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Journal of Bacteriology, May 2008, p. 3244-3255, Vol. 190, No. 9
0021-9193/08/$08.00+0     doi:10.1128/JB.01940-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Functional Characterization and Mutagenesis of the Proposed Behavioral Sensor TlpD of Helicobacter pylori{triangledown}

Tobias Schweinitzer,1,{dagger} Tomoko Mizote,2,{dagger} Naohiro Ishikawa,2 Alexey Dudnik,1 Sakiko Inatsu,2 Sören Schreiber,3 Sebastian Suerbaum,1 Shin-Ichi Aizawa,4 and Christine Josenhans1*,{dagger}

Institute for Medical Microbiology, Hannover Medical School, 30625 Hannover, Germany,1 Department of Physiology, Ruhr-University Bochum, 44780 Bochum, Germany,3 Department of Human Science, Yamaguchi Prefectural University, Yamaguchi 753-8502, Japan,2 Department of Life Sciences, Prefectural University of Hiroshima, Shobara 727-0023, Japan4

Received 13 December 2007/ Accepted 23 January 2008


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ABSTRACT
 
Helicobacter pylori requires flagellar motility and chemotaxis to establish and maintain chronic infection of the human stomach. The pH gradient in the stomach mucus is essential for bacterial orientation and guides the bacterium toward a narrow layer of the mucus, suggesting that H. pylori is capable of energy sensing or taxis. In the present study, H. pylori wild-type behavior in a temporal swimming assay could be altered by electron transport inhibitors, indicating that a connection between metabolism and behavior exists. In order to elucidate mechanisms of behavioral responses of H. pylori related to energy sensing, we investigated the phenotypes of single and multiple mutants of the four proposed chemotaxis sensor proteins. All sensor mutants were motile, but they diverged in their behavior in media supporting different energy yields. One proposed intracellular sensor, TlpD, was crucial for behavioral responses of H. pylori in defined media which did not permit growth and led to reduced bacterial energy levels. Suboptimal energetic conditions and inhibition of electron transport induced an increased frequency of stops and direction changes in the wild type but not in tlpD mutants. Loss of metabolism-dependent behavior in tlpD mutants could be reversed by complementation but not by electron donors bypassing the activity of the electron transport chain, in contrast to the case for the wild type. TlpD, which apparently lacks transmembrane domains, was detected both in the bacterial cytoplasm and at the bacterial periphery. The proposed energy sensor TlpD was found to mediate a repellent tactic response away from conditions of reduced electron transport.


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INTRODUCTION
 
Helicobacter pylori has the ability to survive and multiply in the mucous layer of the human stomach, where it colonizes persistently for many years. Gastric Helicobacter species need complete flagella and full motility to colonize their hosts persistently (5, 13, 23, 42). Both for the first entry of H. pylori into the human gastric mucus and for chronic colonization, H. pylori probably exploits horizontal and vertical substance gradients to reach and stay in its optimal habitat. Motile H. pylori mutants deficient in chemotaxis by knockout mutagenesis of the chemotaxis histidine kinase gene cheA and other chemotaxis proteins have been found to be unable to colonize in mouse and gerbil colonization models (16, 26). Analysis of nanobiopsies taken from the stomach mucus of anesthetized, Helicobacter-infected mice and Mongolian gerbils has shown that Helicobacter actively accumulates in a very narrow zone of the proximal stomach mucus, at a distance from the epithelial surface of between 0 and 15 µm (45, 46). It is guided there by a vertical proton gradient within the gastric mucus. Changes in other chemical gradients in the mucus in vivo did not alter the horizontal distribution of H. pylori, suggesting that pH taxis may be dominant over other chemotactic responses (45). pH taxis, a manifestation of energy-dependent taxis, was therefore shown to be essential for persistent colonization in this model, although H. pylori does not appear to possess Aer-like sensors or PAS domain proteins (50). Knowledge about the tactic properties that enable H. pylori to maintain its preferred niche and about the environmental signals that are made use of for topological guidance during this process is scarce. By whole-genome analyses of H. pylori strains, the key proteins in the H. pylori chemotaxis system have been identified (3, 43, 53), among them four putative chemosensor proteins of the methyl-accepting chemotaxis protein (MCP) family (22, 34). Three of the four described genome-encoded MCP orthologs (HP0082/TlpC, HP0099/TlpA, and HP0103/TlpB) seem to be classical transmembrane sensor proteins which, by amino acid sequence and derived structural predictions, possess two transmembrane domains and a periplasmic sensing domain. One of the putative H. pylori sensors (HP0599/TlpD) possesses the conserved C-terminal MCP domain but appears to lack the periplasmic loop and transmembrane domains. The receptor functions of all four sensor proteins are not well established, nor has a role in energy taxis been described, except for a reported role of HP0103/TlpB in pH taxis (12). There is no similarity of the N-terminal receptor domains of any of the four chemosensors with other well-characterized MCPs, which would allow us to extrapolate receptor specificities.

Bacterial chemoreceptors sense mostly small, outer membrane-permeating substances, e.g., sugars and amino acids (2), and in addition, some of them monitor the energy status of the cell, leading to a behavior termed energy taxis (1, 19, 51). In some bacteria, such as the soil-inhabiting organism Azospirillum brasilense, energy sensing and taxis seem to be dominant with respect to tactic abilities, which may be related to the fact that they have relatively strict metabolic requirements for growth, e.g., with regard to environmental oxygen concentration (1). Other bacteria, such as Escherichia coli, also employ energy taxis, but this seems to be a less dominant response (51), probably since their abilities to generate energy and electron flow from terminal electron acceptors under aerobic and anaerobic conditions are quite versatile. In E. coli, two different modes of energy sensing have been discovered, including redox sensing, performed by the PAS domain protein Aer (7, 50), and sensing of the proton motive force (PMF; membrane proton gradient) by the classical transmembrane MCP Tsr (14). Bacterial energy sensing, oxygen taxis, and pH taxis are thus closely linked via the proton and electron gradients across the inner membrane, and they seem to be governed by closely connected physicochemical and metabolism-dependent mechanisms. H. pylori has been described to show positive chemotaxis toward urea, urea analogs (flurofamide), bicarbonate, mucin, and amino acids (9, 16, 37, 57), some of which could be explained by energy sensing, since those substances can either cause a pH change or be metabolized by H. pylori and provide essential nutrients (10, 35).

The present work describes the mutagenesis, functional characterization, and subcellular localization of the unusual H. pylori chemosensor TlpD, which was found to contribute to energy sensing.


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MATERIALS AND METHODS
 
Bacterial strains and culture conditions. H. pylori strains N6 (15), 88-3887 (a motile variant of the fully sequenced strain 26695) (53), and ATCC 43504 (NCTC11637) were used. Culture conditions were as described previously (38). Alternatively, H. pylori was grown in a biphasic medium consisting of blood agar or serum-containing brucella plates overlaid with brucella broth. E. coli strains used for cloning were DH5{alpha}, MC1061, and TG1 (44). Media used for liquid medium motility analysis (temporal assays) were as described in Results, employing BHI broth including 3% or 10% fetal bovine serum (FBS) and RPMI 1640 defined cell culture medium at pH 7.4 (RPMI 1640 medium buffered by 20 mM HEPES; Invitrogen Inc.), with or without the addition of 3% FBS. RPMI 1640 does not support the proliferation of H. pylori, either in the absence or in the presence of serum (52), but in the presence of serum, the bacterium remains vividly motile, can survive in these media for >12 h, and can be cultured further afterwards in other media. In BHI with serum, a growth-permissive medium for H. pylori, proliferation of bacteria at a maximum rate in exponential phase (µ) of 0.1791 (calculated doubling time, 3.87 h) is usually observed (39). Bacterial growth rates in BHI supplemented with 10% serum were determined by generating a growth curve for bacteria in liquid culture, inoculated with bacteria grown on plates for 20 to 24 h (mid-log growth) to an initial optical density at 600 nm (OD600) of 0.05. Atmospheric conditions were tested to be almost equivalent in terms of growth and ATP production of the bacteria for an H. pylori gas mixture (10% CO2, 5% oxygen, 85% nitrogen), a Helicobacter hepaticus gas mixture (10% CO2, 10% hydrogen, 80% nitrogen), and the ambient atmosphere supplemented with 5% CO2 (data not shown). For routine growth, the H. pylori gas mixture in a Scholzen incubator was used. Swimming assays were performed in the ambient atmosphere enriched with 5% CO2 in order to facilitate the experimental setup. Bacteria were used for the motility or tracking assays only when >70% of the bacteria in suspension were vividly motile.

Techniques of molecular cloning and protein analysis. DNA purification, DNA manipulation, and cloning procedures were performed according to standard protocols. H. pylori was transformed with plasmids by natural transformation or electroporation (15, 24). Plasmids and primers for cloning and sequencing are contained in Tables 1 and 2. PCRs were run in PerkinElmer thermocyclers, using Amersham Taq polymerase or an Expand High Fidelity kit (Roche) for longer fragments (2 kb) or if a high amplification accuracy for protein expression was required.


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  		TABLE 1. Oligonucleotide primers used for sequencing, PCR, and DNA cloning


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  		TABLE 2. Plasmids used for this study

Crude H. pylori flagellar preparations and bacterial lysates were obtained as previously described (6). Crude bacterial fractions (soluble, insoluble, and membrane-associated fractions) were obtained by sonication of the bacterial suspension in 0.9% NaCl (harvested from one-half of a plate grown for approximately 40 h, to late log phase) for 4 min in a Branson sonifier. The supernatants (soluble) and pellets (crude membranes and insoluble proteins) were then separated by centrifugation at 9,000 x g for 30 min at 4°C in an Eppendorf centrifuge. The pellets were carefully separated from the supernatants and resuspended in 200 µl of 0.9% NaCl. Bicinchoninic acid protein assays were performed on the fractions to determine their protein content. The quality of the fractionations was tested by light microscopy, and the material was used for further analyses only when no intact bacterial cells could be observed upon visual inspection. Furthermore, the fractions were controlled for separation of soluble and membrane-associated proteins by Western blotting, using antisera against the H. pylori flagellar membrane protein FlhA. Protein analysis of the fractions was performed using denaturing 11% sodium dodecyl sulfate-polyacrylamide gels (28) and Western immunoblot detection according to the method of Towbin et al. (54). For immunolabeling of H. pylori TlpD on membranes, either a polyclonal rabbit antiserum raised against E. coli Tsr (generously provided by John S. Parkinson; dilution, 1:1,000) or an anti-V5 tag antibody (mouse monoclonal; dilution, 1:5,000) (Invitrogen) was used to detect expression of V5-tagged TlpD. Peroxidase-coupled goat anti-mouse or goat anti-rabbit secondary antibodies (Jackson ImmunoResearch Inc.) were used at a 1:20,000 dilution.

Construction of chemotaxis sensor mutants. Plasmid constructs for the four mutants were generated in E. coli. In brief, PCR-amplified fragments of the genes were cloned into the pUC18 or pILL570 plasmid. Antibiotic resistance cassettes were inserted into the 3' fragments of the genes by inverse PCR amplification of the plasmids. In most cases, deletions in the central regions of the genes were introduced during mutagenesis. A cheA mutant was constructed in parallel as a negative control for taxis.

(i) HP0082 (tlpC). The tlpC gene was PCR amplified from strain KE26695 genomic DNA by using primers OLHP0082_1s and OLHP0082_2s and cloned into pILL570. The plasmid was inversely amplified using primers OLHP0082_3s and -_5s and ligated at EcoRI sites with a kanamycin resistance cassette (aphA3'-III from Campylobacter coli) (pSUS252).

(ii) HP0099 (tlpA). The tlpA gene was amplified (strain KE26695 DNA) using primers OLHP0099_1s and OLHP0099_2s and cloned into the BglII site of pILL570B (the BamHI restriction site was inactivated). A unique BamHI site in the HP0099 gene was used to insert the kanamycin resistance cassette (pSUS255). Similarly, to construct a tlpA deletion mutant with the insertion of a chloramphenicol resistance gene, a PCR-amplified tlpA fragment was first inserted into pBluescript II KS(+) that was cut with EcoRV by blunt end cloning. Next, a chloramphenicol resistance cassette from pBSC103 was excised by EcoRV cleavage and ligated with a blunted unique SphI site within tlpA (at nucleotide position 2048 of tlpA), yielding pHPS807.

(iii) HP0103 (tlpB). The tlpB gene was amplified (strain KE26695 DNA) using primers OLHP0103_1s and OLHP0103_2s. Inverse PCR was carried out with primers OLHP0103_3s and -_4s, and the product was ligated (using EcoRI sites) with the kanamycin resistance cassette (pSUS260).

(iv) HP0599 (tlpD). The tlpD gene from strain KE26695 was cloned as a PCR product (amplified using primers OLHP0599_1s and OLHP0599_2s) into pILL570. The kanamycin cassette was inserted after inverse PCR (EcoRI sites) using primers OLHP0599_3s and OLHP0599_6s (pSUS275).

(v) HP0392 (cheAY2). The cheA gene from H. pylori strain 26695, which is a 3' gene fusion with a cheY domain (16), was PCR amplified (using primers OLHPcheA1 and OLHPcheA2) and cloned into pUC18. The kanamycin cassette was then inserted in the same transcriptional orientation into the unique PstI site in cheA (pSUS131). The resulting mutant is supposed to lack the function of both CheA and CheY2, similar to a previously described mutant (16).

The antibiotic resistance cassettes were inserted in all cases in the same transcriptional orientation as the respective genes to avoid polar effects. All plasmids were introduced into different H. pylori strains (N6, ATCC 43504, and 26695) by natural transformation, and allelic exchange mutants were selected on kanamycin-containing medium. The genotypes of the mutants were verified by PCR with different combinations of oligonucleotide primers (available on request). Four mutant colonies of each strain were tested to be certain about the stability of the respective geno- and phenotypes.

Generation of a triple transducer knockout strain (tlpABC mutant). For construction of a triple mutant lacking functional tlpA, tlpB, and tlpC genes, in a first step the tlpC gene was partially deleted as an unmarked mutation using the counterselectable gene sacB of Bacillus subtilis (11). For this purpose, the kanamycin resistance cassette in pSUS252 was removed (by restriction with EcoRI) and replaced by the kan-sacB construct taken from pKSF (11), using the primers aphA3_M_F and HpFlaAP_M_F (resulting plasmid, pCJ511). Inverse PCR was carried out with primers OLHP0082_5s and OLHP0082_6s, yielding plasmid pCJ512, carrying tlpC with a 509-bp deletion. After transformation with pCJ511, the obtained kanamycin-resistant and sucrose-sensitive mutant was transformed with pCJ512, yielding kanamycin-sensitive, sucrose-resistant mutants with an unmarked deletion in tlpC. Subsequently, tlpB was disrupted in the tlpC deletion mutant by allelic exchange mutagenesis with a kanamycin resistance cassette insertion, using pSUS260, and tlpA was likewise disrupted in the same strain by allelic exchange with a chloramphenicol resistance cassette, using pHPS807. Selection for positive clones was performed on sucrose-, kanamycin-, or kanamycin-chloramphenicol-containing medium.

Construction of a tlpD-V5 shuttle plasmid for complementation in trans. For complementation, the tlpD gene, including its own untranslated 5' region containing putative promoter sequences, was C-terminally fused to a V5 tag sequence (GKPIPNPLLGLDST) with a 7-amino-acid (aa) linker (GGSSAAG) (8) and cloned into the E. coli/H. pylori shuttle plasmid pHel2 (20). Initially, the vector pEF6-V5 (Invitrogen) was amplified by inverse PCR using primers (OLpEF6_R and OLV5_F) fusing the last 5 aa of the 7-aa linker, provided by the primer sequence, to the 5' end of the encoded V5 tag. The PCR product was digested with BglII and ligated with the BamHI-digested PCR product of the tlpD gene. The tlpD gene had been amplified before from H. pylori KE26695 with primers (OLHP0599_V5_F and OLH0599_V5_R) fusing the first 2-aa-encoding sequence of the described 7-aa linker to the 3' end of the gene. The in-frame fusion of the tlpD gene with the V5 tag was checked by sequencing pCJ520. For cloning into plasmid pHel2 (19), required for expression in trans in H. pylori, the HP0599_V5 fusion gene was amplified from the plasmid pCJ520, using primers (OLHP0599_V5_F and OLV5_R) which introduced a stop codon at the 3' end of the gene fusion, and then cloned into the BamHI site of pHel2 (resulting shuttle plasmid, pCJ522). pCJ522 was then introduced into the tlpD::kan mutant of strain N6 by natural transformation, and plasmid-containing clones were selected on kanamycin-chloramphenicol-containing medium and characterized. Several complemented clones grew on chloramphenicol plates, and motile clones were further characterized. All clones contained the correct plasmid. Plasmids and primers for cloning and sequencing are contained in Tables 1 and 2.

Motility testing (semisolid plates). Motility testing was performed with semisolid agar plates consisting of BHI broth supplemented with 0.25% Bacto agar and 3% FBS, both as stab tests and as single-colony motility tests (24). Briefly, for stab testing, bacteria from mid-log-phase plate cultures were harvested and diluted to an equivalent OD600 of 0.5 for each strain in 0.9% NaCl. Five-microliter bacterial suspensions were spotted onto a semisolid plate by piercing it with a thin pipette tip. Concentric taxis halos, appearing by bacteria moving within the agar outward from the stab, were observed, and their diameters were measured after 5 days. For single-colony motility testing, bacteria from a liquid suspension prepared as described above were further diluted 1:105 in 0.9% NaCl. Fifty microliters of this highly diluted suspension was then gently mixed with 6 ml of still-liquid motility agar at 40°C and poured into a petri dish. Plates were left to solidify and were further incubated for 5 days to observe taxis halos around single colonies.

Measurement of behavior (temporal assays) and single-bacterium tracking. Bacteria were prepared from early to mid-log-phase cultures as a suspension with an OD600 of 0.2 to 0.3 in the appropriate medium. Cells were resuspended gently in liquid medium without prior washing, since washing of H. pylori severely diminishes motility, as found by us and others (12, 32). The bacteria were equilibrated in the medium for 10 to 20 min at 37°C in ambient air enriched with 5% CO2. Bacterial suspensions were then observed in small incubation chambers (on coverslips) or in screw-cap cell culture bottles, using either a Zeiss Axiovert microscope outfitted with a 63x lens or an Olympus IX80 inverted microscope equipped with 40x, 63x, and 100x magnification lenses. Motility of single H. pylori bacteria was recorded directly as a sequence of digital frames, using a Cell-R live-imaging microscopy system (Olympus Inc.) including a climate chamber with adjustable humidity, temperature, and CO2 concentration. Digital frames, acquired at 20 frames/s, were converted to digital video sequences of 20 frames/s and approximately 12 s in length. Bacterial tracking and determination of single-bacterium tracking parameters, such as curvilinear velocity (CLV; velocity along a curved trace) and number of stops per unit of time, was then performed semiautomatically using the software TrackIt (Olympus SIS, version 1.2.445). An automatic tracking functionality which works for cells of large size and low velocity is implemented in the software, but this did not function for our bacteria, which were too numerous, too fast, and rather small. Therefore, we used the semiautomatic features of the software for bacterial tracking. We only tracked bacteria which were visible within the same horizontal plane of focus for at least 3 s. The main semiautomatic feature of the software allows the user to manually follow the path of swimming bacteria by mouse clicking on the same individual bacterium on the computer screen in each acquired frame, thereby automatically assembling and recording the complete swimming trace of the bacterium, which the software then displays and saves as a line trace. The traces can then be exported from TrackIT as JPEG graphs (examples are shown in Fig. 1 and 3). The software automatically calculates the lengths of the single line traces, which can then be exported into MS Excel. Bacterial stops were counted manually from the movies. CLV and stops per unit of time were determined in MS Excel, after import of the acquired trace data (trace length and time of tracing) from TrackIT. Mean CLVs and the mean number of stops per unit of time for each condition and each bacterium were then further calculated in MS Excel as required. At least 20 bacterial cells were analyzed in every single experiment and traced, and the average of the behavioral pattern during 3 s for all analyzed bacteria were calculated. Mean values of the above parameters and the respective standard deviations for measurements of equivalent bacterial numbers of each strain (20 to 100 bacteria) were used to compare the different strains statistically (Student's t test). Details of liquid media used for temporal assays are provided in Results or figure legends.


Figure 1
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  		FIG. 1. ATP measurements and tracking analysis (temporal assays) of H. pylori wild type in the absence and presence of metabolic inhibitors. (A) Measurements of intrabacterial ATP levels (see Materials and Methods), performed in different media and in the presence of metabolic inhibitors. R, RPMI medium; RS, RPMI with FBS. RLU, relative light units. (B) Measurements of single bacteria (closed triangles) in two representative experiments with H. pylori N6 wild type. Inhibition of the stopping response was achieved after the addition of 10 µM HQNO (H) or myxothiazol (M). An electron donor (TMPD/ascorbate [TA]) reconstituted stopping behavior in wild-type bacteria pretreated with myxothiazol. Differences between the conditions were statistically significant (P < 0.01). (C and D) Individual traces for wild-type bacteria in RPMI-FBS without (C) or with (D) inhibition by myxothiazol. (E and F) Kinetics of behavioral inhibition of wild-type bacteria with myxothiazol and subsequent reconstitution with TMPD/ascorbate in RPMI medium with (E) or without (F) FBS. Velocities in panels B, E, and F were not inhibited to < 25 µm/s by any of the added substances, while behavior was drastically altered. Bacterial CLVs in panels B, E, and F are displayed on the second y axis (µm/s; graphs with open squares and dotted lines), while numbers of stops (graphs with closed triangles) are indicated on the left y axis. For panels B, E, and F, behavioral measurements (stops) for at least 20 bacteria under each condition were averaged (means and standard deviations are depicted). All movies for tracing the bacteria were taken after 20 min of incubation in medium.


Figure 3
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  		FIG. 3. Temporal assays of H. pylori tlpD single mutant, tlpABC triple mutant, and the complemented tlpD mutant. The figure shows the results of tracking analyses (temporal assays) of tlpD and tlpABC mutant bacteria (strain N6) and of the complemented N6 tlpD mutant strain expressing TlpD-V5 in trans. (A and B) Behavioral analysis. Bacterial velocities in panels A and B are displayed on the second y axis (µm/s; graphs with open squares and dotted lines), while numbers of stops (closed triangles) are indicated on the left y axis. TlpD-V5 complemented the behavioral defect of the tlpD mutant. Differences between the wild type or the tlpABC mutant and the tlpD mutant and between the tlpD mutant and the complemented strain were statistically significant (P < 0.01). M, myxothiazol; TA, TMPD/ascorbate. (C and D) Exemplary traces for individual tlpD and tlpABC bacteria. Movies for tracing the bacteria were acquired after 20 min of incubation in medium.

Inhibitor assays of bacterial sensing. Inhibition of bacterial behavior (stops) in liquid media by metabolic inhibitors was performed in the different media by adding the inhibitors posterior to equilibration in the media, as indicated in the figures. The metabolic inhibitors myxothiazol and 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) were used at MICs determined by temporal assays: inhibitors were added at concentrations of 1 µM, 2.5 µM, 5 µM, 7.5 µM, 10 µM, 15 µM, 20 µM, and up to 120 µM to the media (both BHI and RPMI 1640 were tested, supplemented with 10% and 3% FBS, respectively). CLV and number of stops were measured at all concentrations for at least 20 bacteria (data not shown). The addition of low concentrations of inhibitors (between 1 and 2.5 µM) led to an initial increase in CLV and number of stops. In BHI-FBS without inhibitor, when 70 bacteria were tracked, 3 ± 1.9 stops per 3 s were recorded (99% confidence interval, 2.5 to 3.6), compared to 4.8 ± 2 stops per 3 s for the same medium with 1 µM myxothiazol (99% confidence interval, 3.9 to 5.7). The difference in the numbers of stops between those two conditions was statistically significant (t test; P = 9.4 x 10–6). At inhibitor concentrations exceeding 2.5 µM, a gradual decrease in the number of stops per 3 s was observed (data for concentration-dependent effects not shown). For both inhibitors, 10 µM was the lowest concentration that abolished stopping behavior, while CLV was only moderately decreased under those conditions (Fig. 1). For both inhibitors, a time-dependent decrease in intrabacterial ATP levels was also recorded over the duration of 60 min (data not shown). Myxothiazol (10 µM) led to a reduction of intrabacterial ATP by about 30% after 20 min (Fig. 1A). If not indicated otherwise, a concentration of 10 µM for both inhibitors was used for the assays, since it led to a highly significant change in behavior.

Measurements of internal bacterial ATP levels (ATP assays). As an equivalent of intrabacterial energy loads, intracellular ATP was measured. Bacteria were prepared from early to mid-log-phase cultures as suspensions with OD values of both 0.3 (~108 cells/ml) and 0.03 (~107 cells/ml) in the appropriate media. Cells were taken directly from the freshly grown plates during exponential growth and resuspended gently in liquid medium without prior washing. The bacteria were then incubated for various defined time periods at 37°C before starting the ATP assay. Details of liquid media, supplements, and incubation times used for ATP assays are provided in Results and figure legends. For determining ATP contents of the cells, BacTiter-Glo reagent (an ATP-dependent luciferase-luciferin reagent mixture; Promega Inc., Madison, WI) was added directly to the live bacterial suspensions at a ratio of 1:1 after the desired incubation times. The suspensions were incubated for 5 min at 37°C to lyse the bacteria and to initiate the enzymatic reaction. The emission of photons was measured with a Wallac 1420 Victor3 V multilabel counter instrument (PerkinElmer) in luminescence mode. All experiments were performed at least three times on separate days in quadruplicate measurements. Values were collected and further processed in MS Excel, calculating either the level of luminescence (as relative luminescence units) per cell or the amount of ATP per cell, with regard to OD measurements. The ATP measurements were performed at different time points. Those shown in the results were performed after a 20-min incubation time, a reference time point for comparison with the behavioral assays.

Taxis assay in slide chemotaxis chambers. For taxis assays, we utilized commercial microscope slide chemotaxis chambers, consisting of two separate opposing liquid chambers or reservoirs (volume, 40 µl [each]) divided by a 1-mm narrow liquid transition zone with a 1.4-µl volume (Ibidi GmbH, Martinsried, Germany). The two chambers of the same system were completely filled, according to the manufacturer's instructions, with BHI-FBS medium. A small volume of bacterial suspension in the same medium (OD600 = 0.3) was then added to one reservoir, and after that step, the second reservoir of the same system was supplemented or not (control) with the metabolic inhibitor myxothiazol (final concentration, 10 µM). Great care was taken not to introduce any air bubbles into the whole chamber system. During the incubation, atmospheric gases can enter the chamber by inlet/outlet vents on both sides and in the center, which can be capped by gas-permeable caps to prevent drying. According to the description provided by the manufacturer, a linear gradient between the maximum concentration of a substance (10 µM in this study) and zero, which remains stable for at least 24 h, forms in the small midchamber of the slide. Myxothiazol was used as a substance for taxis assays, since the inhibitor at concentrations below 10 µM (1 µM) had been shown in pretesting to induce stronger stopping behavior than the absence of inhibitor (see above), indicating a repellent response to this substance mediated by inhibition of electron transport. The slides were incubated on a Cell-R microscope stage in ambient atmosphere supplemented with 5% CO2. The experiment, including controls, was repeated three times. The number of bacteria which transmigrated from the first reservoir into the transition chamber or into the second reservoir, with or without inhibitor, were observed and counted after a 2-h incubation. Three fields of view (field size, 221.9 µm by 167.4 µm) at the transition zone between the midchamber and the second reservoir containing the metabolic inhibitor were photographed for each condition, and the bacteria were counted in the second reservoir only.


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RESULTS
 
Inhibition of electron transport chain alters behavioral responses of H. pylori. Since pH tactic or energy-sensing behavior of H. pylori has been found to be essential for orientation in vivo (45), we attempted to assess the contribution of metabolism-dependent sensing to behavioral responses of H. pylori in vitro. The H. pylori respiratory chain possesses only one terminal oxidase, of the Cbb3 type, encoded by a gene cluster (47, 53). In addition, a fumarate reductase for anaerobic energy generation is present (27). We first attempted to mutagenize the cbb3 terminal oxidase subunit genes in order to assess their impact on bacterial growth, motility, and taxis. The Cbb3 terminal oxidase appeared to be essential for bacterial proliferation in our hands, even in the presence of alternative electron acceptors (e.g., fumarate, dimethyl sulfoxide, trimethylamine N-oxide), rendering it impossible to mutate any of the cbb3 cluster genes (data not shown).

One alternative approach to elucidate metabolism-dependent sensing and taxis makes use of metabolic inhibitors of the electron transport chain and was first developed for Azospirillum brasilense and E. coli (1). In those works, behavioral responses toward metabolizable substrates, which were inhibited in the presence of metabolic inhibitors that target the electron transport chain, were attributed to energy-sensing mechanisms. We utilized two electron transport inhibitors, myxothiazol (inhibitor of the cytochrome bc1 terminal oxidase subunit) (55) and HQNO, a quinone analogue and ubiquinone inhibitor (1), to elucidate metabolism-dependent sensing in H. pylori. We initially assessed the effects of different media and metabolic inhibitors on the intracellular ATP levels of H. pylori as a measure of intrabacterial energy yields (see Materials and Methods). Bacterial ATP contents varied corresponding to the media, and both inhibitors reduced the intrabacterial ATP content (Fig. 1A). BHI supplemented with 10% FBS (BHI-FBS), a growth-permissive medium, yielded significantly higher intrabacterial ATP levels than the defined medium RPMI supplemented with 3% FBS (RPMI-FBS). The latter medium does not support H. pylori growth, but the bacterium remains vividly motile for several hours. Subsequently, we performed swimming assays of H. pylori wild-type bacteria in RPMI-FBS. Bacterial suspensions were incubated in ambient atmosphere (air enriched with 5% CO2) at 37°C. It has been reported before that in contrast to Enterobacteriaceae, H. pylori cells display no tumbling behavior but show straight runs with intermittent stops or, in certain media, also reversal of swimming direction (backup movement) (25). In RPMI-FBS in the absence of inhibitors, wild-type bacteria showed a characteristic swimming behavior consisting of runs interrupted by frequent stops connected to direction changes (Fig. 1B and D; Table 3), enabling us to describe their behavior in simple terms (CLV and stopping frequency as a measure of behavior). To these bacterial suspensions, the electron transport inhibitors myxothiazol and HQNO were then added during the incubation to assess their effects on behavior. When wild-type H. pylori was incubated with subsequent addition of increasing concentrations (see Materials and Methods) of either one or the other respiratory inhibitor, the inhibitors altered or abolished behavioral responses in a concentration-dependent manner (see Materials and Methods). At a 10 µM concentration of the inhibitors, stops were completely abolished, causing an exclusively smooth-running phenotype (Fig. 1B, C, and E). Under the same conditions, we measured a significant decrease of intrabacterial ATP content (Fig. 1A). This result suggested that smooth running is the null phenotype of H. pylori behavior when severe intracellular energy loss occurs or when electron transport decreases below a certain threshold. The inhibitors at 10 µM prohibited a behavioral response (stops) of the wild type under any medium conditions, although vivid swimming at high CLV was still recorded (Fig. 1B). The effect upon addition of myxothiazol was visible after a delay of approximately 2 min and then persisted for more than 30 min (Fig. 1E), permitting us to analyze the effects of electron donors bypassing the bc1 complex. The addition of a combination of ascorbate (500 µM) and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) (250 µM), which donate electrons directly to cytochrome c, reconstituted the stopping response after myxothiazol treatment (Fig. 1B). It was described before that the H. pylori terminal oxidase is able to oxidize TMPD (36). Ascorbate and TMPD alone did not entail a significant change in stopping behavior in control experiments using RPMI-FBS without inhibitor (not shown).


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  		TABLE 3. Behavioral analysis of H. pylori wild type and tlp mutants (temporal assay)

We used a third defined medium insufficient to support H. pylori growth (buffered RPMI 1640 without serum) as a control to exclude direct serum effects, such as potential repellent effects through serum components. In this medium, the cellular ATP content was lower than that in BHI-FBS and RPMI-FBS (see below) (Fig. 1A). In RPMI without FBS, a smooth swimming response of the wild type with very few stops was observed (Fig. 1D; Table 3). This corresponded to the null response obtained with serum-containing medium in the presence of 10 µM electron transport inhibitors. We also assessed the effects of electron donors as an equivalent for metabolism-dependent electron transport in RPMI without serum. When TMPD and ascorbate were added to this defined medium, bypassing the need for proper electron transport activity by the bacteria, a stopping response was reconstituted in the wild type under ambient oxygen-5% CO2 conditions, similar to the response observed in RPMI-FBS (Fig. 1D). Although not entirely ruling out direct repellent responses to FBS, we concluded from this experiment that the stopping response in RPMI-FBS depended at least partially on energy and electron flow. Notably, a stopping response in RPMI and RPMI-FBS could not be reconstituted by TMPD and ascorbate in the taxis-negative cheAY2 mutant (null control for behavior, which showed only smooth running) and the tlpD mutant (see below).

Construction and characterization of sensor mutants of H. pylori. We next addressed the role of single H. pylori chemosensors in the metabolism-dependent behavioral responses described above. Four orthologs of MCP-like sensor proteins have been identified in the whole genome sequences of H. pylori (3, 53). Since one of those, TlpD, was predicted to be localized entirely in the cytoplasm and to lack a periplasmic sensing domain, due to the absence of hydrophobic transmembrane domains, we suspected it to participate in the detection of intracellular signals, e.g., signals related to the cellular energy status. Therefore, we chose to mutagenize and characterize tlpD in order to elucidate its possible function in energy sensing. In parallel, mutagenesis of the three genes tlpA, tlpB, and tlpC, encoding canonical MCPs, was performed for comparative purposes. We engineered all mutants in three H. pylori strains, 26695, N6, and ATCC 43504, using an established allelic exchange strategy (see Materials and Methods).

All four sensor mutants showed flagellar rotation and translational motility at similar velocities in liquid medium (Table 3). All of them were also able to form concentric chemotaxis halos of similar diameters by moving within motility plates (termed "taxis halos" below), as previously described by others (12, 32), indicating true tactic abilities. cheAY2 mutants displayed flagellar rotation and smooth swimming only (Table 3); no taxis halo was observed in motility plates (not shown). Morphological assessment of the five different mutants by electron microscopy revealed a regular number (four or five) of flagella per bacterium (data not presented). Analyses of sheared flagellar appendages of the receptor mutants in Western blots developed with specific H. pylori anti-flagellin antisera did not show a difference in FlaA and FlaB flagellin expression in comparison to the wild-type strains (data not shown). Using an antibody against the E. coli Tsr sensor (kindly provided by John S. Parkinson), which recognizes the conserved C-terminal MCP domain of chemosensors, we were able to detect expression of three of the four H. pylori MCP orthologs (TlpD, TlpA, and TlpB) and the loss of their expression in the respective H. pylori mutants (Fig. 2 [for TlpD] and data not shown).


Figure 2
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  		FIG. 2. Expression of TlpD detected in fractionated H. pylori by Western blotting. H. pylori wild-type (strain N6) and tlpABC and tlpD mutant bacteria were fractionated into crude soluble (cytoplasmic [SF]) and insoluble (membrane associated [MF]) fractions by differential centrifugation after ultrasonication. TlpD (arrow) was detected by anti-E. coli Tsr antibody (provided by J. S. Parkinson) in the wild type and the tlpABC triple mutant but not in the tlpD mutant. In the wild type and the tlpABC mutant, TlpD was distributed about equally between the cytoplasmic and membrane-associated fractions.

Mutants in the proposed H. pylori sensor TlpD display unique behavioral responses in liquid medium. In order to define the differences in motility and energy-dependent behavior between the wild type and the four chemotaxis sensor mutants more clearly, tracking assays were performed in different liquid media. Since capillary assays in minimal motility media used for enterobacteria have not been found useful for H. pylori, because cells tend to be largely nonmotile or show very slow motility or trapping effects in anaerobic capillaries with standard nutrient-poor motility buffers, we opted for these temporal assays in liquid medium. Again, defined buffered RPMI, RPMI-FBS, and BHI-FBS media were employed.

Bacterial cells grown to early log phase (see Materials and Methods) were incubated in the appropriate liquid media under ambient atmospheric conditions supplemented with 5% CO2. CLV and the number of stops per 3 s were recorded by single-cell tracking to define the behavioral phenotype. In RPMI in the absence of serum, all strains (wild type as well as tlp and cheAY2 mutants) swam in mostly straight runs, almost without any intermittent stops (Table 3), supporting our hypothesis that straight running is the default phenotype for behavior with reduced metabolic activity or electron transport or in the absence of a functional taxis system in H. pylori. Only the tlpC mutant showed a slightly larger number of stops under those conditions (Table 3). When RPMI was supplemented with 3% FBS (permitting higher bacterial ATP content) (Fig. 1A), the wild type and all mutants except tlpD and cheAY2 showed an increased number of stops and direction changes (Table 3). This behavior reversed to the running state again after 10 to 16 h of incubation in the same medium for all strains, possibly as a consequence of nutrient depletion. When RPMI medium without serum was used, the addition of the electron donors ascorbate and TMPD, which simulate electron transport, led to a reconstitution of high stopping frequencies in the wild type and all mutants, except for the tlpD mutant and the cheAY2 negative control (Fig. 3 and Table 3; data not presented).

Hence, solely the tlpD mutants displayed a pronounced and unique phenotype in defined RPMI containing FBS or electron donors. Whereas the wild type and the other mutants did not differ considerably from each other in this medium and showed approximately 6 stops per 3 s (Table 3) and frequent changes of direction, the tlpD mutant showed straight runs, almost without any stops. This behavior resembled the null phenotype of cheAY2 mutants and the behavior of the wild type in the presence of metabolic inhibitors (see above). The velocities of the wild type and tlpD mutants were almost identical under all conditions (Table 3; Fig. 3). The tlpD phenotype was not a behavioral null phenotype, since the mutant displayed stopping when it was incubated in rich medium (BHI-FBS) (Fig. 4), although much less than the wild type. Stopping behavior in rich medium for the wild type and all mutants could also be abolished by the addition of 10 µM of metabolic inhibitors, indicating that it was dependent on electron transport (not shown). When ambient air supplemented with 5% CO2 was replaced by nitrogen flooding to test for the effect of completely anaerobic and CO2-free conditions (nonpermissive for growth of H. pylori), bacterial motility of all strains and mutants ceased altogether, after maintenance of residual sluggish motility for a few seconds. This motility inhibition was quickly reversible to vivid motility with stops when nitrogen was exchanged again for ambient air, with or without 5% CO2.


Figure 4
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  		FIG. 4. Behavior of H. pylori mutants in two different media. Tracking assays were performed with H. pylori wild-type bacteria (N6) and mutants either lacking TlpD or exclusively expressing TlpD (tlpABC mutant) under different medium conditions. The numbers of stops per 3 s are depicted as a measure of behavior. In nutrient-rich medium (BHI-FBS), TlpD appeared to contribute to sensing/behavior to a minor extent, while the other sensors seemed to play a major role. In nutrient-poor medium with serum (RPMI-FBS; lower ATP levels) TlpD appeared to contribute more dominantly to behavior. Mean values and standard deviations for more than 50 bacterial traces are depicted for each single experiment.

In order to test the hypothesis that TlpD alone is sufficient for the behavior observed in RPMI-FBS, we constructed a triple transducer knockout strain in H. pylori N6, lacking transducers TlpA, TlpB, and TlpC, and only expressing native TlpD (for construction details, see Materials and Methods). This strain still expressed TlpD (Fig. 2) and showed stopping behavior in RPMI-FBS, although fewer stops than those observed for the wild type (Table 3; Fig. 4). When the phenotypes of the tlpABC triple mutant and of the tlpD mutant were compared in rich medium (Fig. 4), TlpABC jointly and TlpD alone contributed to a similar, low extent to stopping, but the wild type was about fourfold more active in this behavior, suggesting that TlpABC and TlpD cooperated in a synergistic manner. The behavior of the triple mutant could again be altered or abolished by the addition of myxothiazol and was reconstituted by ascorbate/TMPD after myxothiazol inhibition (Fig. 3). The tlpABC mutant produced a taxis halo in motility agar (not shown), indicating the presence of functional attractant responses induced through TlpD.

Repellent tactic response in the presence of electron transport inhibitors is mediated predominantly by H. pylori TlpD. In order to assess whether stopping behavior in the presence of metabolic inhibitors leads to a true metabolism-dependent tactic response by H. pylori, we performed taxis assays with microscope slide chemotaxis chambers (see Materials and Methods). Prior to those assays, we had determined that low concentrations of myxothiazol (1 to 2.5 µM) caused wild-type bacteria to perform an increased number of stops in all media (BHI-FBS and RPMI-FBS), indicative of an increased repellent response when electron transport was inhibited (see Materials and Methods). Therefore, we used these conditions to test for tactic responses to metabolic inhibition. The chemotaxis chamber we employed allows the formation of a linear gradient of added test substance in a transition chamber between two larger reservoirs (see detailed description in Materials and Methods). When a gradient between 0 and 10 µM of myxothiazol was established in this chamber for a 2-h period (see Materials and Methods), wild-type bacteria did not transmigrate into the second, inhibitor-containing reservoir (only 1.4 ± 0.2 bacteria counted), while control experiments without inhibitor permitted unhindered passage of bacteria to the second reservoir (63 ± 9.2 bacteria counted). When the same conditions were applied to tlpD mutants, they did readily transit to the second reservoir containing inhibitor (24 ± 4.9 bacteria counted with myxothiazol, compared to 37 ± 8.6 bacteria under control conditions), indicating that the observed repellent response did not work efficiently in the mutant and hence is at least partially dependent on TlpD. For tlpABC mutants, we observed an intermediate phenotype between those of wild-type bacteria and the tlpD mutant, with significantly fewer bacteria transmigrating under myxothiazol conditions (26.3 ± 1.15 bacteria counted) than under control conditions (48 ± 4.8 bacteria counted).

Complementation of H. pylori tlpD in trans and detection of a tagged TlpD protein by fluorescence microscopy and bacterial fractionation. The H. pylori N6 tlpD mutant was complemented by reintroducing tlpD in trans, as a translational fusion to a V5 immunodetection peptide tag cloned in the E. coli/H. pylori shuttle plasmid pHel2 (see Materials and Methods). TlpD-V5 expression of the complemented clones (tlpD/TlpDV5) was assessed by Western blotting and subsequent detection, using an anti-V5-tag monoclonal antibody (Fig. 5A). All clones expressed the TlpD-V5 fusion protein with a molecular mass of approximately 52 kDa, as predicted, which was slightly larger than wild-type TlpD (Fig. 5A, lanes 3 and 6). In order to assess whether the behavioral phenotype of the complemented tlpD mutants was reconstituted back to the wild-type phenotype, we again used a tracking assay with buffered RPMI medium with 3% FBS. A behavioral response with an increased frequency of stops was reconstituted (Fig. 3). The complemented mutants showed slightly lower stop frequencies (mean, 3.42 ± 1.4 stops per 3 s) than did the wild type (Fig. 1 and 3).


Figure 5
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  		FIG. 5. Localization of TlpD in wild-type and complemented H. pylori by fractionation and fluorescence microscopy. (A) Localization of TlpD in wild-type bacteria in comparison to the complemented strain tlpD/TlpD-V5 (both in the strain N6 background), using bacterial fractionation. TlpD-V5 expression was detected using anti-V5 antibody (upper panel) and anti-E. coli Tsr antiserum, while intrinsic native TlpD was detected only using anti-E. coli Tsr antiserum (lower panel). The proportion of overexpressed TlpD-V5 was higher (approximately fivefold) in the soluble fraction (SF) than in the insoluble fraction (MF). Black arrows, native TlpD; open arrows, TlpD-V5. Five micrograms of protein was loaded in each lane. (B) Crude fractionation of cheAY2 mutant, in which TlpD was expressed as in the wild type but membrane association of TlpD was no longer detectable. (C) TlpD-V5 was detected in intact bacteria by immunofluorescence labeling (anti-V5 antibody and secondary goat anti-mouse immunoglobulin G antibody coupled to Alexa 488) of fixed and permeabilized tlpD/TlpD-V5 bacteria (strain N6). Bar, 5 µm.

The expression of a tagged functional TlpD protein in trans now facilitated testing for the localization of the unorthodox sensor protein TlpD, which appears to be devoid of transmembrane domains. Whole bacteria (N6 wild type and N6 tlpD/TlpD-V5) were separated into insoluble (membrane-enriched) and soluble (cytoplasmic) fractions by sonication and centrifugation. The fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to membranes for detecting native and V5-tagged TlpD. Immunodetection of native TlpD on Western blots by anti-E. coli Tsr revealed an about equal distribution of TlpD in the insoluble fractions and soluble fractions in wild-type H. pylori (Fig. 5A), indicating that the protein may be localized at least partially close to the membrane. In TlpD-V5-expressing bacteria, an approximately fivefold overexpression of TlpD-V5 in comparison to nontagged TlpD in the wild type was observed, probably due to a gene copy effect (Fig. 5A). The overexpressed tagged TlpD was not equally distributed between the insoluble and soluble fractions but was more abundant in the soluble, cytoplasmic fraction. While the inactivation of tlpABC (in the triple mutant) appeared not to disturb the localization of native TlpD (Fig. 2), native TlpD was not detected in the membrane fraction in a cheAY2 mutant (Fig. 5B). The distribution of TlpD was further analyzed in intact bacteria in situ by immunofluorescence labeling of fixed and permeabilized TlpD-V5-expressing bacteria (N6 tlpD/TlpD-V5), using an anti-V5 antibody. Very specific detection by immunofluorescence confirmed that TlpD-V5 was uniformly present in the cytoplasm and appeared to be accumulated around the bacterial periphery (Fig. 5C). This membrane-proximal portion of TlpD-V5 did not show a predominantly polar or patchy localized distribution, as shown for other MCP-like proteins, but appeared to be distributed throughout the whole circumference of the bacterium, with labeling, in some cases, of polar or subpolar cap-like structures.


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DISCUSSION
 
In this study, interference between energy metabolism and tactic behavior in H. pylori was investigated, owing to the earlier finding that this bacterium performs pH sensing in vivo (45) and in vitro (12). We found that H. pylori responded to alterations in energetic conditions by altered swimming behavior, and its MCP-like sensor molecule TlpD emerged as an important factor involved in energy sensing.

H. pylori has a limited capacity to generate energy aerobically, which matches its quite restricted habitat in the lower layers of the human gastric mucus (45). It is a microaerophilic capnophilic bacterium and is able to thrive even under very-low-oxygen conditions, when CO2 is available, by fixing CO2 and generating pyruvate (48). It possesses one single terminal oxidase, which is of the Cbb3 type, which presumably is optimized to work under microaerobic conditions. The bacterium can also generate energy aerobically directly from molecular hydrogen with the help of a hydrogenase (40). Moreover, H. pylori is able to use fumarate as an electron acceptor (17, 33). No redox regulator Fnr homolog which might regulate the Cbb3 terminal oxidase has been identified, implying that its expression is constitutive. Addressing energy sensing by analyzing mutants in cbb3 subunit genes was not successful in our hands, since such mutants could not be generated, indicating that Cbb3 is essential for bacterial proliferation. The described alternative metabolic pathways for energy generation under aerobic or anaerobic conditions may be accessory pathways, which provide additional energy for specific purposes, such as motility or enzyme activation (38, 41). The limited range of metabolic capacities available indicates that the environmental requirements of H. pylori for energy generation, including acidity and atmospheric conditions, may be quite strict. This again stresses the importance of sensing capacities that may guide the bacterium to pH and atmospheric conditions optimal for energy generation. In wild-type and all chemosensory mutant bacteria that we tested, and in various media, the behavioral responses of H. pylori were altered or completely abolished by metabolic inhibitors. cheA mutants (cheAY2) served as a taxis negative control in all media and allowed for the conclusion that smooth swimming is the taxis null phenotype of H. pylori. By measuring metabolic activity by determining the ATP content of H. pylori under various conditions, we showed that intrabacterial ATP content in the absence of serum was reduced, similar to the case in the presence of serum combined with metabolic inhibitors, and that reduced ATP levels could be elevated again by the addition of electron donors. This strengthened our hypothesis that metabolism and electron transport are intimately connected to behavioral phenotypes that differed under those conditions. Intermediate energetic (not growth-permissive) conditions supported robust behavioral responses (stopping) in the wild type, in tlpB, tlpA, and tlpC single mutants, and in the tlpABC triple mutant, which could be inhibited by electron transport inhibitors. Bypassing the need for intrinsic metabolic activity by supplying electron donors rescued behavioral abilities in defined medium without serum for all strains and mutants (single sensor mutants as well as the triple tlpABC mutant), except for the tlpD mutant, supporting our hypothesis that a specific activity of the electron transport chain, jointly with TlpD, is important for H. pylori behavior triggered by energy sensing. Stopping behavior and repellent taxis under conditions of reduced electron transport activity were mediated predominantly by TlpD and were at least partially lacking in tlpD mutants, which still possessed the other sensors. These results suggested that low activity of the electron transport chain provides a repellent signal transmitted via TlpD but that energy-dependent taxis is not mediated exclusively via TlpD. In tlpABC mutants, TlpD-dependent stopping responses were largely retained, which rules in favor of an important functionality of TlpD which is quite independent of the other sensors. The energy-dependent behavioral phenotype which is provided by TlpD appeared to be particularly dominant under conditions of low energy yield (e.g., RPMI-FBS medium). tlpD mutants were still able to provide stopping responses in rich medium to a similar extent as the triple tlpABC mutant, indicating that the tlpD phenotype is not a taxis null phenotype. When an all-transducer knockout, lacking all four known H. pylori sensors, was established (T. Schweinitzer, T. Mizote, and C. Josenhans, unpublished data), the stopping phenotype did finally change to smooth swimming in all media. It was also not possible to rescue stopping behavior by TMPD and ascorbate in the all-transducer knockout, which behaved similarly to a cheAY2 mutant.

How could TlpD work in energy sensing? TlpD appeared to be involved predominantly in repellent sensing under conditions of impaired electron transport and was sufficient to produce taxis halos in motility agar. It is not possible yet to conclude whether TlpD itself is a sensor or whether it is aided by other proteins. Studies with E. coli and other bacteria (Bacillus and Halobacterium) unraveled that energy taxis/energy sensing can be mediated by at least the following three different mechanisms: (i) heme-like sensors, such as HemAT (21), which directly bind oxygen-containing heme; (ii) redox sensors (Aer-like, containing PAS domains) which bind flavin adenine dinucleotide (7) and are strongly influenced by the activity of the NADH dehydrogenase I complex (14); and (iii) Tsr-like sensors sensing protons of the bacterial membrane potential (PMF) (14). TlpD, like the energy and redox sensor Aer (7, 18), does not have transmembrane domains. However, TlpD shares only little similarity with Aer or HemAT-like sensors. In contrast to Campylobacter jejuni (31) or enterobacteria, none of the H. pylori sensor proteins, including TlpD, possesses a PAS domain. H. pylori TlpD appears to be a rather unique protein, although similar sensor proteins lacking transmembrane domains have been identified in C. jejuni (Cj448c) (31) and Helicobacter hepaticus (HH0891) (49), which also lacks PAS domain proteins. Functional information about these homologs is as yet lacking. It is quite likely that TlpD mediates sensing of a signal with the help of the electron transport chain. As described for E. coli Aer (14), the NADH dehydrogenase complex I, which has some specific properties in {varepsilon}-proteobacteria and in H. pylori (47), may be involved in the sensing mechanism jointly with TlpD. The possibility that H. pylori TlpD senses the energy status by direct ligand binding cannot be excluded and will be explored in future work. From the present results, PMF, redox, or electron flow as a signal for TlpD-mediated sensing cannot be ruled out (14). A small number of histidine and cysteine residues, potentially able to bind heme or protons, are present in the N-terminal domain of TlpD. Oxygen is probably not the signal sensed with the help of TlpD, since the TlpD-dependent stopping response was observed as long as CO2 was present, even in the complete absence of oxygen (T. Schweinitzer and C. Josenhans, unpublished data). As a second, alternative mechanism to direct substance binding, TlpD may sense a conformational change of the membrane itself or of a protein which is influenced by differential activity of the electron transport chain. A third possibility is an indirect role in sensing, for which TlpD could collaborate with an unidentified (membrane) protein, which may either bind a substance or undergo a conformational change in response to altered energetic conditions. However, a novel protein which fulfills the latter criteria for a TlpD partnering function has not been identified in H. pylori. The H. pylori protein-protein interaction database (http://pim.hybrigenics.com) reports experimental evidence for two interaction partners of TlpD, namely, HP0697 and HP1033. HP1033 localizes in a gene cluster associated with polar expression of flagellar components (38). HP0697, which bears some homology to NAD synthases and also interacts with a hydrogenase accessory protein, HypF (41), may provide some indirect connection of TlpD to metabolic functions and electron transport. These possible interactions linked to flagellar function or energy metabolism need to be investigated further.

By expressing a functional, tagged version of TlpD in H. pylori from a plasmid, we gained some insight into the localization of the unusual sensor, which could also be confirmed by detecting native TlpD in bacterial fractions. In Enterobacteriaceae, the polar clustered localization of chemosensors has been demonstrated using microscopy and biochemical studies (22, 29, 30). In contrast to what has been observed for other chemosensory proteins, we did not observe a clearly polar or clustered localization for TlpD, but an even distribution throughout the whole bacterium, with a uniform zone of accumulation close to the membrane and infrequent polar cap-like structures. Since immunofluorescence detection could be performed only in a setting where TlpD was overexpressed, it is possible that the distribution of tagged TlpD was influenced by overexpression. The membrane-associated portion of wild-type native TlpD was about equal to the portion of the overexpressed TlpD-V5 that associated with the membrane, indicating that membrane localization of TlpD may be determined by the limited abundance of an interaction partner at the membrane. In a tlpABC mutant, native TlpD was still expressed and localized similarly to the wild type, as determined by fractionation, indicating that the other Tlps are not required for stabilizing or localizing TlpD at the membrane.

CheAY2 appeared to contribute to membrane localization of TlpD. Foynes et al. described phenotypes for different single-gene and double mutants in the H. pylori cheA gene (strains N6 and SS1) and the two H. pylori cheY genes (cheY1 and cheY2), using tracking microscopy (16). cheY2 is a cheY gene fusion at the 3' end of the cheA gene (cheAY2) (16). All single and double mutants carrying a mutation in cheY2 showed a phenotype of elongated path lengths compared with the wild type, as well as a higher CLV, similar to the phenotype we observed for the tlpD mutant in defined medium. The single cheY1 mutant, in contrast, had shown shortened path lengths and a lower CLV than the wild type, indicating antagonistic roles for CheY1 and CheY2. The cheY2 mutant phenotype was dominant over the cheY1 mutant phenotype in all cases. These results suggested that the CheY2 domain, in addition to CheA, is apparently involved in increasing the stopping response, which was also enhanced by TlpD. Whether a direct interaction of TlpD with the CheY2 domain occurs will have to be investigated in future work.

Our results suggest that sensing and taxis in H. pylori are at least partially metabolism dependent and require a functional electron transport chain, which may contribute to the deleterious effects of mutations in the taxis system on stomach colonization shown in vivo (26). Three H. pylori sensors, TlpB (12), TlpA (9), and TlpC, have been found to be required for colonization of the mouse or gerbil stomach or at least to contribute to more efficient colonization (4, 12, 32, 42) in vivo. As we show in the present work, TlpD has a dominant function in metabolism-dependent sensing and behavior. Together with TlpB (12), at least two of the four receptors in H. pylori contribute to energy taxis, but since three of those sensors possess periplasmic sensing domains, they are also supposed to provide true chemosensing properties. Presumably, those functions are displayed predominantly under conditions of high metabolic activity, and it will be fascinating to unravel their contribution to taxis in vitro and to vertical or horizontal spread of H. pylori in the stomach habitat.


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ACKNOWLEDGMENTS
 
We are indebted to Mark Johnson, Barry Taylor, Kylie Watts, Lynn Schwarzer, and Eike Niehus for helpful discussions and to two anonymous reviewers for valuable comments. Daniela Goeppel, Susanne Friedrich, Verena Ryan, and Claudia Nauznicov are acknowledged for skillful experimental assistance, and Minato Nakazawa is acknowledged for some statistical analysis.

This work was supported by a Bennigsen-Foerder Award of the Federal State of Nordrhein-Westfalen (Germany) to C. Josenhans and S. Schreiber as well as by grants Jo 344/2-2 and Su 133/2-3 (Gerhard Hess Award) from the Deutsche Forschungsgemeinschaft. Funding by the Urakami Foundation (Japan) to T.M. is gratefully acknowledged.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Medical Microbiology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. Phone: 49 511532 4348. Fax: 49 511532 4355 E-mail: josenhans.christine{at}mh-hannover.de Back

{triangledown} Published ahead of print on 1 February 2008. Back

{dagger} T.S. and T.M. share first authorship; T.M., T.S., and C.J. contributed equally to this study. Back


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Journal of Bacteriology, May 2008, p. 3244-3255, Vol. 190, No. 9
0021-9193/08/$08.00+0     doi:10.1128/JB.01940-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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