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Applied and Environmental Microbiology, April 2008, p. 2095-2102, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.01348-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development of Inducible Systems To Engineer Conditional Mutants of Essential Genes of Helicobacter pylori{triangledown} ,{dagger}

Ivo G. Boneca,1* Chantal Ecobichon,1 Catherine Chaput,1,{ddagger} Aurélie Mathieu,1,3 Stéphanie Guadagnini,2 Marie-Christine Prévost,2 Frédéric Colland,4 Agnès Labigne,1 and Hilde de Reuse1

Institut Pasteur, Unité de Pathogénie Bactérienne des Muqueuses, Department of Microbiology, Paris, France,1 Institut Pasteur, Plate-forme de Microscopie Electronique, Department of Cell Biology and Infection, Paris, France,2 UMR 217 CNRS/CEA, Department of Radiobiology and Radiopathology, Commissariat à l'Energie Atomique, Fontenay aux Roses, France,3 Hybrigenics SA, Paris, France4

Received 18 June 2007/ Accepted 25 January 2008


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ABSTRACT
 
The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacIq-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacIq repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring β-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-β-D-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.


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INTRODUCTION
 
Helicobacter pylori is a naturally competent and transformable species, thus permitting genetic manipulation to construct mutant strains. Researchers have explored the genomic information to study in detail H. pylori's physiology, virulence, and adaptation to the stomach mucosa, as illustrated by several global genetic screens for essential genes either in vitro or in vivo (18, 19, 31) and proteomic (1, 3, 28, 30) and transcriptome (4, 23-25, 38) studies. This wealth of new information on H. pylori has improved the annotation of the genomes by reducing the percentage of genes of unknown function from 40% in 1997 (39) to 30% in 2003 (2). Several groups have been able to demonstrate the essential nature of certain genes by introducing a second copy of them into the chromosome and inactivating the original loci, but such a strategy did not give access to the physiological role of the encoded proteins. The lack of a suitable genetic system to control gene expression in H. pylori has been a handicap to study the roles of essential genes in H. pylori physiology. Attempts to overcome this problem were addressed by Delany and colleagues using the endogenous H. pylori promoter of the iron-regulated pfr gene to induce the expression of HP1043RR (9). However, H. pylori has very few transcriptional regulators that respond to changes in the environment, such as iron, nickel concentrations, and external pH (7, 26, 27, 40). These transcriptional regulators are central for H. pylori response and adaptation to its ecological niche and modulate the expression of an important number of genes (4, 14, 23, 26). Hence, the use of endogenous inducible promoters that respond to iron or nickel concentrations results in pleiotropic effects on H. pylori and would complicate interpretation of conditional mutant phenotypes.

To overcome the lack of suitable endogenous inducible promoters, we developed an inducible system based on the lacIq-pTac system of E. coli using the pHeL2 E. coli-H. pylori shuttle vector (16), in which expression of lacIq and lacZ were under the control of modified endogenous H. pylori promoters. Several plasmids were constructed allowing different levels of gene expression. To validate these tools, we constructed conditional mutants of the genes encoding the essential penicillin-binding protein 1 (PBP1) and PBP3 of H. pylori. Depletion of PBP1 led to premature coccoid formation while depletion of PBP3 led to filamentation, as expected from their predicted functions. Hence, we were able to construct the first conditional mutants of H. pylori.


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MATERIALS AND METHODS
 
Bacteria, cells, and growth conditions.
Escherichia coli MC1061 (5) was used as host for the construction and preparation of plasmids. Chemically competent E. coli was generated as previously described (5). E. coli was cultivated in Luria-Bertani liquid or solid medium supplemented when appropriate with chloramphenicol (30 µg/ml). H. pylori strain N6 (15) was used to construct conditional mutants. Several different isolates of H. pylori were used as the recipient for transformation by in vitro-methylated inducible plasmids (Table 1). H. pylori DNA was extracted from strain 26695 (39) using the QIAamp DNA purification kit (Qiagen). Bacteria were grown microaerobically (CampyGen; Oxoid) at 37°C on blood agar plates or in liquid medium consisting of brain heart infusion (Oxoid) with 0.2% β-cyclodextrin (Sigma) supplemented with antibiotic-antifungal mix (4). H. pylori mutants were grown with 20 µg/ml kanamycin and/or 4 µg/ml chloramphenicol when required.


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  		TABLE 1. Strains and plasmids used in this study

Construction of plasmids.
The shuttle vector pHeL2 (16) was used as a scaffold to construct the expression systems for H. pylori. For each construction, the high-fidelity (HF)-PCR products were amplified from different templates (see below) using the primers described in Table S1 of the supplemental material and as recommended by the manufacturer (Roche). The HF-PCR products and vectors were both digested with the restriction enzymes as recommended by the manufacturers, the linearized plasmids were dephosphorylated with alkaline phosphatase (Roche), and the mixtures were ligated using T4 ligase (Roche). The ligation mixtures were used to transform competent E. coli. After PCR screening, a clone was selected and the insert was sequenced to ensure sequence fidelity.

The lacIq-pTac region was amplified by HF-PCR from plasmid pMAL-C2X (Amersham) using primers 1552 and 1553 (see Table S1 in the supplemental material) and cloned between the BglII and SphI sites of pHeL2. The resulting plasmid was digested with NdeI and BamHI, dephosphorylated, and ligated to the lacZ gene previously amplified by HF-PCR from E. coli chromosomal DNA using primers 1556 and 1557. The resulting plasmid was named pHeL2-TL (Table 1 and Fig. 1; see also Fig. S1 in the supplemental material). All other plasmids were derived from pHeL2-TL (Table 1).


Figure 1
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  		FIG. 1. Maps of plasmids pHeL2-TL, pILL2150, pILL2151, pILL2153, and pILL2157. These plasmids have the same backbone, the pHeL2 vector constructed by Heuermann and Haas (16). pILL2150, pILL2153, and pILL2157 plasmids carry the lacIq gene under the control of the H. pylori endogenous amiE gene promoter cloned in the unique HindIII restriction site. Plasmids pHeL2-TL and pILL2150 (11.15 kb) carry in the MCS between BglII and BamHI a promoterless lacIq gene and the pTac promoter controlling the expression of the reporter lacZ gene. Plasmids pILL2151, pILL2153, and pILL2157 carry in the same MCS between BglII and BamHI the lacZ gene under the control of a modified ureI gene promoter with one (pILL2151 and pIL2153) or two (pILL2157) LacI-binding sites. LacI-binding sites are schematically depicted as black boxes. For more details on the maps refer to the figures in the supplemental material.

pILL2150 was generated from pHeL2-TL as follows: (i) lacIq was amplified by HF-PCR using primers lacI(q)-RBS-PacI-2 and lacI(q)-HindIII and pHeL2-TL as DNA template; (ii) the amiE gene promoter region was amplified from H. pylori strain 26695 DNA using primers pamiE-HindIII and pamiE-PacI; (iii) both PCR products were digested with HindIII and PacI and the pHeL2-TL plasmid was digested with HindIII; (iv) the two PCR fragments were cloned into the unique HindIII site of pHeL2-TL. A clone was selected, and the resulting plasmid, pILL2150, was purified (Fig. 1; see also Fig. S2 in the supplemental material).

pILL2151 and pILL2153 were generated from pHeL2-TL as follows: (i) the ureI promoter region was amplified by HF-PCR using primers pureI-lacI(op)-BglII and pureI-lacI(op)-SpeI and the H. pylori strain 26695 DNA as template; (ii) the PCR product and plasmid pHeL2-TL were digested with BglII and SpeI, dephosphorylated, and ligated; (iii) a clone was selected, and the resulting plasmid, pILL2151, was purified (Fig. 1; see also Fig. S3 in the supplemental material). The pILL2153 plasmid was generated from pILL2151 by cloning the pamiE-lacIq region from pILL2150. The pILL2150 and pILL2151 plasmids were linearized by HindIII digestion. pILL2150 generated two fragments (a 2.85-kb and an 8.3-kb fragment). The 2.85-kb fragment was purified from agarose gel using the QIAquick gel extraction kit (Qiagen) and religated to the HindIII-linearized pILL2151 plasmid. The resulting plasmid, pILL2153, was purified (Fig. 1; see also Fig. S4 and S5 in the supplemental material).

Plasmid pILL2157 was constructed from plasmid pILL2150 as described above for pILL2153, except that the ureI promoter region was amplified using the following primers: pureI-lacI(op)-BglII and pureI-lacI(2op)-NdeI/SpeI. The plasmid and the promoter sequence are illustrated in Fig. 1; see also Fig. S6 and S7 in the supplemental material.

Plasmids pILL2161 and pILL2163 were constructed as follows: pbp1 and ftsI were amplified by HF-PCR using the primer pairs pbp1-NdeI with pbp1-BamHI and ftsI-NdeI with ftsI-BamHI, respectively. The PCR products and plasmid pILL2150 were cloned between the NdeI and BamHI sites.

Construction of mutants.
Transformation by plasmids and gene disruption in H. pylori were performed by natural transformation as previously described (34). H. pylori mutants were constructed by transformation of the different plasmids into strain N6 and selected on blood agar plates containing chloramphenicol (8 µg/ml). Inactivation of the chromosomal copy of each gene was performed by allelic exchange after transformation with a three-fragment assembly product (6, 11) carrying the gene of interest flanking regions and the nonpolar kanamycin aphA-3 cassette (34). Selection was done on blood agar plates containing kanamycin (20 µg/ml) with or without isopropyl-β-D-thiogalactopyranoside (IPTG; 1 mM). The clones from IPTG-containing plates were screened by PCR to ensure that the chromosomal locus of interest had been properly deleted.

In vitro methylation of pILL2150 and pILL2157 and transformation into different H. pylori strains.
The different strains (Table 1) were grown on blood agar plates with antibiotic-antifungal mix (4). In vitro methylation of plasmid DNA was performed as previously described (13) with some modifications. Bacteria were harvested from the plates and washed twice with 1x phosphate-buffered saline (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl). The pellets were resuspended in 500 µl of ice-cold 1x methylation buffer (20 mM Tris-acetate pH 7.9, 50 mM potassium acetate, 15 mM EDTA, 1 mM dithiothreitol), sonicated for 30 seconds on ice, and centrifuged for 5 min at 14,000 rpm at 4°C. Supernatants were collected and used immediately to methylate plasmid DNA (protein extract concentration range, 13 to 19 µg/µl). Methylation of DNA (20 µg) was performed with 260 to 380 µg of protein extracts in 1x methylation buffer with 0.4 mM S-adenosyl-L-methionine (freshly prepared) for 1 h at 37°C. Intact plasmid DNA was purified using the Qiaprep Spin miniprep kit (Qiagen). To transform methylated plasmid DNA, the corresponding recipient strains were grown on blood agar plates and concentrated in peptone water at an optical density at 600 nm (OD600) of 30. Bacteria (15 µl) were mixed with plasmid DNA (1 µg) and plated as a spot for 18 h on nonselective medium (blood agar plates). Each spot was resuspended in 500 µl of peptone water and plated on blood agar plates (150 to 200 µl per plate) supplemented with chloramphenicol (8 µg/ml). In parallel, untreated DNA was used as a negative control of the transformation efficiency. Clones appeared on plates after 5 to 7 days of incubation under microaerophilic conditions.

Transcriptional start point mapping by primer extension.
Total RNA of H. pylori strain 26695 was extracted as previously described (4). Primers H23 and H32 (Table 1) were 5'-radiolabeled with 50 µCi of [{gamma}-32P]ATP (specific activity, 5,000 Ci/mmol; Amersham Biosciences) by T4 DNA kinase (USB) as described by (32). One pmol of each labeled primer was added to 15 µg of H. pylori RNA, heated for 5 min at 95°C, and further incubated at 70°C during 1 h to allow hybridization. Reverse transcription was performed on this template with 10 U of avian myeloblastosis virus reverse transcriptase (Roche Applied Sciences) in the provided reverse transcriptase buffer with 1 mM of each deoxynucleoside triphosphate and 20 U of RNase inhibitor (RNAguard; Pharmacia). The reaction mixture was incubated for 1 h at 42°C, stopped by a 10-min passage at 75°C, and treated with RNase A. After a phenol-chloroform extraction, cDNAs were ethanol precipitated and suspended in 8 µl of sequencing dye (Amersham Biosciences). These samples were subjected to gel electrophoresis on a 6% urea-Tris-Taurine-EDTA gel. Sequencing reaction mixtures were electrophoresed in parallel to these samples; they were obtained by annealing the labeled primers used for the primer extension to DNA of a plasmid carrying the entire urease gene cluster and performing manual sequencing with the T7 sequencing kit (Amersham Biosciences).

Measurement of β-galactosidase activity.
At different time points of growth, the OD600 was measured and 0.5 ml (exponentially growing) or 0.2 ml (stationary phase) of bacterial culture was mixed with PM2 buffer (0.07% β-mercaptoethanol, 100 mM NaH2PO4, 1 mM MgSO4, 0.2 mM MnSO4 at pH 7) up to a total volume of 2 ml. Bacteria were lysed by addition of 50 µl chloroform and 50 µl of sodium dodecyl sulfate at 0.05%. PM2 buffer was added to the bacterial extracts up to a total volume of 2 ml prewarmed at 28°C. At time T0, 0.5 ml of orthonitrophenyl-β-galactopyranoside at 0.4% was added. The reaction was stopped at time Tf with 1 ml of 1 M Na2CO3. The OD was measured at 420 nm and 550 nm. β-Galactosidase activity was calculated as follows: [OD420 – (1.75 x OD550)] x (dilution factor) x 1,000/[OD600 x (Tf – T0)], expressed in Miller units.

Scanning electron microscopy.
Bacteria were washed with phosphate-buffered saline (pH 7.4), prefixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 30 min, and then rinsed in 0.2 M cacodylate buffer. After postfixation in 1% osmium tetraoxide (in 0.2 M cacodylate buffer), bacteria were dehydrated in a series of increasing ethanol concentrations. Specimens were critical point dried using carbon dioxide, then coated with gold, and examined with a JEOL JSM-6700F scanning electron microscope.

Nucleotide sequence accession numbers.
The sequences of plasmids pILL2150 and pILL2157 were deposited at GenBank and are available under the accession numbers EU423134 and EU423135, respectively.


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RESULTS AND DISCUSSION
 
H. pylori only uses glucose as a sugar carbon source and has no cyclic AMP-based catabolic repression system, as in Escherichia coli (22). Hence, the use of inducible systems that respond to other sugars, such as arabinose, widely used in molecular genetics, was unlikely to work for H. pylori. In contrast, establishing an inducible system of expression on the lacIq regulator had the advantage of being completely independent of any endogenous regulation. The only potential limitation would be the accessibility of the IPTG inducer to the H. pylori cytoplasm. However, since 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside had been used successfully to reveal expression of cytoplasmic β-galactosidase as a reporter for promoter regulation (12), we reasoned that IPTG would also be able to access the cytoplasm.

Construction of inducible expression systems.
To construct the inducible expression system for H. pylori, the lacIq pTac system was amplified from pMAL2-c2X and cloned into the pHeL2 E. coli-H. pylori shuttle vector constructed by Heuermann and Haas (16). As a reporter gene, we amplified and cloned the lacZ gene between the SpeI site and the BamHI site of the pHeL2 multicloning site (MCS). The pHeL2-TL plasmid (Fig. 1; see also Fig. S1 in the supplemental material) was introduced by natural transformation into the recipient H. pylori strain N6. β-Galactosidase activity was measured at different time points of growth in the presence of IPTG (Fig. 2 and data not shown). Detection of β-galactosidase activity was dependent on the addition of IPTG. However, the pHeL2-TL plasmid raised two problems: (i) the production of β-galactosidase was low (200 Miller units), and (ii) we observed a residual β-galactosidase activity without addition of IPTG. We reasoned that these two problems were related to a lack of recognition of E. coli promoters by H. pylori RNA polymerase. Nakazawa and colleagues have shown that the problems of promoter recognition are related to differences between E. coli and H. pylori major sigma factors encoded by the rpoD genes (33).


Figure 2
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  		FIG. 2. β-Galactosidase activity of strain N6 carrying either pILL2150, pHeL2-TL, pILL2151, pILL2153, or pILL2157 in the presence or absence of IPTG (1 mM) after 24 h of growth. Experiments were repeated at least three times, and measurements of β-galactosidase activity represent the mean values of triplicates of one representative experiment. Maps of the plasmids are available in Fig. 1 and also in the figures in the supplemental material.

Thus, to solve the leakiness of the pHeL2-TL plasmid, we decided to add a second copy of lacIq under the control of the amiE promoter, generating plasmid pILL2150 (Fig. 1; see also Fig. S2 in the supplemental material). As shown in Fig. 2, β-galactosidase activity was tightly controlled and strictly dependent on the presence of IPTG. Addition of IPTG resulted in a measurable β-galactosidase activity (around 100 Miller units), albeit lower than that with pHeL2-TL. Therefore, plasmid pILL2150 could be used to control the expression of H. pylori essential genes to study the physiological role of their encoded proteins under depletion conditions (absence of IPTG), particularly for genes encoding low-abundance proteins.

Next, we aimed at improving the level of β-galactosidase production, to be able to use the plasmids to control the production of recombinant proteins, protein-protein interaction domains under native conditions, or the expression of antisense RNAs. In plasmid pILL2150, only the second lacIq was under control of an endogenous H. pylori promoter, namely, pamiE. Thus, to replace in pILL2150 the lacIq-pTac region by another endogenous H. pylori promoter, we chose the strong ureI promoter. Transcription start and promoter mapping by primer extension (Fig. 3) of the ureI promoter region led to the design of primers pureI-lacI(op)-BglII and pureI-lacI(op)-SpeI, in which a LacI-binding site was introduced that did not affect the –35 and –10 boxes. We thus amplified the ureI promoter using these primers and cloned it in both pHeL2-TL and pILL2150 by replacing the lacIq-pTac region, leading to pILL2151 and pIL2153, respectively. As shown in Fig. 2, introduction of the LacI-binding site did not affect the pureI functionality (pILL2151) (Fig. 1, map; see also Fig. S3 in the supplemental material) with a high production of β-galactosidase activity (14,000 to 16,000 Miller units). In the presence of LacIq (pILL2153) (Fig. 1, map; see also Fig. S4 and S5 in the supplemental material), β-galactosidase production remained at high levels (8,000 Miller units, 80-fold greater than with pILL2150) but became IPTG dependent.


Figure 3
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  		FIG. 3. Primer extension and mapping of the ureI promoter. Primer extension was performed with two distinct primers, H23 and H32 (lanes 2 and 3). Lanes 1 and 4 are negative controls in which the primers were omitted from the reaction mixture. The sequencing reactions were done using primer H32. Primer extension with H32 (lane 3) allowed determination of the +1 region by using the sequencing reaction (right sequencing reaction), while that with H23 (lane 2) allowed refinement of the +1 position by determining the exact length of the amplified product (H32 left sequencing reaction was used as a molecular ladder). The promoter region of the ureI gene is represented with the precise location of the –35, –10, and +1 positions of H23 and H32 primers and the ATG start codon. Nucleotides in italics correspond to the 5' end of the ureI coding sequence.

However, repression was not efficient enough to allow study of, for example, the effect of overexpressing protein-protein interaction domains on H. pylori growth. In the absence of IPTG, pILL2153 expressed reasonable amounts of β-galactosidase activity (around 2,000 Miller units). Hence, we decided to introduce a second LacI-binding site. Simultaneously, we inverted two nucleotides at positions –3 and –2 from AC to CA to restore the NdeI site, generating plasmid pILL2157 (Fig. 1; see also Fig. S6 and S7 in the supplemental material). As shown in Fig. 2, both the nucleotide inversion and the second LacI-binding site improved substantially the regulation of the lacZ gene and the β-galactosidase production compared to pILL2153. Plasmid pILL2157 still remained leaky, and β-galactosidase activity (250 Miller units) was significant without IPTG. However, the level of β-galactosidase activity was on the same order of magnitude as that of pILL2150 in the presence of IPTG (100 Miller units). During a time course induction of the β-galactosidase activity, activity reached a peak after 24 h and remained stable up to 42 h (see Fig. S8A in the supplemental material). Hence, pILL2157 could be used to construct conditional mutants of highly expressed genes, H. pylori recombinant proteins, interacting protein-protein domains under native conditions, or antisense RNAs, while pILL2150 could be useful for genes expressed at low levels. In recent work, we have been able to show that the amount of an H. pylori protein could be titrated by playing with the IPTG concentration and the plasmid used (either pILL2150 or pILL2157) (41). However, a limiting step for these two plasmids might be the cloning of H. pylori genes that could be toxic for E. coli. As shown in Fig. S8B of the supplemental material, both plasmids are leaky in E. coli, particularly pILL2157. This might reflect the lack of recognition of the amiE gene promoter by E. coli RNA polymerase and, consequently, poor expression in E. coli of the LacI repressor. To overcome potential toxicity problems, it is suggested that E. coli strains that carry a highly expressed LacI repressor be used. Note that both plasmids carry an oriColE1 origin of replication.

Engineering of conditional mutants.
To validate our new genetic tools, we decided to construct conditional mutants of genes though to be essential. We selected the pbp1 and ftsI genes. Indeed, repeated attempts to inactivate these genes with the nonpolar kanamycin cassette were unsuccessful, suggesting their essential nature (Table 2). We cloned these two genes in pILL2150, generating the plasmids pILL2161 and pILL2163 carrying, respectively, the pbp1 and ftsI genes. The resulting plasmids (Table 1) were transformed into H. pylori strain N6 and generated strains carrying two copies of each selected gene (either pbp1 or ftsI). To attempt to inactivate the chromosomal copy of each gene, we amplified the 500-bp flanking regions of each gene and generated a three-fragment assembly product in which the gene of interest was replaced by the nonpolar kanamycin cassette. We transformed the different N6 derivatives carrying the two copies of the pbp gene of interest with the corresponding three-fragment assembly product carrying the corresponding pbp-null allele in the presence or absence of IPTG. As shown in Table 2, selection of kanamycin-resistant clones was dependent on the presence of IPTG on the selective plates. The few clones that grew on plates without IPTG can be explained by promoter mutants that lost tight regulation by LacIq or LacIq-null mutations. We randomly selected eight clones of each conditional mutation and confirmed by PCR amplification the deletion of the chromosomal gene copy. Growth of selected clones was strictly dependent on the addition of IPTG to the plates (data not shown).


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  		TABLE 2. Dependency on IPTG supplementation for chromosomal locus deletion of essential genes in H. pylori

Analysis of conditional mutant morphological phenotypes.
PBP1 and PBP3 encoded by pbp1 and ftsI, respectively, are penicillin-binding proteins predicted to be involved in peptidoglycan assembly and bacterial morphology. We selected a representative clone of pbp1 and ftsI conditional mutants to study the effects of their depletion on H. pylori morphology. Depletion of PBP1 led to premature arrest of growth after two to three generations (Fig. 4). In contrast, PBP3 depletion had no impact on bacterial growth in liquid culture (data not shown). Next, we performed scanning electron microscopy of the conditional mutants under depletion conditions. As shown in Fig. 5, depletion of PBP1 (no induction) led to the premature formation of coccoid bacteria, while depletion of PBP3 led to bacterial filamentation. For example, after PBP3 depletion, bacteria were on average 20 µm long, compared to 2 µm for the parental strain N6. The N6 ftsI{Delta}K2 pILL2163 strain grown with IPTG presented a few slightly longer bacteria (around 3 to 4 µm), indicating that the level of ftsI expression from the pILL2163 plasmid did not totally compensate the deletion of the chromosomal ftsI copy. The observed phenotypes of the conditional mutants are consistent with their predicted function based on homologies with other PBPs (35, 36). Accordingly, inhibition of their activities in H. pylori by different β-lactam antibiotics suggested a role for PBP1 in maintaining spiral/rod morphology and PBP3 in cell division (10). Taken together, we were able to generate the first conditional mutants of H. pylori.


Figure 4
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  		FIG. 4. Growth curve of the pbp1 conditional mutant. Growth of the pbp1 conditional mutant in the presence of IPTG (1 mM) or absence of IPTG was compared to that for wild-type strain N6. In the absence of IPTG, growth arrest occurred after two to three generations. Bacteria were collected at 24 h of growth (arrow) and observed by scanning electron microscopy (see Fig. 6, below). At 30 h, the number of viable bacteria was determined by plating serial dilutions on blood agar plates. Despite PBP1 depletion, premature arrest of bacterial growth, and transformation into coccoid bacteria (see Fig. 6, below), these coccoid bacteria remained viable (2.45 x 108 CFU/ml in the absence of IPTG versus 2.75 x 109 CFU/ml with IPTG). The growth curve is representative of at least four independent experiments.


Figure 5
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  		FIG. 5. Morphological modifications of H. pylori under depletion conditions for PBP1 and PBP3 (FtsI). Bacteria were harvested and treated for scanning electron microscopy after 24 h of growth in liquid medium (see also Fig. 4). At 24 h of growth, the wild-type strains N6 and N6(pILL2150) presented a rod-shaped morphology and unipolar flagella. In the presence of IPTG (1 mM), both the PBP1 and PBP3 conditional mutants presented a rod-shaped morphology comparable to the wild-type N6. On average, the N6{Delta}ftsI pILL2163 strain was slightly longer (3 to 4 µm) than the wild-type strain (2 µm). In the absence of IPTG, the N6{Delta}pbp1 pILL2161 strain was found exclusively as coccoid forms, consistent with a role for PBP1 in rod shape maintenance in H. pylori. Coccoid bacteria maintained their flagella, which were often wrapped around the cell (data not shown). N6{Delta}ftsI pILL2163 was found as long filaments that averaged 20 µm in length, consistent with a role for PBP3 in cell division. Flagella could be observed at one pole of each filament.

Transformation of pILL2150 and pILL2157 in different H. pylori isolates.
H. pylori strains are highly diverse due to free recombination and high rates of mutations (37). To limit DNA exchange, each strain has a unique complement of restriction-modification systems. Restriction-modification barriers have limited the use of pHeL2 (and derivatives) to a few H. pylori strains, none of which colonizes the mouse or gerbil model. Table 3 illustrates the difficulty in introducing pHeL2 into five different genetic backgrounds (four strains that colonize mice and/or gerbils and the sequenced strain 26695). While N6 was efficiently transformed with pILL2150 and pILL2157, none of the other strains was readily able to accept these two plasmids (Table 3). To expand the range of strains capable of accepting these expression vectors, we adapted the in vitro methylation protocol described by Donahue and colleagues (13) to test the efficiency of transformation of pILL2150 and pILL2157. Compared to plasmid DNA directly purified from E. coli, in vitro methylation of plasmid DNA with protein extracts of each recipient strain led to consistent isolation of chloramphenicol-resistant clones, albeit with low frequency (around 10-fold lower) compared to the highly transformable strain N6 (Table 3). Because we estimated the concentration of intact plasmid after in vitro methylation by agarose gel electrophoresis, the lower frequency of transformants is probably related to intrinsic differences in the efficiency of transformation of each strain compared to strain N6. The lower frequency could also be explained by the instability of these plasmids in the different strains. Nevertheless, we were able to purify the pILL2150 plasmid from strain B38 (see Fig. S9 in the supplemental material), indicating the plasmids can be maintained in genetic backgrounds other than N6.


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  		TABLE 3. Efficiency of transformation of pILL2150 and pILL2157 in different genetic backgrounds

Conclusion.
The present results highlight the potential of these newly established expression vectors for H. pylori. For the first time, we have available the genetic tools to construct conditional mutants of H. pylori. Although we have validated pILL2150 as a tool to construct the first H. pylori conditional mutants, both pILL2150 and particularly pILL2157 can serve in construction of conditional mutants also by expressing antisense RNA (8). Such an alternative strategy for gene expression knock-down has the advantage to be based on the addition of the inducer IPTG rather than on its removal from the growth medium. The use of inducible systems could be a starting point to elucidate in more detail the role of virulence factors. In fact, these approaches could give clues on their roles at different time points in the infectious process. Until now, the in vivo roles of virulence factors have been addressed mainly by studying the ability of a mutant strain either to colonize mice or to compete for the same niche with a wild-type strain.

Alternatively, these inducible expression systems for H. pylori could be very useful for expressing recombinant H. pylori proteins under their native conditions, overcoming problems of toxicity and incorrect folding in E. coli. Furthermore, spatial and temporal control of a variety of metabolic and physiological processes is achieved by protein complexes, such as chromosomal segregation and cell division. H. pylori was the first prokaryote for which a comprehensive protein-protein interaction network was established (28). Thus, these plasmids could be used to express trans-interacting domains involved in protein-protein interactions to study the stability of defined protein complexes and their roles in the physiology of H. pylori.

Also, complementation experiments have been done mainly by inserting a second copy of a gene in the rdxA locus, conferring metronidazole resistance to the complemented strain. However, selection of metronidazole-resistant clones depends also on the genetic background of the studied strain; 40% of the strains are naturally metronidazole resistant. Furthermore, sensitive strains can easily become resistant by a point mutation that reduces the efficiency of complementation using the rdxA locus. Complementation experiments with pILL2150 or pILL2157 would allow us to overcome these problems, in particular, by using the in vitro methylation methodology. Finally, despite an increasing interest for the role of small interfering RNAs, their role in H. pylori physiology has been limited, and these two plasmids could be very useful for studying them in H. pylori.


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ACKNOWLEDGMENTS
 
C. Chaput and A. Mathieu were supported by a fellowship from the French Ministry of Science. C. Chaput was also supported by la Fondation pour la Recherche Médicale. I. G. Boneca was supported by a fellowship of the Fundação para a Ciência e Tecnologia (Portugal) and a Bourse Roux from the Institut Pasteur and is an Institut National de la Santé et de la Recherche Médicale research scientist. This work was supported by a grant from Agence Nationale de la Recherche (ANR-05-MIIM-018-02).

We acknowledge Marie Thibonier for being the first to show in the laboratory the potential of pILL2150 in the construction of a conditional mutant of H. pylori.


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FOOTNOTES
 
* Corresponding author. Mailing address: Groupe Biologie et Génétique de la Paroi Bactérienne, Unité de Pathogénie Bactérienne des Muqueuses, Department of Microbiology, Institut Pasteur, 75724 Paris, France. Phone: 33-1-4438-9516. Fax: 33-1-4061-3640. E-mail: bonecai{at}pasteur.fr Back

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

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back

{ddagger} Present address: Department of Cellular Microbiology, Max Planck Institute, Charitéplatz 1, D-10117 Berlin, Germany. Back


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Applied and Environmental Microbiology, April 2008, p. 2095-2102, Vol. 74, No. 7
0099-2240/08/$08.00+0     doi:10.1128/AEM.01348-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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