Novel nickel transport mechanism across the bacterial outer membrane energized by the TonB/ExbB/ExbD machinery

Novel nickel transport mechanism across the bacterialouter membrane energized by the TonB/ExbB/ExbDmachinery

Kristine Schauer,1 Barbara Gouget,2 Marie Carrière,2

Agnès Labigne1 and Hilde de Reuse1*1Unité de Pathogénie Bactérienne des Muqueuses,Département de Microbiologie, Institut Pasteur, 75724Paris Cedex 15, France.2Laboratoire Pierre Süe, CEA-CNRS UMR 9956,CEA/Saclay, 91191 Gif-sur-Yvette, France.

Summary

Nickel is a cofactor for various microbial enzymes, yetas a trace element, its scavenging is challenging. In thecase of the pathogen Helicobacter pylori, nickel isessential for the survival in the human stomach,because it is the cofactor of the important virulencefactor urease. While nickel transport across the cyto-plasmic membrane is accomplished by the nickelpermease NixA, the mechanism by which nickeltraverses the outer membrane (OM) of this Gram-negative bacterium is unknown. Import of iron-siderophores and cobalamin through the bacterial OMis carried out by specific receptors energized by theTonB/ExbB/ExbD machinery. In this study, we show forthe first time that H. pylori utilizes TonB/ExbB/ExbD fornickel uptake in addition to iron acquisition. We haveidentified the nickel-regulated protein FrpB4, homolo-gous to TonB-dependent proteins, as an OM receptorinvolved in nickel uptake. We demonstrate that ExbB/ExbD/TonB and FrpB4 deficient bacteria are unable toefficiently scavenge nickel at low pH. This conditionmimics those encountered by H. pylori duringstomach colonization, under which nickel supply andfull urease activity are essential to combat acidity. Weanticipate that this nickel scavenging system is notrestricted to H. pylori, but will be represented morelargely among Gram-negative bacteria.

Introduction

Nickel is an essential nutrient for microorganisms as it isthe cofactor of at least nine enzymes involved in several

cellular processes such as energy and nitrogen metabo-lism, detoxification and virulence (Mulrooney andHausinger, 2003). The link between nickel and virulence isexemplary in the case of the pathogen Helicobacter pylorithat colonizes about half of the world population andcauses a variety of gastric diseases ranging from gastritisto adenocarcinoma (Atherton, 2006). To colonize thehuman stomach, an acidic environment, H. pylori requiresa constant supply of nickel ions for the activity of theintracellular urease that accounts for up to 6% of thesoluble cellular proteins (Hu and Mobley, 1990) and binds24 nickel ions per active complex (Ha et al., 2001).Urease catalyses the hydrolysis of urea into carbondioxide and ammonia, which are buffering compoundsessential to maintain the pH homeostasis in the bacterialcytoplasm (Stingl et al., 2002). However, to avoid nickeloverload that generates reactive oxygen species andleads to cell toxicity, its intracellular concentration needsto be strictly controlled with sensing, transport and pro-tective mechanisms. H. pylori is equipped with severalproteins involved in these processes, including NikR, anickel responsive pleiotropic transcriptional regulator (VanVliet et al., 2002a; Contreras et al., 2003; Dian et al.,2006), three H. pylori-specific proteins potentially involvedin sequestering of nickel ions, namely Hpn (Gilbert et al.,1995; Ge et al., 2006), Hpn-like and a histidine-rich HspAprotein (Kansau et al., 1996) as well as metal effluxpumps such as CznABC (Stahler et al., 2006). InH. pylori, nickel transport through the cytoplasmic mem-brane is mediated by NixA, a high-affinity and low-capacity nickel transporter (Mobley et al., 1995). NixA isan eight-transmembrane-domain protein that belongs to afamily of prokaryotic and fungal nickel/cobalt secondarytransporters (NiCoTs; TC 2.A.52; Kt = 11.3 � 2.4 nM;Vmax = 1750 � 220 pmol Ni2+ min-1 10-8 cells). Its expres-sion is regulated by NikR (Ernst et al., 2005b; Wolframet al., 2006). As a NixA-deficient mutant retains 50% ofurease activity in unsupplemented medium (Bauerfeindet al., 1996) and a NixA-deficient mutant is still able tocolonize the mouse model (Nolan et al., 2002) severalproteins have been suggested to be additionally involvedin nickel uptake (Hendricks and Mobley, 1997; Davis andMobley, 2005). However, none of them was confirmed toplay a major role in nickel transport.

Accepted 18 December, 2006. *For correspondence.E-mail [email protected]; Tel. (+33) 1 4061 3641; Fax(+33) 1 4061 3640.

Molecular Microbiology (2007) 63(4), 1054–1068 doi:10.1111/j.1365-2958.2006.05578.xFirst published online 18 January 2007

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd

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While the transport of nickel across the cytoplasmicmembrane is well characterized, very little is known abouthow nickel traverses the outer membrane of Gram-negative bacteria to reach the periplasmic space. Lowmolecular mass solutes are thought to diffuseconcentration-gradient-driven through porin channels thatare present in the outer membrane. However, high-affinitybinding to a specific outer membrane receptor protein andactive transport is needed for substrates that are eitherpoorly permeable through the porins or are encounteredat very low concentrations. The energy required for theiraccumulation into the periplasmic space derives from theproton motive force mediated by the inner membraneprotein complex composed of TonB, ExbB and ExbD(Braun and Braun, 2002; Ferguson and Deisenhofer,2004). This system has been associated with the uptakeof cobalamin and iron complexes, as the bioavailability offree cobalt and iron is very low in most cases. Indeed atneutral pH, Fe2+ is rapidly oxidized into insoluble ferric iron(Fe3+), which can only be acquired in complexed form byTonB-mediated transport (Wandersman and Delepelaire,2004). Considering the fact that nickel is only a traceelement in the human body (Hopfer et al., 1989; Sunder-man, 1993), various reported observations led us to askwhether a TonB-energized system might be involved inhigh-affinity nickel uptake in the case of H. pylori.

First, transcriptome analyses and protein–DNA interac-tion assays demonstrated that the tricistronic exbB/exbD/tonB operon (hp1339/1340/1341) coding for the energytransducing machinery is regulated by both the iron andnickel responsive regulators Fur and NikR (Contreraset al., 2003; Merrell et al., 2003; Bury-Moné et al., 2004;Delany et al., 2005; Ernst et al., 2005a). While control ofthis operon by Fur is expected, the additional regulationby NikR suggested a possible overlap between the ironand nickel transport mechanisms across the outermembrane. Noticeable the exbB/exbD/tonB operon com-poses a divergent locus with the gene encoding the NikRprotein.

Second, considering the reduced size of the H. pylorigenome the number of proteins that were annotated to beinvolved in iron transport across the outer membrane issurprising (Tomb et al., 1997). The genome sequence ofH. pylori 26695 shows several orthologues of differentiron-scavenging systems including three outer membraneFe3+ dicitrate receptors (FecA: HP0686, HP0807 andHP1400), three ferric siderophores receptors (FrpB:HP0876, HP0916/5 and HP1512), two periplasmic bindingproteins (CeuE: HP1562 and HP1561) as well as threeorthologues of ExbB/ExbD and two of TonB. These latterproteins are encoded either by a tricistronic operon (exbB/exbD/tonB: hp1339/1340/1341) or in two pairs of sequen-tial genes of exbB/exbD (hp1445/1446 and hp1130/1129)and a separated TonB-like encoding gene (hp0582). Most

interestingly, the expression of a subset of these genesnamely fecA3 (hp1400), frpB4 (hp1512) and ceuE1(hp1561), did not reveal any regulation upon changes iniron concentration and lacked regulation by the ferricuptake regulator Fur (Van Vliet et al., 2002b). On thecontrary, we previously demonstrated that these geneswere regulated by NikR and nickel (Contreras et al., 2003).

Third, Velayudhan et al. (2000) reported that the high-affinity ferrous iron (Fe2+) transporter FeoB (HP0687)rather than the TonB-dependent receptors, was the majorsystem for H. pylori iron acquisition. Indeed, the feoBmutant was strongly defective in import of both forms ofiron Fe2+ and Fe3+ suggesting the coupling of this systemwith a Fe3+ reductase activity. This central role of FeoB iniron uptake was reinforced by the inability of a deficientmutant to colonize the mouse stomach.

In the present work, we show that the TonB/ExbB/ExbD(HP1341/1339/1340) machinery of H. pylori is necessaryfor iron uptake during growth at neutral pH. In addition, theTonB/ExbB/ExbD system is dedicated to nickel accumu-lation at low pH, allowing the bacterium to activate ureaseand to combat acidity. Finally, we report the identificationof an outer membrane receptor, FrpB4 (HP1512), whichpossibly functions in conjunction with the TonB/ExbB/ExbD machinery to transport nickel through the outermembrane. This is the first report on a substrate otherthan iron complexes and cobalamin that is transported bya TonB-mediated mechanism.

Results

Requirement of the TonB/ExbB/ExbD(HP1341/1339/1340) machinery of H. pylori for irontransport

To investigate the function of the TonB/ExbB/ExbD(HP1341/1339/1340) machinery in H. pylori, the entireoperon was targeted in two strains 26695 and X47-2AL.Attempts to delete the three genes and replace them by acassette carrying a non-polar kanamycin-resistance geneusing the standard selection procedure on blood agarplates remained unsuccessful in both strains. As TonB/ExbB/ExbD usually mediates iron import and becauseiron deficiency is lethal for bacteria, we speculated thatthe iron concentration in the selective plates might not becompatible with viability of the deletion mutant. Thus,selection after transformation was performed on platessupplemented with 500 mM FeCl3 in order to provide theFeoB uptake system with sufficient iron. In these condi-tions, deletion mutants of the entire exbB/exbD/tonBoperon could be obtained at high frequencies in bothgenetic backgrounds (Table 1). Growth of H. pylori 26695DexbB/exbD/tonB mutants was monitored in liquid Bru-cella Broth medium containing 0.2% b-cyclodextrin (BBb)

TonB-dependent nickel uptake 1055

© 2007 The AuthorsJournal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1054–1068

supplemented with up to 2000 mM FeCl3 by measuringoptical density (OD) after 14 h. Growth of the DexbB/exbD/tonB mutant increased in an iron concentration-dependent manner to reach wild-type growth at 200 mMFeCl3 (Fig. 1A). Growth of the wild-type strain but not ofthe DexbB/exbD/tonB mutant was inhibited at 2000 mMFeCl3, demonstrating that the DexbB/exbD/tonB mutant isless susceptible to the toxicity of high iron concentrations.Complementation of the DexbB/exbD/tonB mutant wasachieved by reinsertion of the entire exbB/exbD/tonBsequence into the original locus using the

chloramphenicol-resistance cassette for selection. TheexbB/exbD/tonB-complemented strain presented growthbehaviour similar to that of the wild-type strain (Fig. 1A,Table 1). At pH 5, growth of the deletion mutant wassimilar to that of the wild-type strain, possibly due tohigher concentration of Fe2+ under this condition andtransport by FeoB (Fig. 1B). This showed for the first timethat the TonB/ExbB/ExbD machinery is required for irontransport in H. pylori.

To determine whether each gene of the tricistronicoperon was required for iron import, exbB (hp1339), exbD

Table 1. Growth at neutral pH under standard conditions or with FeCl3 supplementation of all strains used in the study.

Strainhp number of thedeleted genes Genotype

Growth instandardconditions

Growth withadded iron

26695 Wild type + +X47-2AL Wild type + +26695 DexbB/D/tonB 1339/1340/1341 D(exbB/exbD/tonB)WKm – +X47-2AL DexbB/D/tonB 1339/1340/1341 D(exbB/exbD/tonB)WKm – +26695 C-exbB/exbD/tonB 1339/1340/1341 D(exbB/D/tonB)WexbB/D/tonB-Cm + +26695 DexbB 1339 DexbBWKm – +26695 DexbD 1340 DexbDWKm – +26695 DtonB 1341 DtonBWKm – +26695 DexbB/exbD 1339/1340 D(exbB/exbD)WKm – +26695 DexbD/tonB 1340/1341 D(exbD/tonB)WKm – +26695 exbB-TAP exbB-TAP-Km + +26695 DfrpB4 1512 DfrpB4WCm + +

The wild-type and mutant strains utilized are indicated in the first column, the numbers of H. pylori-deleted open reading frames as assigned in26695 strain ( Tomb et al., 1997) in the second column and the resulting genotypes are indicated in the two last columns. Bacteria were growneither on standard blood agar plates or on blood agar plates supplemented with 0.5 mM FeCl3 for a period of 4 days. +, growth; –, no growth.

Fig. 1. Growth of the H. pylori wild-type strain (black bars), DexbB/exbD/tonB mutant strain (light grey bars) and theexbB/exbD/tonB-complemented strain (dark grey bars) in medium supplemented or not with Fe3+ (FeCl3) at pH 7 (A) and at pH 5 (B). Allcultures were inoculated at an initial OD of 0.05 and OD was measured after 14 h growth. Error bars represent the standard deviation of threeindependent experiments.

1056 K. Schauer et al.


(hp1340) and tonB (hp1341) were replaced individually orin combination of pairs in strain 26695 by a non-polarkanamycin cassette. Each of the individual deletions andall the combinations resulted in a dependency on addediron for growth suggesting a decrease in iron uptake(Table 1). Thus, all three proteins of the tricistronic operonwere specifically required for the TonB-dependent irontransport under these conditions.

Strong nickel and weak iron dependent changes inprotein levels of the ExbB/ExbD/TonB complex inH. pylori

The expression of the genes encoding the TonB/ExbB/ExbD machinery that we found to be involved in iron uptakewere reported to be under the control of Fur and in additionto be regulated by nickel and NikR (Contreras et al., 2003;Bury-Moné et al., 2004; Delany et al., 2005; Ernst et al.,2005a). We wanted to further investigate the regulation ofthis operon by nickel and iron ions by monitoring theirexpression at the protein level. Therefore, exbB (hp1339),the first gene of the tricistronic exbB/exbD/tonB operon,was C-terminally fused with a tandem affinity purification(TAP) tag that can be detected with a specific antibody. TheTAP tag sequence (Rigaut et al., 1999) was inserted infront of a non-polar chloramphenicol selection cassetteand introduced into H. pylori strain 26695 by homologousrecombination at the original exbB locus. Growth of theexbB-tagged strain was compared with that of the wild type

in the presence and absence of iron overload and wasfound to be similar indicating that the function of theTonB/ExbB/ExbD system was not affected by the tag(Table 1). The exbB-TAP containing strain was grown for14 h in BBb in the presence of NiCl2, nickel dicitrate, FeCl3or ferric dicitrate at increasing concentrations (NiCl2 at 1,20, 100 mM; nickel dicitrate at 1, 20, 100 mM; FeCl3 at 200,1000, 2000 mM; ferric dicitrate at 100, 200, 300 mM). Thehighest ion concentrations used corresponded to the upperlimit at which cell viability was not strongly affected ingrowth assays. ExbB protein levels were analysed byimmunoblotting (using the anti-PA antibody directedagainst the tag region of the fusion protein) of equalamounts of crude extracts from cells harvested aftergrowth under each condition (Fig. 2). Consistent with pre-vious transcriptional analyses, the amount of ExbB proteinwas diminished during growth with added nickel (Fig. 2A)or iron (Fig. 2B). However, the effect of nickel was signifi-cantly stronger than that of iron with almost completereduction of ExbB synthesis at sublethal nickel concentra-tions. These results suggested that in H. pylori the TonB/ExbB/ExbD machinery has a major role in the nickelhomeostasis in addition to its function in iron uptake.

Reduced nickel accumulation in the DexbB/exbD/tonBmutant

The strong nickel-dependent regulation of theexbB/exbD/tonB operon prompted us to ask whether

Fig. 2. Western blots of H. pylori bacteriaexpressing the ExbB-tagged protein grownunder neutral pH at increasing concentrationsof nickel ions (A) and iron ions (B). Undereach blot, Coomassie-stained gels are shownas loading controls. The predicted MW of theExbB-TAP is 33 kDa. The data are fromindividual assays, but are representative ofthree independent experiments. MWstandards are indicated on the left-hand sideof the blot.



this system might be additionally involved in nickeluptake. Cellular nickel concentrations of the wild-type, theDexbB/exbD/tonB mutant and the exbB/exbD/tonB-complemented strains were measured by inductivelycoupled plasma mass spectrometry (ICP-MS). We analy-sed exponentially growing bacteria (6 H) in BBb mediumat pH 7 or pH 5 without added nickel or in the presence of1 or 5 mM NiCl2. The unsupplemented BBb medium wasfound to contain 0.8–1.2 mM nickel, a value in the samerange as previously measured (0.2 mM total nickel)(Wolfram et al., 2006). Under these conditions the cellspresented a low basal nickel content (9.7 � 1.8 mmol g-1

proteins). Addition of nickel to the growth medium resultedin increased nickel accumulation in the wild-type strain(Fig. 3, black bars). Interestingly, nickel accumulation wasstrongly enhanced when bacteria were grown at pH 5 ascompared with pH 7. This enhancement of intracellularnickel at pH 5 was 1.8-fold in unsupplemented medium,threefold in medium with 1 mM NiCl2 and 3.6-fold with5 mM NiCl2. At neutral pH, the nickel content of the DexbB/exbD/tonB mutant was not significantly different from thatof wild-type cells, besides a slight reduction in nickelcontent at 5 mM NiCl2 (Fig. 3A). In contrast, the DexbB/exbD/tonB mutant grown at pH 5 was severely impaired inits capacity to accumulate nickel, with 1.6 times less intra-cellular nickel than the wild-type strain during growth inunsupplemented medium, 5.1 times less with 1 mM NiCl2and 9.3 times less with 5 mM NiCl2 (Fig. 3A, light greybars). Accordingly, the nickel content of the DexbB/exbD/

tonB mutant under every nickel concentration at pH 5 waslower than the respective one at pH 7 in contrast to whatwas observed with the wild-type strain. In both conditions,the nickel content of the DexbB/exbD/tonB mutant slightlyincreased at increasing nickel concentrations. This sug-gested that low amounts of nickel could be accumulatedindependently of the TonB/ExbB/ExbD machinery andthat this occurred predominantly at neutral pH.

The exbB/exbD/tonB-complemented strain recovered anickel accumulation capacity that was comparable to thatof the wild-type strain in all the conditions tested (Fig. 3A,dark grey bars). The presence of 1 mM nitrilotriacetic acid(NTA) in the acidic growth medium that chelates nickeland other divalent ions prevented the strong nickel accu-mulation for every strain (Fig. 3A). These results demon-strated that the DexbB/exbD/tonB mutant is impaired in itscapacity to accumulate nickel ions at pH 5 suggesting thatH. pylori uses a TonB-dependent transport system fornickel uptake under acidic conditions.

The outer membrane protein, FrpB4 (HP1512) isrequired for nickel accumulation

As the TonB/ExbB/ExbD machinery provides energy forthe transport through a specific outer membrane receptor,we searched for a nickel-regulated outer membraneprotein (OMP). Previous studies demonstrated that theOMP FrpB4 (HP1512) annotated to be involved in ferriciron uptake is solely regulated by nickel and NikR (Van

Fig. 3. Nickel content of H. pylori wild-type or isogenic mutants grown at pH 5 or pH 7 without nickel, with increasing NiCl2 concentrations(1 mM and 5 mM) or in medium supplemented with 1 mM NTA. The nickel content was measured in cell lysates using ICP-MS and expressedin mmol of nickel per g of proteins. Measurements were performed in triplicates.A. Nickel content of the wild-type strain (black bars), of the DexbB/exbD/tonB mutant (light grey bars), and the complemented exbB/exbD/tonBstrain (dark grey bars).B. Nickel content of the wild-type strain (black bars) and of the DfrpB4 mutant (grey bars). Note that both panels show the same data for thewild-type strain.



Vliet et al., 2002b; Contreras et al., 2003). An isogenicDfrpB4 mutant was constructed in H. pylori strain 26695replacing the monocistronic frpB4 open reading frame bya chloramphenicol-resistance cassette (Table 1). As theFrpB4 protein was predicted to be involved in iron uptake,the DfrpB4 mutant was tested for growth on sole ironsources that are known to support H. pylori growth ashaemin, haemoglobin, human lactoferrin and ferric dici-trate (Dhaenens et al., 1999). H. pylori is not known to useother siderophores. Under each condition tested, growthof the DfrpB4 mutant did not differ from that of the wild-type strain, revealing that FrpB4 is not essential underiron-limited conditions (Table 1 and data not shown).Alternatively, we asked whether FrpB4 could be involvedin nickel transport by measuring the ability of the DfrpB4mutant to accumulate nickel. Bacteria were grown for 6 hat pH 7 or pH 5 in BBb medium without or with 1 or 5 mMNiCl2, and intracellular nickel content was measured byICP-MS. At pH 7, the DfrpB4 mutant accumulated slightlyless nickel than the wild-type strain; however, this differ-ence was not significant (Fig. 3B). In contrast, at pH 5 theDfrpB4 mutant presented a strong reduction in nickelaccumulation as compared with the wild-type strain underthe same conditions (Fig. 3B). Intracellular nickel contentof the DfrpB4 mutant was 3.2 times less in unsupple-mented medium, 6.2 times less with 1 mM NiCl2 and 6.8times less with 5 mM NiCl2 as compared with the wild-typestrain (Fig. 3B). The reduction in nickel accumulation atpH 5 of the DfrpB4 mutant was equivalent to that mea-sured with the DexbB/exbD/tonB mutant (Fig. 3A and B).Similarly to the DexbB/exbD/tonB mutant, intracellularnickel content of the DfrpB4 mutant was lower at pH 5than at pH 7, and this residual nickel accumulation wasconcentration-dependent. These results showed that theFrpB4 protein, in addition to the ExbB/ExbD/TonBcomplex, is involved in nickel accumulation at pH 5.

The DexbB/exbD/tonB and the DfrpB4 mutants aredeficient in nickel transport

To test whether the diminished nickel content of theDexbB/exbD/tonB and the DfrpB4 mutant strains wasassociated with reduced nickel uptake, we directly mea-sured transport of radioactive 63NiCl2 in whole bacteria.First the test conditions were established using differentbuffers and growth media. To maintain full bacterial viabil-ity at both low and neutral pH, we measured transport inBBb medium like in the nickel accumulation assays pre-sented above. Exponentially growing bacteria were equili-brated for 15 min in BBb medium adjusted to pH 5 orpH 7. Uptake was initiated by addition of 63NiCl2 to a finalconcentration of 50 nM in the presence of 5 mM unlabelledNiCl2. Samples were taken at different time pointsbetween 30 s and 15 min, vacuum filtrated, washed in the

presence of cold NiCl2 to eliminate unspecific binding andthe radioactivity on the filter was counted. We usedlysates of wild-type cells instead of whole cells as acontrol, and we observed the absence of unspecific nickelbinding to cell components (Fig. 4, grey diamonds). AtpH 5, wild-type bacteria accumulated 195 pmol 63Ni per gprotein in the first minute and reached 835 pmol 63Ni per gprotein after 15 min (Fig. 4, black diamonds). Consistentlywith the nickel content measurement presented above,the uptake rate of the wild-type strain was significantlylower at pH 7, being 71 pmol 63Ni per g protein after 1 minand 128 pmol 63Ni per g protein after 15 min (Fig. 4, whitediamonds). A DnixA mutant did not show any transportactivity under acidic conditions (Fig. 4, open square).Similarly, no nickel transport was measured with theDexbB/exbD/tonB (Fig. 4, open circles) and the DfrpB4(Fig. 4, open triangles) mutant strains, as radioactivenickel remained at a background level similar to that of theDnixA mutant. Complementation of the exbB/exbD/tonBoperon restored transport activity to wild-type levels(Fig. 4, black circles). These results demonstrated that theTonB/ExbB/ExbD machinery and FrpB4 accomplishnickel transport at pH 5.

FrpB4 presents the characteristics of a TonB-dependenttransporter

FrpB4 is a 97.4 kDa protein that was located at the cellsurface of H. pylori by proteinase K digestion experiments(Sabarth et al., 2005). Sequence analysis of FrpB4

Fig. 4. 63Ni uptake experiments in whole bacteria. Wild-typebacteria were measured at pH 5 (black diamonds) or pH 7(white diamonds). The measurement at pH 5 is shown for theDexbB/exbD/tonB mutant (open circles), the complementedexbB/exbD/tonB strain (black circles), the DfrpB4 mutant (opentriangles), the DnixA mutant (open squares) as well as for wild-typelysates (grey diamonds), a control of unspecific binding.Measurements were performed in duplicates and are representativefor two independent experiments. Error bars represent the standarddeviation.



(Fig. 5) predicted (i) a signal peptide of 21 residues (1–21,grey letters), (ii) a 104-residue-long N-terminal TonB-plugdomain (residues 37–141, italic letters) and (iii) a TonB-dependent receptor domain from residue 600 to theC-terminus of the protein. The HHpred program thatsearches for homologous proteins with known structuresidentified the six crystallized TonB-dependent outer mem-brane transporters as proteins with the highest similarity.The prediction of the secondary structure of FrpB4 isrepresented in Fig. 5. The plug domain is predicted tocontain the conserved four stranded b-sheets hb1-hb4and two amphipathic a-helices ha1 and ha2. Thesequence GKVTTKG between residue 29 and 35resembles the TonB-box (Fig. 5, underlined), similar to theGVTTLGK motif in the pyochelin receptor (FptA fromPseudomonas aeruginosa), both presenting the function-ally important b-strand. For the rest of the protein, anti-parallel b-strands that are separated by alternating shortturns and long loops are predicted. Such a structureresembles that of a pore-like transmembrane b-barreldomain. In TonB-dependent receptors, short turns areknown to connect the b-strands in the periplasmic spaceand long loops to protrude from the bacterial surface inorder to catch the substrate. Aromatic residues present atthe edges of the b-strands are highlighted (Fig. 5, redletters). Notably, the predicted FrpB4 barrel domain issignificantly longer than that of the crystallized outermembrane iron transporters. Therefore, prediction of theb-strand topology in the region between residues 330–

550 is ambiguous and the prediction of unusual long loopsbetween b-strands 7–8 and/or 9–10 needs to be con-firmed experimentally. Taken together, our data and thisstructure analysis converge to establish that FrpB4 is anew type of TonB-dependent outer membrane receptorthat transports nickel.

Effect of reduced nickel transport on urease activity

H. pylori predominantly utilizes nickel as the cofactor ofthe metalloenzyme urease, which is essential to resistacidity and to colonize the host stomach. Urease activityis known to be strongly enhanced at pH 5, a conditionmimicking the environment of the gastric mucus, as com-pared with that measured for bacteria grown at neutral pH(Bury-Moné et al., 2004; van Vliet et al., 2004). As TonB/ExbB/ExbD and FrpB4 were found to be involved in nickeluptake, we decided to examine how mutations in therespective genes are reflected in urease activity underdifferent conditions and hence may influence virulence.

When grown at neutral pH in the absence of addednickel, urease activity of the DexbB/exbD/tonB mutant didnot significantly differ from that of the wild-type strainunder the same condition (Fig. 6A). Urease activity of theDfrpB4 mutant grown at pH 7 was 0.7 � 0.3 U, whichcorresponds to a twofold reduction in activity as comparedwith the wild-type strain (1.9 � 0.4 U) (Fig. 6A). Supple-mentation of the growth medium with 1 or 5 mM NiCl2 ledto a dose-dependent increase in urease activity in the

Fig. 5. Secondary structure prediction of the FrpB4 protein. The amino acids of the predicted signal peptide are indicated in grey letters andthose of the TonB-plug domain in italics. The PSIPRED prediction of helixes is represented by green barrels and the prediction of b-strands asyellow arrows. Blue bars show the confidence of the prediction. The assigned TonB-box GKVTTKG is underlined and the conserved fourstranded b-sheets hb1-hb4 and two amphipathic a-helices ha1 and ha2 of the plug, as well as the 22 antiparallel b-strands are labelled.Aromatic residues at the edges of predicted b-strands are in red. H (helix), E (strand), C (coil).



wild-type as well as in the DexbB/exbD/tonB and theDfrpB4 mutant strains, and the slight reduction in ureaseactivity of the DfrpB4 mutant disappeared (Fig. 6A). Lowernickel concentrations had no effect on urease activity (notshown). These results were consistent with the nickelaccumulation tests presented above (Fig. 3) and demon-strated that at neutral pH the DexbB/exbD/tonB andDfrpB4 mutants could accumulate nickel in a TonB-independent manner and, hence, could increase ureaseactivity.

During growth at pH 5 in the absence of added nickel(Fig. 6B), urease activity of the wild-type strain increased14.4-fold to reach 27.4 � 3.6 U consistently with previousobservations (Bury-Moné et al., 2004; van Vliet et al.,2004). On the contrary, the DexbB/exbD/tonB mutantshowed a basal urease activity at pH 5 (2.9 � 0.5 U) thatwas 9.5 times lower than that of the wild-type strain. Eachof the individual mutants of the exbB/exbD/tonB operonwere tested and showed comparable low urease activity(data not shown). Complementation of the exbB/exbD/tonB operon restored the urease activity to wild-typelevels (32.3 � 6.7 U). Urease activity of the DfrpB4mutant (1.7 � 0.5 U) was comparable to that of theDexbB/exbD/tonB strain, hence 16 times lower that that ofthe wild type. The effect of nickel ions was investigated bysupplementing the acidic growth medium with 1 or 5 mMNiCl2. In the presence of 1 mM NiCl2 urease activityincreased in all strains, however, being six to seven timesless in the DexbB/exbD/tonB and DfrpB4 mutant strainsthan in the wild-type strain. In the presence of 5 mM NiCl2,all strains reached comparable levels of urease activity,suggesting saturation of urease activity under acidic con-ditions with higher nickel supplementation. To testwhether urease activity was specifically enhanced bynickel ions at low pH, we performed the same assay on

cells grown in acidic BBb that was pre-treated with anickel-specific complex former dimethylglyoxime (DMG)or supplemented with 5 mM NTA. Under nickel free con-ditions, acid activation of urease in the wild-type strainwas strongly reduced (Fig. 6B). These data demonstratedthat the TonB/ExbB/ExbD machinery and the OMP FrpB4are needed for urease activity at low pH and low nickelconcentrations.

Discussion

In this study we have described the specific function of theTonB/ExbB/ExbD machinery in H. pylori (HP1341/1339/1340) in both iron and nickel uptake. Our data suggestthat depending on the environmental pH, the TonB systemis used according to the needs for iron and nickel ofH. pylori and the bioavailability of each metal ion. Further-more, we have identified the OMP FrpB4 (Hp1512) as aprobable TonB-dependent nickel receptor. We proposethat H. pylori accumulates nickel from nickel poor, acidicenvironments by a TonB energized mechanism that issimilar to the iron siderophore uptake. As nickel is a traceelement in the human stomach, this uptake system isessential for nickel-dependent urease activity of thispathogen.

Dual role of the TonB/ExbB/ExbD (HP1341/1339/1340)system in iron and nickel uptake

The role of TonB/ExbB/ExbD in iron uptake in H. pyloriwas first questioned when a tonB (hp1341) deletionmutant of H. pylori strain NCTC 11637 was shown topresent wild-type transport capacity for iron (Velayudhanet al., 2000). In contrast to those results we showed in thisstudy that deletion of the exbB/exbD/tonB operon

Fig. 6. Urease activity of H. pylori wild-type and isogenic mutants grown under different conditions. Strains were wild-type (black bars),DexbB/exbD/tonB mutant (light grey bars), DfrpB4 mutant (dark grey bars), and complemented exbB/exbD/tonB strain (white patterned bars).A. Growth medium at pH 7 without nickel supplementation or with increasing NiCl2 concentrations (1 mM and 5 mM).B. Growth medium at pH 5, without nickel or with increasing NiCl2 concentrations (1 mM and 5 mM), DMG or NTA indicate that the medium wastreated with DMG or supplemented with NTA. Note that the scales of the two panels are different. Error bars represent the standard deviationof three independent experiments.



(hp1339/1340/1341) in strains 26695 and X47-2ALresulted in strong iron starvation, demonstrating theinvolvement of this system in iron uptake. Testing strain26695, the same phenotype was observed with individualmutants of each gene of the operon, including tonB, indi-cating that there is no cross-complementation by either ofthe two paralogous copies of exbB (hp1445 and hp1130)and exbD (hp1129 and hp1446) or by the predicted TonB-like gene (hp0582). This demonstrated the specificrequirement of the entire tricistronic exbB/exbD/tonBoperon in iron uptake under these conditions. The contra-dictory phenotype observed for the DtonB mutant in thestrain NCTC 11637 (Velayudhan et al., 2000) may arisefrom redundancy of the ExbB/ExbD/TonB systems in thisstrain, for which genomic sequence data are notavailable. Alternatively, the respective TonB/ExbB/ExbDsystem of NCTC11637 might not be involved in ironuptake but only in nickel uptake (see below).

In addition to iron uptake, we demonstrated that theTonB/ExbB/ExbD system is required for nickel transport inH. pylori, using measurements of cellular nickel contentand 63Ni transport assays. Notably, TonB-dependentnickel uptake was unambiguously observed at pH 5.Under this condition, nickel uptake was stronglyenhanced in the wild-type strain as compared with pH 7.At neutral pH, the contribution of the TonB/ExbB/ExbDmachinery for nickel uptake is questionable, because theDexbB/exbD/tonB mutant behaved like the wild-typestrain. Further studies including analysis of the role of theparalogous ExbB, ExbD and TonB-like proteins will beconducted to better understand the nickel uptake mecha-nism at neutral pH.

FrpB4, a novel outer membrane receptor involved innickel uptake

We showed in this study that the DfrpB4 mutant wasstrongly deficient in nickel accumulation and nickel trans-port activity at pH 5. Notably, the behaviour of the DfrpB4mutant was very similar to that of a TonB/ExbB/ExbDdeficient strain. Sequence analysis revealed that FrpB4presents the characteristics of a TonB-dependent outermembrane receptor. Two very recent studies analysed insome details the previously reported NikR and nickel-dependent regulation of FrpB4 by real-time quantitativePCR and NikR–DNA interaction studies, and showed thatfrpB4 promoter was directly regulated by nickel-boundNikR (Davis et al., 2006; Ernst et al., 2006). Davis et al.report some hints of decreased intracellular nickel con-centration in a DfrpB4 mutant as the deregulation of somenickel-related genes and, similar to us, a reduction ofurease activity at pH 7 (Davis et al., 2006). This indirectstudy supports our results from direct measurement ofnickel transport and nickel accumulation. Considering the

outer membrane localization of FrpB4 (Sabarth et al.,2005; Ernst et al., 2006), the homology to other TonB-dependent transporters and the similarity of the pheno-types of the DfrpB4 and DexbB/exbD/tonB mutants, wepropose that FrpB4 transports nickel across the outermembrane in conjunction with the TonB/ExbB/ExbDmachinery.

Within the epsilon proteobacteria, FrpB4 homologuesare present in the H. pylori strains J99 and HPAG1 and inthe closely related Helicobacter acinonychis species(strain Sheeba) but are absent in the non-gastric patho-gens Helicobacter hepaticus, Campylobacter jejuni andthe non-pathogenic bacterium Wolinella succinogenes,for which complete genomic sequences are available(Alm et al., 1999; Baar et al., 2003; Suerbaum et al.,2003; Eppinger et al., 2006). The high degree of conser-vation between the homologues (90% identical aminoacids on the entire length of the open reading frames,JHP1405, HPGAG1400 and HAC0072 in strains J99,HPAG1 and Sheeba respectively) strongly suggests anidentical function in nickel uptake.

The role of TonB-dependent nickel transport is reflectedin urease activity

According to its central role in acid resistance, ureaseactivity is tightly controlled in a complex manner (Stingland De Reuse, 2005; Pflock et al., 2006). Urease geneexpression is regulated transcriptionally by NikR (Delanyet al., 2005; Ernst et al., 2005b; Abraham et al., 2006) andthe two-component acid responsive system ArsRS (Pflocket al., 2005). In addition, urease activity is controlled post-transcriptionally by the amount of bioavailable nickel andthe incorporation of this ion into the apo-urease throughthe action of accessory proteins (reviewed in Stingl andDe Reuse, 2005). As nickel ions affect urease both tran-scriptionally and post-transcriptionally, we examined howreduced nickel accumulation in the DexbB/exbD/tonB andDfrpB4 mutants influenced urease activity.

At neutral pH, urease activity of the DexbB/exbD/tonBand DfrpB4 mutants did not differ from the wild-typestrain besides a twofold reduction in the DfrpB4 mutantwithout added nickel. This slight reduction in the DfrpB4mutant is consistent with a role in nickel scavengingunder low nickel concentration. In contrast, urease activ-ity of the DexbB/exbD/tonB mutant strains did not differfrom that of the wild type. This might indicate that underneutral conditions, FrpB4 is partially energized by theparalogues of ExbB, ExbD, TonB. Additionally, alteredgene regulation in the DexbB/exbD/tonB mutant due toits strong iron deficiency may modify nickel uptake.Supplementation of the growth medium with nickelincreased urease activity in a TonB/ExbB/ExbD- andFrpB4-independent manner, in agreement with a minor



role of TonB-dependent nickel transport at neutral pH.Importantly, these results showed that the TonB/ExbB/ExbD machinery or the FrpB4 protein did not perturbateeither the synthesis of urease or the incorporation ofnickel into urease apo-enzyme.

At pH 5, the TonB/ExbB/ExbD system and FrpB4were required for acid activation of urease. In theDexbB/exbD/tonB and DfrpB4 mutants, diminishedurease activity correlates with less nickel accumulationindicating that acid activation of urease is mainly nickel-dependent. Accordingly, chelation of nickel in the growthmedium was found to strongly reduce acid activation ofurease in the wild-type strain. Our results showed thatthe nickel bioavailability for H. pylori is enhanced atacidic pH, a condition under which full urease activity iscrucial and which mimics the stomach environment.Supplementation of the growth medium with 5 mM nickelions led to a saturation of urease activity in all strainssimilar to that observed at pH 7. Interestingly, the levelof nickel-saturated urease activity was higher at pH 7than at pH 5 (87.2 U versus 40.8 U for the wild-typestrain) probably reflecting the stress that the bacteriumencounters at pH 5 that leads to an overall reduction inprotein synthesis and an altered energy homeostasis(Bauerfeind et al., 1997).

Central role of TonB in the adaptation of H. pylori to thespecific gastric environment

Based on our data we propose a model, in which theTonB/ExbB/ExbD machinery is the central element in theadaptation of H. pylori to the fluctuating needs for nickeland iron during the frequent pH changes of the gastricenvironment (Fig. 7). At pH 7, the insoluble Fe3+ form ispredominating and the TonB/ExbB/ExbD machinery isessential to acquire comlexed iron across the outermembrane. After binding to a periplasmic binding protein,Fe3+ passes the cytoplasmic membrane through an ABC-transporter (such as FecD/E). Under this condition, theTonB system does not play a major role in nickel uptake,hence, nickel is weakly accumulated and urease activity isbasal. At pH 5, Fe2+ becomes the predominant ironsource, enters the periplasm by diffusion through non-selective porins and is transported across the cytoplasmicmembrane by the FeoB transporter. At this low pH, theTonB/ExbB/ExbD system accomplishes nickel uptakethrough the outer membrane receptor FrpB4. From theperiplasmic space nickel can by transported by the NixApermease or another not yet identified transporter. In thecytoplasm, nickel is incorporated into urease leading to itsactivation that maintains the intracellular pH homeostasis.

Fig. 7. Model for the ExbB/ExbD/TonB function in H. pylori. At pH 7, at environmental low nickel concentrations, the TonB complex is mainlyused for transport of ferric iron (Fe3+) bound to a siderophore (blue box) across the outer membrane (OM). After binding to a periplasmicbinding protein (PBP), Fe3+ passes the cytoplasmic membrane (CM) through an ABC-transporter (such as FecD/E). Nickel is not accumulatedat pH 7, hence, urease activity is basal. At pH 5, ferrous iron (Fe2+) becomes the predominant iron source, passes the OM by diffusion throughporins and is taken up across the CM by the FeoB transporter. Under these conditions, TonB-dependent transport is used for the uptakethrough the OM of nickel that is complexed to a postulated nickelophore (pink box). In the periplasmic space (PP), nickel might bind to aspecific protein and then be transported across the CM by the NixA permease or another inner membrane transporter. In the cytoplasm, nickelis incorporated into urease leading to its activation. On the right side of the figure, conditions of nickel overload result in passive diffusion ofnickel through porins followed by transport across the IM either via NixA or another metal transporter ultimately leading to enhanced ureaseactivity.



The utilization of the TonB/ExbB/ExbD system in accor-dance with the pH-dependent needs for iron and nickelenables H. pylori to adapt to the frequent changes of thegastric environment.

Hypothetical nickel-specific metallophores

The concentration of nickel in the human serum is verylow (3–10 nM; Hopfer et al., 1989; Sunderman, 1993).Indeed, the stomach was found to be the organ with thelowest nickel body burden in the mouse model (Nielsenet al., 1993). In the human body, nickel is not likely to existin the free ionic form (Ni2+) but rather occurs in complexwith proteins such as albumin and nickeloplasmin or withsmall organic molecules such as polypeptides andL-histidine (Van Soestbergen and Sunderman, 1972; Osk-arsson and Tjalve, 1979). Therefore, nickel uptake froman environment in which the effective free nickel concen-tration is low might require a nickel-specific chelatingagent. By analogy to the iron siderophore, we postulatethe existence of a nickel-binding metallophore tentativelynamed nickelophore. The existence of a nickel-specificsiderophore has been proposed recently from crystalstructure analyses of NikA (Cherrier et al., 2005). NikA isthe periplasmic nickel-binding protein of the NikABCDEhigh-affinity nickel transporter belonging to the ABC trans-porter family first characterized in E. coli (Wu andMandrand-Berthelot, 1986) with no homologue inH. pylori. Careful analyses of the bound NikA ligand sug-gested that NikA does not coordinate Ni2+ directly butrequires a metallophore that displays significant structuralsimilarities to EDTA. Indeed, a metallophore specific fornickel that is induced by low pH could serve as an ampli-fier of the subtle chemical differences among differentmetal ions and activate urease when it is needed.

Nickel TonB-dependent uptake system probably notunique to H. pylori

In an elegant study, Rodionov et al. (2006) performed alarge-scale in silico comparative genomic analyses tosearch for prokaryotic nickel and cobalt uptake transport-ers in 200 prokaryotic genomes including proteobacteriaand archaea. Based on genomic linkage to genes encod-ing NikR regulators and nickel metalloenzymes or nickelpermeases as well as the presence of regulatory ele-ments, they identified genes coding for uncharacterizedOMPs similar to the TonB-dependent receptors inBradyrhizobium japonicum, Dechloromonas aromatica,Oligotropha caboxidovorans pHCG3, Rhodopseudomo-nas palustris and Rubrivivax gelatinosus. They hypoth-esized that these OMPs might serve in TonB-dependentnickel transport across the outer membrane (Rodionovet al., 2006). Our data correlate with this prediction and

imply that TonB-dependent nickel transport might be alargely distributed mechanism to acquire nickel.

In conclusion, our data indicate that H. pylori importsnickel by a TonB/ExbB/ExbD-dependent mechanismthrough the OMP FrpB4. This is the first report on anickel-specific, energy-dependent uptake system throughthe bacterial outer membrane. We anticipate that thissystem is not restricted to H. pylori but will be representedmore largely among the Gram-negative bacteria possiblydepending on the nickel availability in their ecologicalniche.

Experimental procedures

Bacterial strains and growth conditions

The H. pylori strains employed in this study were 26695(Tomb et al., 1997) and X47-2AL (Ermak et al., 1998) as wellas their respective mutants (Table 1). H. pylori was grown onblood agar base 2 (Oxoid) plates supplemented with 10%defibrinated horse blood and an antibiotics/fungicide cocktailconsisting of vancomycin (12.5 mg ml-1), polymyxin B(0.31 mg ml-1), trimethoprim (6.25 mg ml-1) and fungizone(2.5 mg ml-1). Kanamycin (30 mg ml-1) or chloramphenicol(30 mg ml-1) were added for selection when needed. Forgrowth in liquid medium, Brucella broth (Difco) supplementedwith 0.2% b-cyclodextrin (Sigma) plus the antibiotic/fungicidecocktail was used, this medium (BBb) was adjusted to eitherpH 7 or 5 (by adding HCl). The total iron content of the BBbmedium was estimated to be 14–20 mM (Bereswill et al.,2000; Bijlsma et al., 2002). To follow growth of H. pylorimutant strains, liquid medium containing the indicated NiCl2(Sigma) or FeCl3 (Sigma) concentrations was inoculated atan initial OD of 0.05 with a liquid preculture without addedmetal. Iron and nickel dicitrate were prepared by combiningsodium citrate (Sigma) and FeSO4 or NiSO4 in a 100:1 molarratio. In order to grow bacteria on sole iron sources BBb agarwas supplemented with 15 mM deferoxamine mesylate (des-feral, Sigma) and one of the following iron sources: 5 mMhaemin, 10 mM haemoglobin, 50 mg ml-1 human lactoferrinand 200 mM ferric dicitrate (all from Sigma). The nickel-specific complex former DMG (log K(Ni) = 14.6, Sigma) wasused to deplete the medium from nickel as described previ-ously (Scott et al., 2002), and the chelator NTA (Sigma,logK(Ni) = 11.54; logKFe(II) = 8.84; logKFe(III) = 15.87) was usedat the indicated final concentrations. After 14 h incubation,growth was monitored by measuring the OD at 600 nm. Allgrowth experiments were performed at least in triplicates.Plates and flasks were incubated under microaerobic condi-tions (Campygen gas pack, Oxoid) at 37°C. Escherichia colistrain MC1061 (Casadaban and Cohen, 1980) grown at 37°Con solid or liquid Luria–Bertani medium (Miller, 1992) wasused for subcloning and as a host for the preparation of theplasmids employed to transform H. pylori.

Molecular techniques and construction of H. pylorimutant strains

DNA manipulations were carried out following standard pro-cedures (Sambrook and Russel, 2001). Chromosomal DNA



of H. pylori strain 26695 was used as a template for all PCRamplifications and the primers are listed in Table S1. PCRwas carried out according to the manufacturer’s recommen-dations using the Expand High Fidelity DNA Polymerase Kit(Roche). DNA cassettes to target specific genomic loci ofH. pylori were constructed following two strategies. TheDfrpB4 mutant, the exbB-TAP fusion strain and the exbB/exbD/tonB complementation strain were constructed using athree-step PCR procedure adapted from Derbise et al.(2003). In a first step, the H. pylori genomic DNA was used toamplify the 500 bp fragments flanking the target sequencewith primer pairs UpF/UpR and DownF/DownR. For eachpair, one primer contained at its 5′ end a 20 bp regionhomologous to the extremity of one of the antibiotic resis-tance cassette, either aphA-3� (Skouloubris et al., 1998) or achloramphenicol-resistance gene derivative (pCM4 Car-tridge, Pharmacia) (Table S1). In the case of exbB-TAP theTAP tag from pBS1479 (Rigaut et al., 1999) was inserted infront of the antibiotic resistance cassette to allow a C-terminaltagging of a target protein. For the exbB/exbD/tonB comple-mentation strain the full-length exbB/exbD/TonB sequence(1600 bp) was utilized as 5′ region. The two flanking frag-ments were mixed with the antibiotic resistance cassette inan equimolar ratio and the resulting product was amplifiedwith the UpF/DownR primers. This PCR product was eitherused directly for transformation experiments or subclonedinto vector pUC18 (Yanisch-Perron et al., 1985). For all exbB,exbD, tonB single, double and triple null mutants, the respec-tive 5′ regions were amplified and the products were clonedwith EcoRI/SacI upstream of the aphA-3 cassette intopUC18. The respective 3′ region was amplified and clonedwith BamHI/PstI downstream the aphA-3� cassette. MidiQiagen columns were employed for large-scale plasmidpreparations. H. pylori mutants were obtained by naturaltransformation as previously described (Bury-Mone et al.,2003) with approximately 2 mg of plasmid DNA or PCRproduct. Clones that had undergone allelic exchange wereselected after 4–5 days of growth on plates containing theappropriate antibiotic. Genomic DNA of the recombinantH. pylori mutants was isolated using QIAamp DNA Mini Kit(Qiagen). All insertions into the chromosome were verified byPCR and sequenced with an ABI 310 automated DNAsequencer (Perkin-Elmer).

Immunoblotting

Immunoblotting was performed on equal amounts of crudeextracts (sonicates) according to standard protocols. Theprotein amounts of the crude extracts were calibrated with theBradford Assay (Bio-Rad) using BSA as a standard. TheH. pylori-tagged protein (exbB-TAP) was specifically detectedwith a protein A-specific peroxidase coupled a-peroxidaseantibody (Sigma) and was detected using the ECL reagent(Pierce).

Nickel content measurements by ICP-MS

Overnight cultures of H. pylori bacteria were diluted to an ODof 0.5 in fresh BBb medium without added nickel or with 1 or5 mM NiCl2 each adjusted to pH 7 or pH 5. Bacteria were

incubated for 6 H, then 6 ml of culture was centrifuged at400 g at 4°C for 5 min through 0.3 ml of a 1:2 mixture of thesilicone oils AR20/AR200 (SPCI) in order to separate thecells from the medium. Cells were lysed with 400 ml 0.2 MNaOH/1% SDS for 60 min at 90°C. Protein contents werecalibrated with standard series of cell cultures using the Brad-ford Assay for protein determination (Bio-Rad). The sampleswere acidified with ultrapure 65% nitric acid (Normatomquality grade; Prolabo) and diluted in ultrapure water to 2%.Nickel concentrations were measured by ICP-MS using anX7 series quadrupole instrument (Thermo Electron Corpora-tion, Cergy-Pontoise, France). Calibration curves wereobtained by analysis of a range of SPEX certiPrep nickelstandards (Metuchen), and yttrium was used as an internalstandard (1 mg l-1). The measurement of each strain in eachcondition was performed in triplicates.

Nickel uptake assay

The nickel transport assay was adapted from Bauerfeindet al. (1996). Overnight cultures of wild-type and mutantH. pylori strains were diluted to an OD 0.5 in BBb and furtherincubated for 4–6 h to ensure exponential growth of thebacteria. Cells were harvested by centrifugation, washed withmedium and resuspended to an OD of 0.5–1 in fresh BBbmedium that was adjusted to pH 5 or pH 7 and the cells left toequilibrate for 15 min. Uptake was initiated by the addition ofradioactive 63NiCl2 (5.5 mCi ml-1; 0.585 mg of Ni ml-1; Amer-sham) to a final concentration of 50 nM in the presence of5 mM cold NiCl2. Samples of 200 ml were taken at 0.5, 1, 2, 5,10 and 15 min, immediately vacuum filtrated (20 lb in-2)through 0.45 mM pore-size cm filters (∅ = 2.5; Millipore) andwashed with 10 ml of 50 mM Tris-HCl (pH 7.0) containing1 mM cold NiCl2 to avoid unspecific binding. Two series ofexperiments were performed and each time point was mea-sured in duplicates. Filters were dried and subsequently intro-duced into scintillation vials with 4.1 ml of ScintillationCocktail (Filter Count LCS; Packard). Radioactive counting ofeach sample was performed during 10 min using a window of30 to 700 in a Beckman LS3801 scintillation counter. Controlscomprised counting of the filters after incubation in theabsence of bacteria or with lysed cells. No radioactive nickelwas measured under these conditions showing the absenceof unspecific binding to the filters and more importantly to thecell components.

Sequence analyses of FrpB4 (HP1512)

Sequence analyses of FrpB4 were carried out with thePfam domain search (http://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl), the secondary structure prediction with thePSIPRED protein structure prediction server (McGuffin et al.,2000) and the similarity search with HHpred program (Sodinget al., 2005).

Measurement of urease activity in H. pylori strains

Urease activities were measured on crude extracts preparedby sonication as previously described (Cussac et al., 1992;Skouloubris et al., 1997). One unit (U) of urease activity was



http://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl

http://www.sanger.ac.uk/cgi-bin/Pfam/swisspfamget.pl

defined as the amount of enzyme required to hydrolyse1 mmol of urea (producing 2 mmol of ammonia) per min permg of total protein. The amount of ammonia released wasdetermined from a standard curve. Protein concentration wasdetermined with a commercial version of the Bradford Assay(Sigma Chemicals) using bovine serum albumin as astandard.

Acknowledgements

The authors are grateful to Cécile Wandersman, KerstinStingl and Jost Enninga for helpful discussions and criticalreview of the manuscript. We also thank Isabelle Martin-Verstraete and Philippe Champeil for their judicious advicefor the nickel uptake experiments as well as Clarisse Marietand Francine Carrot for the ICP-MS analyses. K.S. receivesfinancial support from the German Academic ExchangeService (DAAD).

References

Abraham, L.O., Li, Y., and Zamble, D.B. (2006) The metal-and DNA-binding activities of Helicobacter pylori NikR.J Inorg Biochem 100: 1005–1014.

Alm, R.A., Ling, L.S., Moir, D.T., King, B.L., Brown, E.D.,Doig, P.C., et al. (1999) Genomic-sequence comparison oftwo unrelated isolates of the human gastric pathogen Heli-cobacter pylori. Nature 397: 176–180.

Atherton, J.C. (2006) The pathogenesis of Helicobacterpylori-induced gastro-duondenal diseases. Annu RevPathol: Mech Dis 1: 63–96.

Baar, C., Eppinger, M., Raddatz, G., Simon, J., Lanz, C.,Klimmek, O., et al. (2003) Complete genome sequenceand analysis of Wolinella succinogenes. Proc Natl Acad SciUSA 100: 11690–11695.

Bauerfeind, P., Garner, R.M., and Mobley, L.T. (1996) Allelicexchange mutagenesis of nixA in Helicobacter pyloriresults in reduced nickel transport and urease activity.Infect Immun 64: 2877–2880.

Bauerfeind, P., Garner, R., Dunn, B.E., and Mobley, H.(1997) Synthesis and activity of Helicobacter pylori ureaseand catalase at low pH. Gut 40: 25–30.

Bereswill, S., Greiner, S., Van Vliet, A.H.M., Waidner, B.,Fassbinder, F., Schiltz, E., et al. (2000) Regulation offerritin-mediated cytoplasmic iron storage by the ferricuptake regulator homolog (Fur) of Helicobacter pylori.J Bacteriol 182: 5948–5953.

Bijlsma, J.J.E., Waidner, B., Van Vliet, A.H.M., Hugues, N.J.,Hag, S., Bereswill, S., et al. (2002) The Helicobacter pylorihomologue of the ferric uptake regulator is involved in acidresistance. Infect Immun 70: 606–611.

Braun, V., and Braun, M. (2002) Active transport of iron andsiderophore antibiotics. Curr Opin Microbiol 5: 194–201.

Bury-Mone, S., Skouloubris, S., Dauga, C., Thiberge, J.M.,Dailidiene, D., Berg, D.E., et al. (2003) Presence of activealiphatic amidases in Helicobacter species able to colonizethe stomach. Infect Immun 71: 5613–5622.

Bury-Moné, S., Thiberge, J.M., Contreras, M., Maitournam,A., Labigne, A., and De Reuse, H. (2004) Responsivenessto acidity via metal ion regulators mediates virulence in the

gastric pathogen Helicobacter pylori. Mol Microbiol 53:623–638.

Casadaban, M., and Cohen, S.N. (1980) Analysis of genecontrol signals by DNA fusions and cloning in E. coli. J MolBiol 138: 179–207.

Cherrier, M.V., Martin, L., Cavazza, C., Jacquamet, L.,Lemaire, D., Gaillard, J., and Fontecilla-Camps, J.C. (2005)Crystallographic and spectroscopic evidence for high affin-ity binding of FeEDTA (H2O)- to the periplasmic nickeltransporter NikA. J Am Chem Soc 127: 10075–10082.

Contreras, M., Thiberge, J.M., Mandrand-Berthelot, M.A.,and Labigne, A. (2003) Characterization of the roles ofNikR, a nickel-responsive pleiotropic autoregulator of Heli-cobacter pylori. Mol Microbiol 49: 947–963.

Cussac, V., Ferrero, R.L., and Labigne, A. (1992) Expressionof Helicobacter pylori urease genes in Escherichia coligrown under nitrogen-limiting conditions. J Bacteriol 174:2466–2473.

Davis, G.S., and Mobley, H.L.T. (2005) Contribution of dppAto urease activity in Helicobacter pylori 26695. Helico-bacter 10: 416–423.

Davis, G.S., Flannery, E.L., and Mobley, H.L. (2006) Helico-bacter pylori HP1512 is a nickel-responsive NikR-regulatedouter membrane protein. Infect Immun 74: 6811–6820.

Delany, I., Ieva, R., Soragni, A., Hilleringmann, M., Rappuoli,R., and Scarlato, V. (2005) In vitro analysis of protein–operator interactions of the NikR and fur metal-responsiveregulators of coregulated genes in Helicobacter pylori.J Bacteriol 187: 7703–7715.

Derbise, A., Lesic, B., Dacheux, D., Ghigo, J.M., and Carniel,E. (2003) A rapid and simple method for inactivating chro-mosomal genes in Yersinia. FEMS Immunol Med Microbiol38: 113–116.

Dhaenens, L., Szczebara, F., Van Nieuwenhuyse, S., andHusson, M.O. (1999) Comparison of iron uptake in differentHelicobacter species. Res Microbiol 150: 475–481.

Dian, C., Schauer, K., Kapp, U., McSweeney, S.M., Labigne,A., and Terradot, L. (2006) Structural basis of the nickelresponse in Helicobacter pylori: crystal structures ofHpNikR in Apo and nickel-bound states. J Mol Biol 361:715–730.

Eppinger, M., Baar, C., Linz, B., Raddatz, G., Lanz, C., Keller,H., et al. (2006) Who ate whom? Adaptive Helicobactergenomic changes that accompanied a host jump from earlyhumans to large felines. PLoS Genet 2: e120.

Ermak, T.H., Giannasca, P.J., Nichols, R., Myers, G.A.,Nedrud, J., Weltzin, R., et al. (1998) Immunization of micewith urease vaccine affords protection against Helicobacterpylori infection in the absence of antibodies and is medi-ated by MHC class II-restricted response. J Exp Med 188:2277–2288.

Ernst, F.D., Bereswill, S., Waidner, B., Stoof, J., Mader, U.,Kusters, J.G., et al. (2005a) Transcriptional profiling ofHelicobacter pylori Fur- and iron-regulated geneexpression. Microbiol 151: 533–546.

Ernst, F.D., Kuipers, E.J., Heijens, A., Sarwari, R., Stoof, J.,Penn, C.W., et al. (2005b) The nickel-responsive regulatorNikR controls activation and repression of gene transcrip-tion in Helicobacter pylori. Infect Immun 73: 7252–7258.

Ernst, F.D., Stoof, J., Horrevoets, W.M., Kuipers, E.J.,Kusters, J.G., and van Vliet, A.H. (2006) NikR mediates



nickel-responsive transcriptional repression of the Helico-bacter pylori outer membrane proteins FecA3 (HP1400)and FrpB4 (HP1512). Infect Immun 74: 6821–6828.

Ferguson, A.D., and Deisenhofer, J. (2004) Metal importthrough microbial membranes. Cell 116: 15–24.

Ge, R., Watt, R.M., Sun, X., Tanner, J.A., HE, Q.Y., Huang,J.D., and Sun, H. (2006) Expression and characterizationof a histidine-rich protein, Hpn: potential for Ni2+ storage inHelicobacter pylori. Biochem J 393: 285–293.

Gilbert, J., Ramakrishna, J., Sunderman, F., Jr, Wright, A.,and Plaut, A. (1995) Protein Hpn: cloning and character-ization of a histidine-rich metal-binding polypeptide in Heli-cobacter pylori and Helicobacter mustelae. Infect Immun63: 2682–2688.

Ha, N.C., Oh, S.T., Sung, J.Y., Cha, K.A., Lee, M.H., and Oh,B.H. (2001) Supramolecular assembly and acid resistanceof Helicobacter pylori urease. Nat Struct Biol 8: 505–509.

Hendricks, J.K., and Mobley, H.L. (1997) Helicobacter pyloriABC transporter: effect of allelic exchange mutagenesis onurease activity. J Bacteriol 179: 5892–5902.

Hopfer, S.M., Fay, W.P., and Sunderman, F.W., Jr (1989)Serum nickel concentrations in hemodialysis patients withenvironmental exposure. Ann Clin Lab Sci 19: 161–167.

Hu, L.T., and Mobley, H.L. (1990) Purification and N-terminalanalysis of urease from Helicobacter pylori. Infect Immun58: 992–998.

Kansau, I., Guillain, F., Thiberge, J.M., and Labigne, A.(1996) Nickel binding and immunological properties of theC-terminal domain of the Helicobacter pylori GroES homo-logue (HspA). Mol Microbiol 22: 1013–1023.

McGuffin, L.J., Bryson, K., and Jones, D.T. (2000) ThePSIPRED protein structure prediction server. Bioinformat-ics 16: 404–405.

Merrell, D.S., Thompson, L.J., Kim, C.C., Mitchell, H., Tomp-kins, L.S., Lee, A., and Falkow, S. (2003) Growth phase-dependent response of Helicobacter pylori to ironstarvation. Infect Immun 71: 6510–6525.

Miller, J.H. (1992) A Short Course in Bacterial Genetics: ALaboratory Manual and Handbook for Escherichia coli andRelated Bacteria. Cold Spring Harbor, NY: Cold SpringHarbor Laboratory.

Mobley, H.L., Garner, R.M., and Bauerfeind, P. (1995) Heli-cobacter pylori nickel-transport gene nixA: synthesis ofcatalytically active urease in Escherichia coli independentof growth conditions. Mol Microbiol 16: 97–109.

Mulrooney, S.B., and Hausinger, R.P. (2003) Nickel uptakeand utilization by microorganisms. FEMS Microbiol Rev 27:239–261.

Nielsen, G.D., Andersen, O., and Jensen, M. (1993) Toxico-kinetics of nickel in mice studied with the gamma-emittingisotope 57Ni. Fundam Appl Toxicol 21: 236–243.

Nolan, K.J., McGee, D.J., Mitchell, H.M., kolesnikow, T.,Harro, J.M., O’Rourke, J., et al. (2002) In vivo behaviour ofa Helicobacter pylori SS1 nixA mutant with reduced ureaseactivity. Infect Immun 70: 685–691.

Oskarsson, A., and Tjalve, H. (1979) Binding of 63Ni bycellular constituents in some tissues of mice after theadministration of 63NiCl2 and 63Ni(CO)4. Acta PharmacolToxicol (Copenh) 45: 306–314.

Pflock, M., Kennard, S., Delany, I., Scarlato, V., and Beier, D.(2005) Acid-induced activation of the urease promoters is

mediated directly by the ArsRS two-component system ofHelicobacter pylori. Infect Immun 73: 6437–6445.

Pflock, M., Kennard, S., Finsterer, N., and Beier, D. (2006)Acid-responsive gene regulation in the human pathogenHelicobacter pylori. J Biotechnol 126: 52–60.

Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M.,and Séraphin, B. (1999) A generic protein purificationmethod for protein complex characterization and proteomeexploration. Nat Biotechnol 17: 1030–1032.

Rodionov, D.A., Hebbeln, P., Gelfand, M.S., and Eitinger, T.(2006) Comparative and functional genomic analysis ofprokaryotic nickel and cobalt uptake transporters: evidencefor a novel group of ATP-Binding cassette transporters.J Bacteriol 188: 317–327.

Sabarth, N., Hurvitz, R., Schmidt, M., Zimny-Arndt, U., Jung-blut, P.R., Meyer, T.F., and Bumann, D. (2005) Identifica-tion of Helicobacter pylori surface proteins by selectiveproteinase K digestion and antibody phage display.J Microbiol Methods 62: 345–349.

Sambrook, J., and Russel, D.W. (2001) Molecular Cloning: ALaboratory Manual. Cold Spring Harbor, NY: Cold SpringHarbor Laboratory.

Scott, D.R., Marcus, E.A., Weeks, D.L., and Sachs, G. (2002)Mechanisms of acid resistance due to the urease system ofHelicobacter pylori. Gastroenterology 123: 187–195.

Skouloubris, S., Labigne, A., and De Reuse, H. (1997) Iden-tification and characterization of an aliphatic amidase inHelicobacter pylori. Mol Microbiol 25: 989–998.

Skouloubris, S., Thiberge, J.M., Labigne, A., and De Reuse,H. (1998) The Helicobacter pylori UreI protein is notinvolved in urease activity but is essential for bacterialsurvival in vivo. Infect Immun 66: 4517–4521.

Soding, J., Biegert, A., and Lupas, A.N. (2005) The HHpredinteractive server for protein homology detection and struc-ture prediction. Nucleic Acids Res 33: W244–W248.

Stahler, F.N., Odenbreit, S., Haas, R., Wilrich, J., Van Vliet,A.H., Kusters, J.G., et al. (2006) The novel Helicobacterpylori CznABC metal efflux pump is required for cadmium,zinc, and nickel resistance, urease modulation, and gastriccolonization. Infect Immun 74: 3845–3852.

Stingl, K., and De Reuse, H. (2005) Staying alive overdosed:how does Helicobacter pylori control urease activity. Int JMed Microbiol 295: 307–315.

Stingl, K., Altendorf, K., and Bakker, E.P. (2002) Acid sur-vival of Helicobacter pylori: how does urease activitytrigger cytoplasmic pH homeostasis? Trends Microbiol 10:70–74.

Suerbaum, S., Josenhans, C., Sterzenbach, T., Drescher, B.,Brandt, P., Bell, M., et al. (2003) The complete genomesequence of the carcinogenic bacterium Helicobacterhepaticus. Proc Natl Acad Sci USA 100: 7901–7906.

Sunderman, F.W., Jr (1993) Biological monitoring of nickel inhumans. Scand J Work Environ Health 19 (Suppl. 1):34–38.

Tomb, J.F., White, O., Kerlavage, A.R., Clayton, R.A., Sutton,G.G., Fleischmann, R.D., et al. (1997) The completegenome sequence of the gastric pathogen Helicobacterpylori. Nature 388: 539–547.

Van Soestbergen, M., and Sunderman, F.W., Jr (1972) 63 Nicomplexes in rabbit serum and urine after injection of 63NiCl 2. Clin Chem 18: 1478–1484.



Van Vliet, A.H.M., Poppelaars, S.W., Davies, B.J., Stoof, J.,Bereswill, S., Kist, M., et al. (2002a) NikR mediates nickel-responsive transcriptional induction of urease in Helico-bacter pylori. Infect Immun 70: 2846–2852.

Van Vliet, A.H.M., Stoof, J., Vlasblom, R., Wainwright, S.A.,Hugues, N.J., Kelly, D.J., et al. (2002b) The role of theferric uptake regulator (Fur) in regulation of Helicobacterpylori iron uptake. Helicobacter 4: 237–244.

Velayudhan, J., Hughes, N.J., McColm, A.A., Bagshaw, J.,Clayton, C.L., Andrews, S.C., and Kelly, D.J. (2000) Ironacquisition and virulence in Helicobacter pylori: a majorrole for FeoB, a high-affinity ferrous iron transporter. MolMicrobiol 37: 274–286.

van Vliet, A.H.M., Kuipers, E.J., Stoof, J., Poppelaars, S.W.,and Kusters, J.G. (2004) Acid-responsive gene induction ofammonia-producing enzymes in Helicobacter pylori ismediated via a metal-responsive repressor cascade. InfectImmun 72: 766–773.

Wandersman, C., and Delepelaire, P. (2004) Bacterial ironsources: from siderophores to hemophores. Annu RevMicrobiol 58: 611–647.

Wolfram, L., Haas, E., and Bauerfeind, P. (2006) Nickelrepresses the synthesis of the nickel permease NixA ofHelicobacter pylori. J Bacteriol 188: 1245–1250.

Wu, L.F., and Mandrand-Berthelot, M.A. (1986) Genetic andphysiological characterization of new Escherichia colimutants impaired in hydrogenase activity. Biochimie 68:167–179.

Yanisch-Perron, C., Vieira, J., and Messing, J. (1985)Improved M13 phage cloning vectors and host strains:nucleotide sequences of the M13mp18 and pUC19vectors. Gene 33: 103–119.

Supplementary material

The following supplementary material is available for thisarticle online:Table S1. Oligonucleotides used in this study.

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