first cluster of genes encompasses P∼ArsR-dependent target operons regulated by ArsR in a phosphorylation-dependent manner, upon mild acidification of the periplasm through ArsS signalling (omp11, carbonic anhydrase, hypA, ureAB).
- A second cluster of genes contains target operons that are regulated by more harsh acidic conditions, promoting acidification of the cytoplasm. Their regulation is P∼ArsR-dependent and phosphorylation of the regulator similarly promotes high affinity DNA binding to their promoters. However, they are not deregulated in arsS deletion mutants, and may therefore rely on a different (cytoplasmic) acid signal transducer to promote the phosphotransfer needed to activate ArsR. This group includes other genes central to the acid acclimation process such as amiE, amiF, and others.
- Finally, a third group of genes includes targets of unphosphorylated ArsR (including the arsRS operon) and whose regulation is not necessarily pH dependent. The latter group contains genes of unknown function that appear to be essential for viability.
Although a unifying consensus sequence has not been defined yet, it appears that the DNA elements recognized by either unphosphorylated or phosphorylated ArsR may be characterized by distinctive features, such as extension of the footprint and nucleotide sequence –. This substantiates the hypothesis of a bipartite ArsR regulon, which controls through distinct SIMs transcription of different sets of genes, according to the phosphorylation status of the response regulator (Figure S1). Additional complexity is provided by the position of the binding site in the target promoter, according to which ArsR can act as inducer or repressor of transcription.
Very interestingly, a recent work identified FlgS, the aforementioned cytosolic NtrB-like histidine kinase belonging to the flagellar biosynthesis module, as being also essential for survival of H. pylori at low pH . Although it is not known whether FlgS is able to trigger ArsR phosphorylation upon acidification of the cytoplasm, it may represent a good candidate as a cytosolic acid sensor feeding into the ArsR regulon.
The Metal Homeostasis Origon
In many bacterial pathogens, including H. pylori, metal starvation triggers the expression of virulence factors, which enables them to compete with the host for these essential nutrients. On the other hand, metal ions are toxic if present intracellularly in high amounts. Therefore, their homeostasis must be tightly controlled . Three systems are dedicated to this fundamental task in H. pylori: the CrdRS two-component system, the ferric uptake regulator (Fur) involved in iron homeostasis , and a homolog of the Ni-responsive NikR regulator of E. coli . Whilst the only identified genes targets of the CrdRS system appear to be involved in copper resistance , Fur and NikR have been described as pleiotropic regulators.
Fur regulates genes involved in both Fe2+ uptake – and detoxification , . Distinctively, the metal ion cofactor can act as co-repressor (holoFur repressed genes) or as inducer (apoFur repressed genes) , . Thus, the same information (presence or absence of Fe2+ in the cell) translates into two opposite transcriptional outputs. In accordance with its pleiotropic role, exemplified by the observed competitive colonization defects of fur mutants , Fur is an abundant protein and binds to ∼200 target loci in vivo, including genes coding for other regulators (rpoN, flgR, flgS, cheA, nikR) . Consequently, hundreds of genes deregulated by fur deletion have been reported, although not all appear to be direct targets of the regulator , . This suggests that Fur has a very central role in the H. pylori TRN.
On the other hand, NikR mediates regulation of Ni2+ homeostasis in the cell, central to the activity of the nickel-enzyme urease. In contrast to Fur, apoNikR is unable to bind DNA, and Ni2+ coordination at high-affinity metal binding sites drives allosteric changes promoting the DNA binding activity of holoNikR –. According to the position of the operator elements, NikR can act as a positive or negative regulator of transcription ,  (see also legend to Figure 4).
Fur regulates transcription of metal transporters and siderophores (e.g., frpB and fecA paralogues), which need to be repressed upon iron repletion –, as well as detoxifying genes that need to be promptly derepressed under the same Fe2+ replete conditions (e.g., pfr bacterioferritin and sod superoxide dismutase) , . Fur generally acts as repressor, but it may also act positively on transcription on certain genes (flaB, oor) . Similarly, NikR can act as positive or negative regulator of transcription, according to the position of the operator elements , . With respect to the promoter, binding upstream appears to induce transcription of the ureAB operon in cultures grown in Ni2+ replete cultures . Conversely, binding within the core promoter represses transcription, as shown for the fecA3 and frpB4 genes, encoding outer membrane proteins , , , the nixA permease gene , and the exbBD-tonB operon, that is involved in energization of outer membrane transport complexes involved in metal uptake , and itself part of the Fur regulon , , , . Note that both Fur and NikR are autoregulated, and that they control reciprocally their transcription levels, through direct binding to their respective promoters . Several operons such as exbBD-tonB are under the control of both Fur and NikR.
Together, Fur and NikR operate a bifan motif (BM; Figure 1D), which connects the metal responses in a horizontal and symmetric coordination logic with no obvious hierarchy (Figure 4). These motifs occur when multiple responses need to be coordinated to answer to mutual stimuli , as expected for metal ions, which play a key role in many different physiological processes. The metal homeostasis regulatory logic is further complicated by a multicomponent loop (MCL; Figure 1B), which directs reciprocal regulation of Fur and NikR . In this scenario, regulation of shared targets can switch to a FFL motif as soon as the intracellular concentrations of metals reach threshold levels to cofactor the regulator DNA binding activity of either Fur or NikR. This provides a great flexibility to the circuit, especially in virtue of the capacity of Fur to bind specific promoters also in the apo-form .
Notably, regulatory connections emanate from the metal ion homeostasis module towards TFs and target genes encompassed in all other origons, making it a central “core” processor of the H. pylori TRN. Accordingly, fur deletion mutants are impaired in their acid tolerance , , and Fur in vivo targeting of ArsR has been reported in ChIP-chip experiments , substantiating a direct (and hierarchically important) role of Fur in the regulation of acid acclimation. The same study also revealed that protein levels of Fur increase in stationary phase, suggesting that this regulator is involved in the reported growth phase–dependent regulation of genes encoding metallo-proteins and iron-trafficking factors , , . Similarly to Fur, NikR might be also involved in the regulation of several acid acclimation genes, including the urease operon , , possibly through increased bioavailability of the Ni2+ ion under low pH conditions, or through pH-responsive DNA binding activity, which has recently been reported . In addition, the heat shock response origon is linked to the metal homeostasis circuit, as the heat shock operons are deregulated in nikR deletion mutants . This deregulation is possibly due to indirect effects, arising from altered intracellular Ni2+ concentrations occurring in the nikR mutant strain that may interfere with the chaperone function of GroESL, which can also act as a nickel storage protein . However, a search for genomic targets using a NikR consensus sequence  identifies two binding sites upstream of the hspR and hrcA encoding operons, suggesting that regulation of these stress response TFs may be directly subjected to NikR transcriptional control. Finally, in analogy to Neisseria meningitidis , Fur appears also to have a positive role on a subset of targets , including genes important for chemotaxis and motility, interactions with the host (fibfibBP), or redox equilibrium (oor). This suggests an intriguing direct link between the metal response module, virulence, and the flagellar circuit, which certainly deserves further investigation.
Conclusion: A Shallow and Densely Interconnected TRN
The systematic analysis of origons strongly suggests that H. pylori adopts a shallow multilayer TRN, displaying a low level of hierarchy. The four main origons do not appear to exist as segregated regulatory modules. In fact, separate environmental inputs are interpreted by different combinations of small sets of TFs or associated proteins. For example, the flagellar FlgS histidine kinase also controls the regulation of acid response genes through ArsR. This is of broad interest, as the specificity and the biological significance of crosstalk between signal transduction pathways are vividly debated in the prokaryotic community , . Even more striking is the metal response module, which feeds into all other origons. In particular, Fur represents a key regulator, possessing the features of a regulatory “hub” in H. pylori, a role played by pleiotropic regulators such as Crp, Fnr, H-NS, and Fis in E. coli and other bacteria , .
Another peculiar feature is the prime use of negative interactions that endow the H. pylori TRN to constantly monitor the environment and to quickly respond only when a regulatory signal has to be transduced. Moreover, we notice that long regulatory cascades of sequentially expressed TFs, involved in bacterial differentiation (e.g., biofilm formation and sporulation) , are virtually absent in this TRN. Together, the data indicate that the TRN of H. pylori is unambiguously built to maintain homeostasis. It is not tailored to adapt to many environmental stimuli, and apparently not flexible enough to react to metabolic signals encountered outside of the gastric niche. Possibly, the shallow network design has evolved by selecting for particular transcriptional interactions to respond to prevalent environmental inputs, in line with studies of evolutionary dynamics of bacterial TRNs, which postulate that motif tinkering allows for specific responses .
Several important aspects concerning the TRN remain to be elucidated. Robust discriminative binding consensus motifs are missing for many H. pylori TFs. Moreover, responses to oxidative and osmotic stresses have been overlooked to date, and open questions remain concerning the regulation of virulence factors and toxins promoting the inflammatory response in the host. Certain genes, including members of the cag pathogenicity island coding for a type IV secretion system found in hypervirulent strains, are specifically expressed upon adhesion to host cells . For a comprehensive understanding of the H. pylori TRN, it will be imperative to identify the TFs that are involved in sensing the contact with the host cells. Similarly, the regulatory circuits driving transition form the exponential to the stationary phase , , as well as differentiation into and out of the coccoid form, which has been associated with the elusive epidemiology of H. pylori , , need to be identified. Poorly characterized but essential regulators, such as the atypical orphan response regulators HP1043  and HP1021 , , representing the 20% of TFs not covered by the TRN model, may contribute to these circuits. The recent finding of natural antisense transcripts and putative small non-coding RNAs in H. pylori ,  indicates that non-coding RNA regulation also may contribute to the adaptive responses of the bacterium.
In conclusion, we would like to convey the idea that the low number of transcriptional regulators, together with the considerable bulk of molecular tools and literature, set H. pylori as a very tractable model organism to dissect and characterize transcriptional network structures involved in virulence regulation and host–pathogen interactions. In addition, specific motifs that confer peculiar response dynamics (such as the incoherent type-2 FFL of the heat shock origon) can have interesting potential as regulatory building blocks in synthetic biology.
Recent papers have provided an integrated study of the proteome, the metabolic network, and the transcriptome of the human pathogen Mycoplasma pneumoniae at the systems level . In particular, Güell and colleagues demonstrated that in M. pneumoniae complex regulatory responses are maintained despite the low number of transcriptional regulators found, via extensive use of noncoding RNAs and transcriptional units that allow complex regulation mechanisms . Finally, recent work by Sharma et al.  in H. pylori discovered an unexpected wealth of antisense transcripts and small RNAs, which may act as potential regulators of cis- and trans-encoded target mRNAs. This adds to the concept that infectious agents with reduced genome sizes may be excellent model organisms in which to attempt dissection of pathogenic regulatory networks at the systems level.
Binary matrix of regulatory interactions for the main H. pylori TFs. The zipped folder includes two Excel files, one for pre-2004 versions and one for later versions.
(0.50 MB ZIP)
The acid acclimation origon. The acid acclimation origon is wired in two SIMs, controlled respectively, by the phosphorylated (P∼ArsR; dark green) or the unphosphorylated (ArsR; red) form of the response regulator. The phospho-transfer event is mediated by the transmembrane histidine kinase ArsS (light green) and may be promoted in the cytoplasm by FlgS (light blue) under harsh acidic conditions. The color code of operons reflects the respective signal transduction pathway, ArsS- or FlgS-dependent (light green and light blue, respectively), or P∼independent (red). TF-DNA interactions and direct transcriptional control are depicted by black lines; arrowheads denote positive regulation; hammerheads indicate negative regulation. Ectopic regulation of Fur and NikR, feeding in from the metal homeostasis origon, is depicted.
(0.40 MB PDF)
Zipped folder containing files for the BioTapestry Editor Java plug-in (bioTapestryEditor.jnlp) and the H. pylori TRN model (HpTRN_ModelS1.btp).
(0.02 MB ZIP)
Instructions for opening and browsing the H. pylori TRN model with BioTapestry editor and a caption for the model.
(0.03 MB DOC)
The authors wish to thank F. Agriesti, S. Romagnoli and D. Roncarati for fruitful discussions and critical reading of the manuscript.
- 1. Marshall BJ (2001) One hundred years of discovery and rediscovery of Helicobacter pylori and its association with peptic ulcer disease. In: Mobley HL, Mendz GL, Hazell SL, editors. Helicobacter pylori: physiology and genetics. Washington (D.C.): ASM Press. pp. 19–24.
- 2. Marshall BJ, Warren JR (1983) Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1: 1311–1315.
- 3. Parsonnet J, Hansen S, Rodriguez L, Gelb AB, Warnke RA, et al. (1994) Helicobacter pylori infection and gastric lymphoma. N Engl J Med 330: 1267–1271.
- 4. de Reuse H, Bereswill S (2007) Ten years after the first Helicobacter pylori genome: comparative and functional genomics provide new insights in the variability and adaptability of a persistent pathogen. FEMS Immunol Med Microbiol 50: 165–176.
- 5. Josenhans C, Beier D, Linz B, Meyer TF, Suerbaum S (2007) Pathogenomics of helicobacter. Int J Med Microbiol 297: 589–600.
- 6. Tomb JF, White O, Kerlavage AR, Clayton RA, Sutton GG, et al. (1997) The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388: 539–547.
- 7. Scarlato V, Delany I, Spohn G, Beier D (2001) Regulation of transcription in Helicobacter pylori: simple systems or complex circuits? Int J Med Microbiol 291: 107–117.
- 8. Balazsi G, Oltvai ZN (2005) Sensing your surroundings: how transcription-regulatory networks of the cell discern environmental signals. Sci STKE 2005: pe20.
- 9. Balaji S, Babu MM, Aravind L (2007) Interplay between network structures, regulatory modes and sensing mechanisms of transcription factors in the transcriptional regulatory network of E. coli. J Mol Biol 372: 1108–1122.
- 10. Babu MM, Lang B, Aravind L (2009) Methods to reconstruct and compare transcriptional regulatory networks. Methods Mol Biol 541: 163–180.
- 11. Seshasayee AS, Bertone P, Fraser GM, Luscombe NM (2006) Transcriptional regulatory networks in bacteria: from input signals to output responses. Curr Opin Microbiol 9: 511–519.
- 12. Balazsi G, Barabasi AL, Oltvai ZN (2005) Topological units of environmental signal processing in the transcriptional regulatory network of Escherichia coli. Proc Natl Acad Sci U S A 102: 7841–7846.
- 13. Alon U (2007) Network motifs: theory and experimental approaches. Nat Rev Genet 8: 450–461.
- 14. Madan Babu M, Teichmann SA, Aravind L (2006) Evolutionary dynamics of prokaryotic transcriptional regulatory networks. J Mol Biol 358: 614–633.
- 15. Madan Babu M, Balaji S, Aravind L (2007) General trends in the evolution of prokaryotic transcriptional regulatory networks. Genome Dyn 3: 66–80.
- 16. Longabaugh WJ, Davidson EH, Bolouri H (2009) Visualization, documentation, analysis, and communication of large-scale gene regulatory networks. Biochim Biophys Acta 1789: 363–374.
- 17. Schulz A, Schumann W (1996) hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes. J Bacteriol 178: 1088–1093.
- 18. Narberhaus F (1999) Negative regulation of bacterial heat shock genes. Mol Microbiol 31: 1–8.
- 19. Servant P, Mazodier P (2001) Negative regulation of the heat shock response in Streptomyces. Arch Microbiol 176: 237–242.
- 20. Spohn G, Delany I, Rappuoli R, Scarlato V (2002) Characterization of the HspR-mediated stress response in Helicobacter pylori. J Bacteriol 184: 2925–2930.
- 21. Homuth G, Domm S, Kleiner D, Schumann W (2000) Transcriptional analysis of major heat shock genes of Helicobacter pylori. J Bacteriol 182: 4257–4263.
- 22. Spohn G, Scarlato V (1999) The autoregulatory HspR repressor protein governs chaperone gene transcription in Helicobacter pylori. Mol Microbiol 34: 663–674.
- 23. Spohn G, Danielli A, Roncarati D, Delany I, Rappuoli R, et al. (2004) Dual control of Helicobacter pylori heat shock gene transcription by HspR and HrcA. J Bacteriol 186: 2956–2965.
- 24. Roncarati D, Danielli A, Spohn G, Delany I, Scarlato V (2007) Transcriptional Regulation of Stress Response and Motility Functions in Helicobacter pylori Is Mediated by HspR and HrcA. J Bacteriol 189: 7234–7243.
- 25. Mogk A, Homuth G, Scholz C, Kim L, Schmid FX, et al. (1997) The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis. Embo J 16: 4579–4590.
- 26. Mangan S, Alon U (2003) Structure and function of the feed-forward loop network motif. Proc Natl Acad Sci U S A 100: 11980–11985.
- 27. Chastanet A, Fert J, Msadek T (2003) Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria. Mol Microbiol 47: 1061–1073.
- 28. Eaton KA, Suerbaum S, Josenhans C, Krakowka S (1996) Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect Immun 64: 2445–2448.
- 29. Foynes S, Dorrell N, Ward SJ, Stabler RA, McColm AA, et al. (2000) Helicobacter pylori possesses two CheY response regulators and a histidine kinase sensor, CheA, which are essential for chemotaxis and colonization of the gastric mucosa. Infect Immun 68: 2016–2023.
- 30. Josenhans C, Suerbaum S (2002) The role of motility as a virulence factor in bacteria. Int J Med Microbiol 291: 605–614.
- 31. Alm RA, Ling LS, Moir DT, King BL, Brown ED, et al. (1999) Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397: 176–180.
- 32. Niehus E, Gressmann H, Ye F, Schlapbach R, Dehio M, et al. (2004) Genome-wide analysis of transcriptional hierarchy and feedback regulation in the flagellar system of Helicobacter pylori. Mol Microbiol 52: 947–961.
- 33. Ye F, Brauer T, Niehus E, Drlica K, Josenhans C, et al. (2007) Flagellar and global gene regulation in Helicobacter pylori modulated by changes in DNA supercoiling. Int J Med Microbiol 297: 65–81.
- 34. Macnab RM (2003) How bacteria assemble flagella. Annu Rev Microbiol 57: 77–100.
- 35. Spohn G, Scarlato V (1999) Motility of Helicobacter pylori is coordinately regulated by the transcriptional activator FlgR, an NtrC homolog. J Bacteriol 181: 593–599.
- 36. Brahmachary P, Dashti MG, Olson JW, Hoover TR (2004) Helicobacter pylori FlgR is an enhancer-independent activator of sigma54-RNA polymerase holoenzyme. J Bacteriol 186: 4535–4542.
- 37. Pereira LE, Brahmachary P, Hoover TR (2006) Characterization of Helicobacter pylori sigma54 promoter-binding activity. FEMS Microbiol Lett 259: 20–26.
- 38. Jimenez-Pearson MA, Delany I, Scarlato V, Beier D (2005) Phosphate flow in the chemotactic response system of Helicobacter pylori. Microbiology 151: 3299–3311.
- 39. Shen-Orr SS, Milo R, Mangan S, Alon U (2002) Network motifs in the transcriptional regulation network of Escherichia coli. Nat Genet 31: 64–68.
- 40. Yu H, Luscombe NM, Qian J, Gerstein M (2003) Genomic analysis of gene expression relationships in transcriptional regulatory networks. Trends Genet 19: 422–427.
- 41. Zaslaver A, Mayo AE, Rosenberg R, Bashkin P, Sberro H, et al. (2004) Just-in-time transcription program in metabolic pathways. Nat Genet 36: 486–491.
- 42. Kalir S, Mangan S, Alon U (2005) A coherent feed-forward loop with a SUM input function prolongs flagella expression in Escherichia coli. Mol Syst Biol 1: 2005 0006.
- 43. Douillard FP, Ryan KA, Caly DL, Hinds J, Witney AA, et al. (2008) Posttranscriptional regulation of flagellin synthesis in Helicobacter pylori by the RpoN chaperone HP0958. J Bacteriol 190: 7975–7984.
- 44. Josenhans C, Niehus E, Amersbach S, Horster A, Betz C, et al. (2002) Functional characterization of the antagonistic flagellar late regulators FliA and FlgM of Helicobacter pylori and their effects on the H. pylori transcriptome. Mol Microbiol 43: 307–322.
- 45. Colland F, Rain JC, Gounon P, Labigne A, Legrain P, et al. (2001) Identification of the Helicobacter pylori anti-sigma28 factor. Mol Microbiol 41: 477–487.
- 46. Pereira L, Hoover TR (2005) Stable accumulation of sigma54 in Helicobacter pylori requires the novel protein HP0958. J Bacteriol 187: 4463–4469.
- 47. Roncarati D, Spohn G, Tango N, Danielli A, Delany I, et al. (2007) Expression, purification and characterization of the membrane-associated HrcA repressor protein of Helicobacter pylori. Protein Expr Purif 51: 267–275.
- 48. Scott DR, Marcus EA, Wen Y, Oh J, Sachs G (2007) Gene expression in vivo shows that Helicobacter pylori colonizes an acidic niche on the gastric surface. Proc Natl Acad Sci U S A 104: 7235–7240.
- 49. Sachs G, Weeks DL, Melchers K, Scott DR (2003) The gastric biology of Helicobacter pylori. Annu Rev Physiol 65: 349–369.
- 50. Tsuda M, Karita M, Morshed MG, Okita K, Nakazawa T (1994) A urease-negative mutant of Helicobacter pylori constructed by allelic exchange mutagenesis lacks the ability to colonize the nude mouse stomach. Infect Immun 62: 3586–3589.
- 51. Weeks DL, Gushansky G, Scott DR, Sachs G (2004) Mechanism of proton gating of a urea channel. J Biol Chem 279: 9944–9950.
- 52. Scott DR, Marcus EA, Weeks DL, Sachs G (2002) Mechanisms of acid resistance due to the urease system of Helicobacter pylori. Gastroenterology 123: 187–195.
- 53. Pflock M, Kennard S, Finsterer N, Beier D (2006) Acid-responsive gene regulation in the human pathogen Helicobacter pylori. J Biotechnol 126: 52–60.
- 54. 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.
- 55. Pflock M, Dietz P, Schar J, Beier D (2004) Genetic evidence for histidine kinase HP165 being an acid sensor of Helicobacter pylori. FEMS Microbiol Lett 234: 51–61.
- 56. Pflock M, Kennard S, Delany I, Scarlato V, Beier D (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.
- 57. Wen Y, Feng J, Scott DR, Marcus EA, Sachs G (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.
- 58. Forsyth MH, Cao P, Garcia PP, Hall JD, Cover TL (2002) Genome-wide transcriptional profiling in a histidine kinase mutant of Helicobacter pylori identifies members of a regulon. J Bacteriol 184: 4630–4635.
- 59. Merrell DS, Goodrich ML, Otto G, Tompkins LS, Falkow S (2003) pH-regulated gene expression of the gastric pathogen Helicobacter pylori. Infect Immun 71: 3529–3539.
- 60. Pflock M, Finsterer N, Joseph B, Mollenkopf H, Meyer TF, et al. (2006) Characterization of the ArsRS regulon of Helicobacter pylori, involved in acid adaptation. J Bacteriol 188: 3449–3462.
- 61. Wen Y, Feng J, Scott DR, Marcus EA, Sachs G (2009) The pH-responsive regulon of HP0244 (FlgS), the cytoplasmic histidine kinase of Helicobacter pylori. J Bacteriol 191: 449–460.
- 62. Giedroc DP, Arunkumar AI (2007) Metal sensor proteins: nature's metalloregulated allosteric switches. Dalton Trans 3107–3120.
- 63. Bereswill S, Lichte F, Vey T, Fassbinder F, Kist M (1998) Cloning and characterization of the fur gene from Helicobacter pylori. FEMS Microbiol Lett 159: 193–200.
- 64. van Vliet AH, Poppelaars SW, Davies BJ, Stoof J, Bereswill S, et al. (2002) NikR mediates nickel-responsive transcriptional induction of urease expression in Helicobacter pylori. Infect Immun 70: 2846–2852.
- 65. Waidner B, Melchers K, Stahler FN, Kist M, Bereswill S (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.
- 66. Delany I, Pacheco AB, Spohn G, Rappuoli R, Scarlato V (2001) Iron-dependent transcription of the frpB gene of Helicobacter pylori is controlled by the Fur repressor protein. J Bacteriol 183: 4932–4937.
- 67. van Vliet AH, Stoof J, Vlasblom R, Wainwright SA, Hughes NJ, et al. (2002) The role of the Ferric Uptake Regulator (Fur) in regulation of Helicobacter pylori iron uptake. Helicobacter 7: 237–244.
- 68. Danielli A, Romagnoli S, Roncarati D, Costantino L, Delany I, et al. (2009) Growth phase and metal-dependent transcriptional regulation of the fecA genes in Helicobacter pylori. J Bacteriol 191: 3717–3725.
- 69. Bereswill S, Greiner S, van Vliet AH, Waidner B, Fassbinder F, et al. (2000) Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori. J Bacteriol 182: 5948–5953.
- 70. Ernst FD, Homuth G, Stoof J, Mader U, Waidner B, et al. (2005) Iron-responsive regulation of the Helicobacter pylori iron-cofactored superoxide dismutase SodB is mediated by Fur. J Bacteriol 187: 3687–3692.
- 71. Delany I, Spohn G, Rappuoli R, Scarlato V (2001) The Fur repressor controls transcription of iron-activated and -repressed genes in Helicobacter pylori. Mol Microbiol 42: 1297–1309.
- 72. Carpenter BM, Gancz H, Gonzalez-Nieves RP, West AL, Whitmire JM, et al. (2009) A single nucleotide change affects fur-dependent regulation of sodB in H. pylori. PLoS One 4: e5369.
- 73. Gancz H, Censini S, Merrell DS (2006) Iron and pH homeostasis intersect at the level of Fur regulation in the gastric pathogen Helicobacter pylori. Infect Immun 74: 602–614.
- 74. Danielli A, Roncarati D, Delany I, Chiarini V, Rappuoli R, et al. (2006) In vivo dissection of the Helicobacter pylori Fur regulatory circuit by genome-wide location analysis. J Bacteriol 188: 4654–4662.
- 75. Ernst FD, Bereswill S, Waidner B, Stoof J, Mader U, et al. (2005) Transcriptional profiling of Helicobacter pylori Fur- and iron-regulated gene expression. Microbiology 151: 533–546.
- 76. Abraham LO, Li Y, Zamble DB (2006) The metal- and DNA-binding activities of Helicobacter pylori NikR. J Inorg Biochem 100: 1005–1014.
- 77. Benanti EL, Chivers PT (2007) The N-terminal arm of the Helicobacter pylori Ni2+-dependent transcription factor NikR is required for specific DNA binding. J Biol Chem 282: 20365–20375.
- 78. Zambelli B, Danielli A, Romagnoli S, Neyroz P, Ciurli S, et al. (2008) High-affinity Ni2+ binding selectively promotes binding of Helicobacter pylori NikR to its target urease promoter. J Mol Biol 383: 1129–1143.
- 79. Contreras M, Thiberge JM, Mandrand-Berthelot MA, Labigne A (2003) Characterization of the roles of NikR, a nickel-responsive pleiotropic autoregulator of Helicobacter pylori. Mol Microbiol 49: 947–963.
- 80. Ernst FD, Kuipers EJ, Heijens A, Sarwari R, Stoof J, et al. (2005) The nickel-responsive regulator NikR controls activation and repression of gene transcription in Helicobacter pylori. Infect Immun 73: 7252–7258.
- 81. Delany I, Ieva R, Soragni A, Hilleringmann M, Rappuoli R, 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.
- 82. Bijlsma JJ, Waidner B, Vliet AH, Hughes NJ, Hag S, et al. (2002) The Helicobacter pylori homologue of the ferric uptake regulator is involved in acid resistance. Infect Immun 70: 606–611.
- 83. van Vliet AH, Stoof J, Poppelaars SW, Bereswill S, Homuth G, et al. (2003) Differential regulation of amidase- and formamidase-mediated ammonia production by the Helicobacter pylori fur repressor. J Biol Chem 278: 9052–9057.
- 84. Merrell DS, Thompson LJ, Kim CC, Mitchell H, Tompkins LS, et al. (2003) Growth phase-dependent response of Helicobacter pylori to iron starvation. Infect Immun 71: 6510–6525.
- 85. Choi YW, Park SA, Lee HW, Kim DS, Lee NG (2008) Analysis of growth phase-dependent proteome profiles reveals differential regulation of mRNA and protein in Helicobacter pylori. Proteomics 8: 2665–2675.
- 86. van Vliet AH, Ernst FD, Kusters JG (2004) NikR-mediated regulation of Helicobacter pylori acid adaptation. Trends Microbiol 12: 489–494.
- 87. Li Y, Zamble D (2009) The pH-responsive DNA-binding activity of Helicobacter pylori NikR. Biochemistry.
- 88. Kansau I, Guillain F, Thiberge JM, Labigne A (1996) Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA). Mol Microbiol 22: 1013–1023.
- 89. Delany I, Rappuoli R, Scarlato V (2004) Fur functions as an activator and as a repressor of putative virulence genes in Neisseria meningitidis. Mol Microbiol 52: 1081–1090.
- 90. Laub MT, Goulian M (2007) Specificity in two-component signal transduction pathways. Annu Rev Genet 41: 121–145.
- 91. Skerker JM, Perchuk BS, Siryaporn A, Lubin EA, Ashenberg O, et al. (2008) Rewiring the specificity of two-component signal transduction systems. Cell 133: 1043–1054.
- 92. Martinez-Antonio A, Janga SC, Thieffry D (2008) Functional organisation of Escherichia coli transcriptional regulatory network. J Mol Biol 381: 238–247.
- 93. Kim N, Marcus EA, Wen Y, Weeks DL, Scott DR, et al. (2004) Genes of Helicobacter pylori regulated by attachment to AGS cells. Infect Immun 72: 2358–2368.
- 94. Cellini L, Allocati N, Angelucci D, Iezzi T, Di Campli E, et al. (1994) Coccoid Helicobacter pylori not culturable in vitro reverts in mice. Microbiol Immunol 38: 843–850.
- 95. Eaton KA, Catrenich CE, Makin KM, Krakowka S (1995) Virulence of coccoid and bacillary forms of Helicobacter pylori in gnotobiotic piglets. J Infect Dis 171: 459–462.
- 96. Delany I, Spohn G, Rappuoli R, Scarlato V (2002) Growth phase-dependent regulation of target gene promoters for binding of the essential orphan response regulator HP1043 of Helicobacter pylori. J Bacteriol 184: 4800–4810.
- 97. Pflock M, Bathon M, Schar J, Muller S, Mollenkopf H, et al. (2007) The orphan response regulator HP1021 of Helicobacter pylori regulates transcription of a gene cluster presumably involved in acetone metabolism. J Bacteriol 189: 2339–2349.
- 98. Muller S, Pflock M, Schar J, Kennard S, Beier D (2007) Regulation of expression of atypical orphan response regulators of Helicobacter pylori. Microbiol Res 162: 1–14.
- 99. Xiao B, Li W, Guo G, Li B, Liu Z, et al. (2009) Identification of small noncoding RNAs in Helicobacter pylori by a bioinformatics-based approach. Curr Microbiol 58: 258–263.
- 100. Xiao B, Li W, Guo G, Li BS, Liu Z, et al. (2009) Screening and identification of natural antisense transcripts in Helicobacter pylori by a novel approach based on RNase I protection assay. Mol Biol Rep 36: 1853–1858.
- 101. Ochman H, Raghavan R (2009) Systems biology. Excavating the functional landscape of bacterial cells. Science 326: 1200–1201.
- 102. Güell M, van Noort V, Yus E, Chen WH, Leigh-Bell J, et al. (2009) Transcriptome complexity in a genome-reduced bacterium. Science 326: 1268–1271.
- 103. Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, et al. (2010) The primary transcriptome of the major human pathogen Helicobacter pylori. Nature 464: 250–255.
- 104. Ernst FD, Stoof J, Horrevoets WM, Kuipers EJ, Kusters JG, et al. (2006) NikR-mediates nickel-responsive transcriptional repression of the Helicobacter pylori outer membrane proteins FecA3 (HP1400) and FrpB4 (HP1512). Infect Immun 6821–6828.
- 105. Davis GS, Flannery EL, Mobley HL (2006) Helicobacter pylori HP1512 is a nickel-responsive NikR-regulated outer membrane protein. Infect Immun 74: 6811–6820.
- 106. Wolfram L, Haas E, Bauerfeind P (2006) Nickel represses the synthesis of the nickel permease NixA of Helicobacter pylori. J Bacteriol 188: 1245–1250.
- 107. Schauer K, Gouget B, Carriere M, Labigne A, de Reuse H (2007) Novel nickel transport mechanism across the bacterial outer membrane energized by the TonB/ExbB/ExbD machinery. Mol Microbiol 63: 1054–1068.