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Infection and Immunity, January 2008, p. 153-160, Vol. 76, No. 1
0019-9567/08/$08.00+0     doi:10.1128/IAI.00791-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Critical Role of RecN in Recombinational DNA Repair and Survival of Helicobacter pylori{triangledown}

Ge Wang and Robert J. Maier*

Department of Microbiology, University of Georgia, Athens, Georgia 30602

Received 10 June 2007/ Returned for modification 20 July 2007/ Accepted 10 October 2007


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ABSTRACT
 
Homologous recombination is one of the key mechanisms responsible for the repair of DNA double-strand breaks. Recombinational repair normally requires a battery of proteins, each with specific DNA recognition, strand transfer, resolution, or other functions. Helicobacter pylori lacks many of the proteins normally involved in the early stage (presynapsis) of recombinational repair, but it has a RecN homologue with an unclear function. A recN mutant strain of H. pylori was shown to be much more sensitive than its parent to mitomycin C, an agent predominantly causing DNA double-strand breaks. The recN strain was unable to survive exposure to either air or acid as well as the parent strain, and air exposure resulted in no viable recN cells recovered after 8 h. In oxidative stress conditions (i.e., air exposure), a recN strain accumulated significantly more damaged (multiply fragmented) DNA than the parent strain. To assess the DNA recombination abilities of strains, their transformation abilities were compared by separately monitoring transformation using H. pylori DNA fragments containing either a site-specific mutation (conferring rifampin resistance) or a large insertion (kanamycin resistance cassette). The transformation frequencies using the two types of DNA donor were 10- and 50-fold lower, respectively, for the recN strain than for the wild type, indicating that RecN plays an important role in facilitating DNA recombination. In two separate mouse colonization experiments, the recN strain colonized most of the stomachs, but the average number of recovered cells was 10-fold less for the mutant than for the parent strain (a statistically significant difference). Complementation of the recN strain by chromosomal insertion of a functional recN gene restored both the recombination frequency and mouse colonization ability to the wild-type levels. Thus, H. pylori RecN, as a component of DNA recombinational repair, plays a significant role in H. pylori survival in vivo.


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INTRODUCTION
 
DNA repair is a fundamental process in all free-living organisms and is used by pathogenic bacteria as one of the defense mechanisms that allow them to survive in their hosts. Well-known pathways of DNA repair include postreplication mismatch repair (MutSLH) (30), base excision repair (glycosylases) (29), and nucleotide excision repair (UvrABCD) (35). In addition, homologous recombination is one of the key mechanisms responsible for the repair of DNA double-strand breaks (DSBs) (5). DSBs occur as a result of a variety of physical or chemical insults that modify the DNA. Damage at a single-strand site, if not repaired immediately, leads to a DSB. More commonly, if a replication fork meets damaged bases that cannot be replicated, the fork can collapse, leading to a DSB. In Escherichia coli, 20 to 50% of replication forks require recombinational repair to overcome damage (4).

Helicobacter pylori, a pathogenic bacterium infecting over 50% of humans, is the etiological agent of gastritis, peptic ulcers, and gastric cancer (7). In physiological conditions (i.e., in vivo), H. pylori is thought to frequently suffer both oxidative and acid stress, leading to DNA damage (31, 50). Several DNA repair pathways in H. pylori have been found to be important for its survival and pathogenesis. For example, H. pylori mutants that cannot repair oxidized pyrimidines due to loss of the DNA glycosylase EndoIII (nth gene product) function are more sensitive to the mutagenic and lethal effects of activated macrophages, and they show an attenuated colonization capacity (31). The H. pylori MutY glycosylase was shown to function in avoidance of the mutagenic effect of 8-oxo-guanine (8-oxoG) by removing adenine from an 8-oxoG · A mispair, and the loss of MutY leads to attenuation of the colonization ability (8, 18, 26). However, genes encoding some other DNA glycosylases (e.g., tag, alkA, and mutM found in other bacteria) are not present in the H. pylori genome. H. pylori encodes homologues of all four members of the nucleotide excision repair pathway (UvrA, UvrB, UvrC, and UvrD), all of which are well conserved in bacteria. Notably, H. pylori does not have the postreplication mismatch repair system, which most likely contributes to the high mutation frequencies observed (1). Instead, H. pylori MutS (a member of the MutS2 family) has been shown to play a role in maintaining genome integrity by suppressing homologous and homeologous DNA recombination (33) and by repairing oxidative DNA damage (8-oxoG) (49).

Recombinational DNA repair requires a large number of proteins that act at various stages of the process (5, 24). The most crucial step in DNA recombination is DNA strand exchange catalyzed by RecA (synapsis). With the involvement of RuvAB helicases, the RecA-promoted strand transfer produces a branched DNA molecule termed the Holliday junction, which is subsequently resolved by RuvC resolvase (postsynapsis). The processes of synapsis and postsynapsis appear to be highly conserved in organisms ranging from bacteria to humans. However, there are many differences between species in the initial step of recombinational repair (presynapsis), which involves recognition of the DNA damage site, generation of 3' single-stranded DNA ends that can be used for annealing with the homologous sequence, and recruitment of RecA. In E. coli, there are two separate pathways, RecBCD and RecFOR, for initiation of DNA recombination (24). Several other genes (recJ, recQ, and recN) are also required for recombination, although their functions are unclear (11, 41). Recently, RecN, RecO, and RecF were found to be localized to distinct foci on the DNA in Bacillus subtilis cells after induction of DSBs (22). These proteins form active repair centers at DSBs and recruit RecA, initiating homologous recombination. RecN was shown to play an important role in repairing DSBs, probably coordinating alignment of the broken segments with intact duplexes to facilitate recombination (27). The roles of RecN, as well as other components of DNA recombinational repair, in protection against oxidative damage in Neisseria gonorrhoeae have recently been demonstrated (42, 43).

A limited number of genes that are involved in recombinational repair are present in the H. pylori genome. H. pylori RecA is critical in DNA recombination and repair (13, 47). Genes for RuvABC proteins are present in H. pylori; thus, H. pylori may be able to restore Holliday junctions in a way similar to the way observed for E. coli. A ruvC mutant of H. pylori was sensitive to oxidative stress conditions and to DNA-damaging agents and was unable to establish a long-term infection in a mouse model (25). Notably, many genes coding for the components of DNA recombinational repair that are involved in the presynapsis stage, such as RecBCD, RecF, RecO, and RecQ, are missing in the H. pylori genome. Thus, H. pylori likely has a unique mechanism for DNA recombinational repair, particularly for the presynapsis stage.

In this study, we characterized a recN mutant of H. pylori. We showed that this H. pylori recN mutant is much more sensitive to various types of DNA-damaging agents than the wild type, indicating that RecN plays an important role in DNA repair in H. pylori. Loss of RecN function results in a significant decrease in the DNA recombination frequency, suggesting that the DNA repair performed by RecN involves homologous recombination. We demonstrated that under oxidative stress conditions H. pylori recN mutant cells accumulate much more fragmented DNA than wild-type cells, providing direct evidence that RecN functions in DNA recombinational repair. Finally, an H. pylori recN mutant clearly displays an attenuated ability to colonize mouse stomachs, highlighting the importance of recombinational DNA repair in survival of H. pylori within its host.


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MATERIALS AND METHODS
 
H. pylori culture conditions. H. pylori was cultured on brucella agar (Difco) plates supplemented with 10% defibrinated sheep blood or 5% fetal bovine serum (BA plates). Chloramphenicol (50 µg/ml) or kanamycin (40 µg/ml) was added to the medium used for mutants. Cultures of H. pylori were grown microaerobically at 37°C in an incubator using continuously controlled levels of oxygen (5% [partial pressure] O2, 5% CO2, 90% N2).

Construction of H. pylori recN mutant. A 1.76-kb fragment containing the H. pylori recN gene was amplified by PCR from genomic DNA of strain 26695 using primers recNF (5'-ACGACCACCAAGCTCTTAC-3') and recNR (5'-TAGACGCGTTCAAGCGTTT-3'). The PCR product was cloned into the pGEM-T vector to generate pGEM-recN. Subsequently, a chloramphenicol acetyltransferase cassette was inserted into the recN sequence of pGEM-recN at the Eco47III sites. The disrupted recN gene was then introduced into H. pylori wild-type strains by natural transformation via allelic exchange, and chloramphenicol-resistant colonies were isolated. Disruption of the gene in the genome of the mutant strain was confirmed by PCR that showed that there was an increase in the expected size of the PCR product and by direct sequencing of the PCR fragment.

Construction of H. pylori recN complementation strain. A 2.76-kb fragment covering the HP1394-recN sequence and the upstream promoter sequence was amplified by PCR from genomic DNA of strain 26695 using primers recNCF (5'-TTGACAGAAACAACCAAAGC-3') and recNR (5'-TAGACGCGTTCAAGCGTTT-3'). The PCR product was cloned into the pGEM-T vector to generate pGEM-recN+. Subsequently, a kanamycin resistance cassette (Kan) was inserted behind the recN gene in the plasmid, yielding pGEM-recN+-Kan. The insert portion of this recombinant plasmid was then excised and ligated into plasmid pEU39, yielding pEU-recN+-Kan. Plasmid pEU39 (from TIGR) contains a 2.04-kb fragment of H. pylori genomic DNA covering the HP0405 open reading frame. Previously, our laboratory showed that disruption of HP0405 in H. pylori resulted in no obvious phenotype. In plasmid pEU-recN+-Kan, HP0405 was disrupted into two pieces flanking recN+-Kan. When this plasmid was used to transform H. pylori X47 recN:Cm (with selection for Cmr Kanr), the intact recN gene (together with HP1394 and its promoter) and the Kan cassette were inserted into the genome at the HP0405 locus. This produced a merodiploid strain, X47 recN:Cm-recN+-Kan, which contains the original interrupted recN gene and an intact copy of recN at an unrelated site.

Assessment of the susceptibility to mitomycin C. H. pylori strains were grown on BA plates to late log phase, and the cells were suspended in phosphate-buffered saline (PBS) at a concentration of ~108 cells/ml. The cell suspensions were treated with 0, 50, 100, or 200 ng/ml mitomycin C for 20 min. The samples were serially diluted and spread on BA plates. After 4 days of incubation in a microaerobic atmosphere (5% [partial pressure] O2) at 37°C, colonies were counted.

Air survival assay. H. pylori strains were grown on BA plates to late log phase, and the cells were suspended in PBS at a concentration of ~108 cells/ml. The cell suspensions were incubated at 37°C under normal atmospheric conditions (21% O2) with moderate shaking. Samples were then removed at various time points (2, 4, 6, 8, and 10 h), serially diluted, and spread onto BA plates. Colony counts were recorded after 4 days of incubation in a microaerobic atmosphere (5% [partial pressure] O2) at 37°C.

Assessment of sensitivity to low-pH conditions. H. pylori strains were grown on BA plates to late log phase, and the cells were suspended in buffer (20 mM Tris-HCl, 150 mM NaCl) at different pH values (pH 7.0, 5.0, or 3.0) at a concentration of ~108 cells/ml. The cell suspensions were incubated under microaerobic conditions (5% O2) at 37°C for 1 h. The samples were serially diluted and plated to determine CFU counts (after 4 days of incubation under microaerobic growth conditions). The percentage of cell survival at pH 5.0 or 3.0 relative to that in pH 7.0 was calculated.

DNA transformation assay to assess recombination frequency. The donor DNA used in this study included (i) a 330-bp PCR fragment of the H. pylori rpoB gene containing a site-specific mutation (at the center of the fragment) conferring rifampin resistance, (ii) a linear DNA fragment containing a kanamycin resistance cassette (1.4 kb) flanked by H. pylori acnB gene sequences (about 550 bp on each side of the Kan cassette), and (iii) shuttle plasmid pHP1.

H. pylori strains were grown on BA plates to late log phase, and the cells were suspended in PBS at a concentration of ~ 108 cells/ml (recipient cells for transformation). Thirty microliters of the suspension was mixed with 100 ng of donor DNA and spotted onto a BA plate. After incubation for 18 h under microaerobic conditions at 37°C, the transformation mixture was harvested and suspended in 1 ml PBS. Portions (100 µl) of the suspension (or appropriate dilution) were plated onto either BA plates or BA plates containing a selective antibiotic (20 µg/ml rifampin or 40 µg/ml kanamycin, depending on the donor DNA used). The plates were incubated for 4 days under microaerobic conditions at 37°C, and the numbers of colonies were counted. The transformation frequency was determined by dividing the number of resistant colonies by the total number of CFU. In a normalized DNA transformation assay, the frequency of transformation was expressed as the number of transformants per 108 recipient cells. As negative controls, H. pylori strains with no DNA added were tested under the same assay conditions; no antibiotic-resistant colonies were observed.

DNA fragmentation analysis by electrophoresis. The wild-type or recN mutant cells were grown on BA plates under microaerobic conditions (5% O2) to mid-log phase (for about 1 day). To elicit DNA damage, the plates were exposed to atmospheric conditions (air, 21% O2) for 6 h. The cells on the plates were either directly used for DNA fragmentation analysis or incubated further under the microaerobic conditions for 4 h, followed by DNA fragmentation analysis. The cells grown continuously under the microaerobic conditions (i.e., without exposure to air) were used as controls.

Analysis of DNA fragmentation was performed as described by Zirkle and Krieg (53), with the following minor modifications. Cells were suspended in PBS to an optical density at 600 nm of 0.5. Then 500 µl of the sample was centrifuged for 1 min at 15,000 x g, the pellet was washed in Tris-EDTA buffer (50 mM Tris HCl, 5 mM EDTA; pH 8) at 4°C, and the final pellet was suspended in 10 µl of Tris-EDTA buffer. The resulting suspension was then added to 50 µl of 1% low-melting-point agarose at 37°C. The agarose and cells were mixed thoroughly, and 60-µl blocks were made by pipetting the mixture onto Parafilm. After solidification, the blocks were placed in a lysing solution (0.25 mM EDTA, 0.5% Sarkosyl, 0.5 mg/ml proteinase K) and incubated at 55°C for 1 h, followed by overnight incubation at room temperature. The next day, the blocks were washed three times (10 min each) in cold Tris-EDTA buffer. Agarose plugs were then submerged in a 0.8% agarose gel. Samples were subjected to gel electrophoresis for 7 h at 30 V. Gels were then stained for 30 min with ethidium bromide (0.5 µg/ml), destained in H2O, and then visualized under UV light.

Mouse colonization. Mouse colonization assays were performed essentially as described previously (49, 51). Briefly, wild-type strain X47 or X47 recN mutant cells were harvested after 48 h of growth on BA plates (37°C, 5% oxygen) and suspended in PBS to an optical density at 600 nm of 1.7. The headspace in each tube was sparged with argon gas to minimize oxygen exposure, and the tube was tightly sealed. The bacterial suspensions were administered to C57BL/6J mice (3 x 108 H. pylori cells/mouse) twice, using the oral deliveries made 2 days apart. Three weeks after the first inoculation, the mice were sacrificed, and the stomachs were removed, weighed, and homogenized in argon-sparged PBS (40) to avoid O2 exposure. Stomach homogenate dilutions were plated on BA plates supplemented with bacitracin (100 µg/ml), vancomycin (10 µg/ml), and amphotericin B (10 µg/ml), and the plates were rapidly placed in an incubator containing O2 at a sustained partial pressure of 5%. After incubation for 5 to 7 days, the fresh H. pylori colonies were enumerated and the data were expressed as CFU per gram of stomach.


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RESULTS
 
Bioinformatic analysis of H. pylori RecN homolog and construction of a recN mutant. In the published H. pylori genome sequence (48), HP1393 was annotated as a recN gene homolog. Upstream of the recN gene is an open reading frame (HP1394) coding for a hypothetical protein, and downstream is a gene (HP1392) encoding a fibronectin/fibrinogen binding protein that could be involved in biosynthesis and degradation of cell surface polysaccharides and lipopolysaccharides (Fig. 1). To study the physiological role of RecN, we constructed a recN mutant by inserting a chloramphenicol resistance cassette at the Eco47III restriction sites in the recN gene. This cassette replaced the 15-bp fragment between the two Eco47III sites (Fig. 1). Correct insertion of the cassette within the recN gene in the H. pylori genome was confirmed by PCR, which showed the expected increase in the size of the PCR product (data not shown). recN mutants were constructed for several different strains, such as 26695, J99, and X47. As X47 is a strain well adapted for the mouse colonization assay, most of the in vitro assays and all of the in vivo assays reported here were done with X47 and its isogenic recN mutant.


Figure 1
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  		FIG. 1. Organization of the recN gene and surrounding genes in the H. pylori genome and DNA constructs for generating the recN mutant. HP1394 encodes a hypothetical protein, and HP1392 encodes a fibronectin/fibrinogen binding protein. The gray bar in the N-terminal portion of recN represents the SMC domain encompassing the internal ATP/GTP binding site. A fragment containing the recN gene (1,760 bp) was PCR amplified using the recNF/recNR primer pair and cloned into pGEM-T to obtain pGEM-recN. A chloramphenicol resistance cassette (CAT) (800 bp) was then inserted within the recN fragment at the Eco47III sites, and plasmid pGEM-recN:CAT was used for transformation of wild-type H. pylori to create the recN mutant.

The gene product of H. pylori recN (1,575 bp) displays 22% amino acid identity (42% similarity) with the homolog in E. coli. E. coli RecN is strongly induced by DNA damage (SOS response) and has been implicated in repair of DSBs (12, 32). In the N-terminal portion of the RecN sequence there is a conserved domain related to the SMC (structural maintenance of chromosome) family proteins that are involved in chromosome scaffolding and partitioning (15). An ATP/GTP binding site motif is present in the SMC domain of the RecN protein.

H. pylori recN mutant is highly sensitive to DNA-damaging agents. To investigate the role of RecN in DNA repair, we examined the sensitivity of the recN mutant to various types of DNA-damaging agents. Mitomycin C causes predominantly DNA strand cross-links, leading ultimately to DNA DSBs, which require DNA recombination for repair. We determined the mitomycin C sensitivities of the wild-type X47 strain and the isogenic recN mutant. The results (Fig. 2) indicated that the recN mutant is much more sensitive to mitomycin C than the wild-type strain. For example, after treatment with 100 ng/ml mitomycin C for 20 min, the viability of the wild type was 5 orders of magnitude greater than that of the recN mutant. The same experiment was also performed with H. pylori strains 26695 and J99 and their isogenic recN mutant strains. The results were similar to those obtained for the X47 strain and its recN mutant (data not shown).


Figure 2
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  		FIG. 2. Sensitivity of H. pylori wild-type strain X47 ({blacklozenge}) and its isogenic recN mutant ({blacksquare}) to mitomycin C. H. pylori cell suspensions in PBS were treated with different concentrations of mitomycin C (indicated on the x axis) for 20 min. The numbers of surviving CFU were determined after 4 days of incubation under microaerobic conditions. The data are the means ± standard deviations of three experiments.

Oxidative stress is a major stress condition that H. pylori encounters in its physiological niche. To examine the sensitivity of H. pylori strains to oxidative stress, we performed an air survival assay, and the results (i.e., survival curves) are shown in Fig. 3. After exposure of approximately 108 cells to air for 10 h, about 106 viable cells of the wild-type strain could be recovered. In contrast, all of the recN mutant cells were killed (no viable cells were recovered) by exposure to air for 8 h. As an additional control for the assay, cell suspensions in PBS were incubated under microaerobic conditions (5% O2, 5% CO2, and 90% N2, with occasional shaking). Under these conditions, no significant difference in cell survival between the wild-type and recN strains was observed at any time point (not shown). The results clearly showed that the recN mutant is significantly more sensitive to atmospheric oxygen than the wild type.


Figure 3
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  		FIG. 3. Survival of H. pylori wild-type strain X47 ({blacklozenge}) and its isogenic recN mutant ({blacksquare}) upon exposure to air. H. pylori cell suspensions in PBS were incubated at 37°C under normal atmospheric conditions (21% [partial pressure] oxygen). Samples were removed at the times indicated on the x axis and were used for plate counting in a 5% oxygen environment. The data are the means ± standard deviations of three experiments. Based on a statistical analysis (Student t test), the differences in cell survival between the wild type and the recN strain are significant (P < 0.01) for all the time points except the 2-h time point.

Low pH is another stress condition that H. pylori encounters in its physiological niche. The sensitivity of the recN mutant to low-pH conditions was characterized. The wild-type H. pylori and recN mutant cells were treated for 1 h by suspending them in buffer at different pH values (pH 7.0, 5.0, and 3.0), and the survival was determined. As shown in Table 1, treatment at pH 5.0 for 1 h did not have a significant effect on the survival of the wild-type cells, while the same treatment killed approximately one-half of the recN mutant cells. About 40% of the wild-type H. pylori cells survived the pH 3.0 treatment for 1 h, whereas all of the recN mutant cells were killed by the same treatment.


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  		TABLE 1. Acid sensitivity of H. pylori wild-type strain X47 and its isogenic recN mutant

H. pylori recN mutant has a reduced frequency of DNA recombination. The results obtained for the sensitivity to DNA-damaging agents described above strongly suggest that RecN is involved in H. pylori DNA repair. To investigate if this repair function is due to a DNA recombination process, we tested whether loss of RecN function had an adverse effect on DNA recombination. The frequency of DNA recombination can be experimentally determined by DNA transformation. Natural transformation of H. pylori involves uptake of DNA into the cell, followed by recombination into the genome at the site of the homologous sequence (allelic exchange). Assuming that the wild-type H. pylori strain and its isogenic recN mutant strain are equally competent for DNA uptake, the frequency of natural transformation is an indicator of DNA recombination frequency.

We used two different types of DNA to examine DNA transformation of H. pylori. A specific A-to-G mutation in the H. pylori rpoB gene (rpoB3 allele) confers rifampin resistance (52). A 330-bp PCR fragment containing this specific mutation at the center of the fragment was used to transform H. pylori strains by using rifampin resistance as a selective marker. The other type of DNA used for transformation was the sequence of the H. pylori acnB gene (a 1.1-kb housekeeping gene), in which a 1.4-kb kanamycin resistance cassette (Kan) was inserted at the center (acnB:Kan). A plasmid that did not contain any H. pylori gene sequence (pHP1) was used as a control, as transformation with this plasmid did not involve DNA recombination (the plasmid remained extrachromosomal).

The results of the transformation analysis are shown in Table 2. First, we validated the use of the small DNA fragment of the rpoB3 allele for transformation to distinguish it from spontaneous mutations that lead to rifampin resistance. The spontaneous Rifr mutation frequency in H. pylori is 10–8 (52), whereas the transformation frequency using the rpoB3 fragment was 10–4. Under the transformation assay conditions used in this study, no single spontaneous Rifr colony (i.e., no DNA added) was observed. Compared to the transformation frequency for the wild-type X47 strain (4.68 x 10–4), the transformation frequency for the recN mutant was 10-fold lower (4.34 x 10–5). Using acnB:Kan as donor DNA, wild-type H. pylori had a transformation frequency of 630 x 10–8. In contrast, the transformation frequency for the recN mutant was only 12 x 10–8, which is 50-fold lower than that of the wild type. For both types of DNA donor (rpoB3 and acnB:Kan), the recN strain transformation frequencies were significantly lower than those of the wild type, according to a Student t test (P < 0.01). In the control experiments using plasmid pHP1 as the donor DNA, there was no significant difference in the transformation frequency between the wild type and the recN mutant. The transformation assay was also performed once with H. pylori strain 26695 and its isogenic recN strain; the results were similar to those obtained with the X47 strain and its recN mutant (data not shown). These results indicate that RecN plays a significant role in the DNA recombination process in H. pylori.


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  		TABLE 2. Transformation frequencies with different types of donor DNA

H. pylori recN mutant cells under oxidative stress contain more fragmented DNA. To further investigate the role of RecN in DNA repair, we examined the level of fragmentation of genomic DNA in the recN mutant cells compared to that in the wild-type cells (Fig. 4). H. pylori cells were grown on BA plates under the microaerobic conditions (5% O2) to mid-log phase (for about 1 day). When the genomic DNA was examined at this time point, both the wild-type and recN mutant strains appeared to contain intact genomic DNA (Fig. 4, lanes 1 and 2). This indicated that there is no significant DNA damage in H. pylori cells in vitro under optimal or near-optimal growth conditions. Next, we subjected cells to oxidative stress by exposing them to atmospheric conditions (21% O2) for 6 h. Based on the results shown in Fig. 3, this treatment should have resulted in losses of viability of about 1 order of magnitude for the wild-type strain and 6 orders of magnitude for the recN mutant. After this treatment, the cells were either directly analyzed by the DNA fragmentation assay or incubated further under the microaerobic conditions for 4 h (to allow DNA repair), which was followed by a DNA fragmentation analysis. In each case, the wild-type H. pylori cells contained a small amount of fragmented DNA (Fig. 4, lanes 3 and 5). In contrast, the recN mutant cells contained significantly larger amounts of fragmented DNA; this was shown by large smears (size range, 1 to 10 kb) on the agarose gel (lanes 4 and 6). Using densitometric scanning, the intensity of DNA fragments whose sizes ranged from 1 to 10 kb in the recN cells was determined to be ninefold higher than the intensity in the wild-type cells.


Figure 4
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  		FIG. 4. Agarose gel electrophoresis showing genomic DNA fragmentation in cells of H. pylori X47 (WT) and its isogenic recN mutant. Lanes 1 and 2, cells not exposed to air; lanes 3 and 4, cells exposed to air for 6 h; lanes 5 and 6, cells exposed to air for 6 h and then incubated under microaerobic conditions for 4 h. Fragmented DNA was visualized as smears on the agarose gel. The sizes of DNA standards are indicated on the right. The experiments were repeated three times with similar results.

H. pylori recN mutant displays an attenuated ability to colonize the mouse stomach. To determine whether loss of RecN activity has a significant effect on H. pylori colonization of the host, the relative abilities of the wild-type X47 and X47:recN mutant strains to colonize the mouse stomach were compared (Fig. 5). In one experiment, each strain was injected into nine C57BL/6J mice, and the colonization of H. pylori cells in the mouse stomachs was examined 3 weeks after inoculation (Fig. 5A). H. pylori was recovered from all nine mice that had been inoculated with the wild-type strain, and the average level was 1.06 x 106 CFU/g stomach. In contrast, the average number of H. pylori cells recovered from the mice that were inoculated with the recN mutant strain was 1.1 x 105 CFU/g stomach (Fig. 5A). According to a Wilcoxon rank test analysis, the lower colonization values for the mutant strain were significantly less than the colonization values for the wild type at the 99% confidence level (P < 0.01). The entire mouse colonization assay was performed an additional time, and a similar difference between the wild type and the mutant strain was observed (Fig. 5B). These results suggest that RecN plays a significant role in survival and colonization in the host.


Figure 5
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  		FIG. 5. Mouse colonization assays with H. pylori X47 and its isogenic recN mutant. The mice were inoculated with H. pylori two times (2 days apart) using a dose of 1.5 x 108 viable cells. The colonization of H. pylori in mouse stomachs was examined 3 weeks after the first inoculation. The data are presented as scatter plots of the number of CFU per gram of stomach as determined by plate counting. Each symbol indicates the CFU count for one stomach. The baseline (2.7 log10 CFU/g) is the detection limit of the assay, which represents a value less than 500 CFU/g of stomach. Panels A and B show the results for two independent experiments. WT, wild type.

Complementation of the recN strain. To ensure that the phenotypes (both in vitro and in vivo) observed for the recN strain were completely attributed to inactivation of recN, we introduced a functional copy of the recN gene back into the recN strain. Strain X47 recN:Cm-recN+-Kan contains a mutated recN gene at the original locus and an intact recN gene at an unrelated site (see Materials and Methods). The sensitivity of this strain to DNA-damaging agents was shown to be similar to that of the wild type. For example, both the wild type and the complemented strain could survive treatment with 200 ng/ml mitomycin C for 20 min or exposure to air for 10 h (data not shown), while all cells of the recN mutant strain were killed by these treatments (Fig. 2 and 3). Complementation of the recN function also restored the transformation frequency to the wild-type level, as determined by using the rpoB3 fragment as the DNA donor (Table 2).

The complemented strain was also examined to determine its mouse colonization ability. In an additional mouse colonization experiment, groups of mice (12 animals for each strain) were inoculated with wild-type strain X47, with the recN mutant, or with the complemented strain. Six mice from each group were sacrificed after 3 weeks, and the other six mice were sacrificed after 5 weeks. The viable counts of the strains recovered from the mouse stomachs at each time point were determined (Table 3). Consistent with the previous results (Fig. 5), the average level of the wild-type strain was about 1 x 106 CFU per g of stomach, and similar levels were detected at 3 and 5 weeks postinoculation. The average levels of the recN mutant strain at both time points (3 and 5 weeks postinoculation) were significantly lower than those of the wild type. In contrast, the average levels of the complemented strain at both time points were similar to those of the wild type (Table 3). This indicates that complementation of the recN strain restored its ability to colonize mouse stomachs.


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  		TABLE 3. Assessment of mouse colonization at 3 or 5 weeks after inoculation with H. pylori wild-type strain X47, the recN mutant, or the complemented strain


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DISCUSSION
 
In its physiological environment, H. pylori induces strong host inflammatory responses that involve recruitment of neutrophils, lymphocytes, and macrophages; these cells release free radicals that damage DNA. Although H. pylori is equipped with a variety of enzymes that detoxify reactive oxygen species, these enzymes are not sufficient to avoid damage to the genomic DNA. Indeed, the DNA of the pathogen was shown to be a target for host-generated oxidative stress based on studies of H. pylori nth strains that are unable to repair oxidized pyrimidines (31). Thus, a highly competent DNA repair system is especially important for survival of a pathogen like H. pylori in its host. However, published H. pylori genome sequences have revealed that a limited number of genes are involved in DNA repair. Interestingly, some major components of the DNA recombination machinery, such as RecA and RuvC, have been shown to be important for H. pylori survival (25, 47).

DNA DSBs pose a major threat to the structural and functional integrity of the genome. Homologous DNA recombination is a key mechanism used to repair DNA DSBs and to prevent replication fork collapse. Repair of DSBs in eukaryotic cells involves the orchestrated recruitment of various repair proteins to the sites of DSBs (5). Two members of this repair complex belong to the SMC family of proteins, which are key players in a variety of chromosome dynamics, from chromosome condensation and cohesion to transcriptional repression and DNA repair (16). SMC family proteins have a structural characteristic, an extensive coiled-coil domain located between globular domains at the N and C termini, that brings together Walker A and B motifs associated with ATP binding (15). Bacterial RecN protein is related to this family. E. coli RecN is strongly induced during the SOS response and was shown to be involved in RecA-mediated recombinational repair of DSBs (27). In B. subtilis, RecN was shown to be recruited to DSBs at an early time point during repair (21, 22, 37). In vitro, RecN was shown to bind and protect 3' single-stranded DNA ends in the presence of ATP (36).

In this study, we showed that an H. pylori recN mutant is much more sensitive to mitomycin C (Fig. 2), an agent that predominantly causes DNA DSBs, indicating that RecN plays an important role in DSB repair in H. pylori. Under physiological conditions, DSBs often result from exposure of cells to oxidative agents (6). The H. pylori recN mutant is highly sensitive to exposure to atmospheric oxygen (Fig. 3), as well as to other forms of oxidative stress (such as hydrogen peroxide and paraquat [data not shown]). The results indicate that RecN confers protection against oxidative damage in H. pylori. Similar roles of RecN in protection against oxidative damage have recently been demonstrated for N. gonorrhoeae (42, 43). Our results also showed that an H. pylori recN mutant is much more sensitive to low pH than the wild-type strain, suggesting that RecN is also involved in repair of acid-induced DNA damage. This could be relevant to the organisms's physiological condition, as H. pylori appears to colonize an acidic niche on the gastric surface (39).

Loss of H. pylori RecN function results in a significant decrease in the DNA recombination frequency, suggesting that RecN is a critical factor in DNA recombinational repair. Similarly, mutant strains in which the major components of DNA recombination machinery were disrupted (recA, ruvC) had greatly decreased DNA recombination frequencies (13, 25). In contrast, loss of UvrD or MutS in H. pylori resulted in an increase in DNA recombination frequency in H. pylori (19, 33). Suppression of DNA recombination by UvrD or MutS may play a role in maintaining genome integrity. Recombinational repair could be mutagenic due to homeologous recombination or could cause rearrangement due to recombination with direct repeat sequences (see below). In addition, recombinational repair systems are much more complex and require more energy to operate than nucleotide excision repair (NER) and base excision repair (BER) systems. Thus, UvrD, a component of an NER system, and MutS, a likely component of a BER (8-oxoG glycosylase) system (49), both suppress DNA recombination. Both NER and BER systems would be expected to continuously function in low-stress conditions. Under severe-stress conditions when large amounts of DSBs are formed, RecN perhaps recognizes DSBs and recruits RecA, facilitating recombinational repair.

H. pylori displays exceptional genetic variability and intraspecies diversity (45). Allelic diversity is obvious as almost each (unrelated) isolate of H. pylori has a unique sequence when a fragment of only several hundred base pairs of either housekeeping or virulence genes is compared among strains (10, 20, 46). A key mechanism for generating diversity is thought to be DNA recombination (23). In vivo, there appears to be continuous DNA damage from oxidative and acid stress in H. pylori cells. Some of this damage could result in DNA DSBs and replication fork collapse that require repair via DNA recombination processes. We propose that the requirement for recombinational DNA repair could be a driving force contributing to the genetic diversity of H. pylori. H. pylori is naturally competent for uptake of exogenous DNA, and a special apparatus homologous to type IV secretion systems (comB locus) has a dedicated role in DNA uptake (17). Unlike several other bacterial species, H. pylori does not seem to require specific DNA sequences for uptake of related DNA (38). Thus, small DNA segments could be taken up from neighboring cells of the same strain (homologous recombination) or from cocolonizing strains (homeologous recombination) and then used to repair damaged genes by recombination. Using mathematical modeling approaches with sequence data from 24 pairs of sequential H. pylori isolates, Falush et al. (9) estimated that the mean size of imported fragments was only 417 bp, which is much shorter than the sizes observed for other bacteria. In this study, we used a 330-bp DNA fragment (rpoB3) to assess the transformation frequency. The wild-type frequency of DNA recombination was 4 orders of magnitude higher than the spontaneous Rifr mutation frequency (10–4 versus10–8). Therefore, DNA recombination provides a mechanism to generate new genotypes much more rapidly than mutation alone can provide (2, 14).

In normal laboratory growth conditions, an H. pylori recN mutant does not show a growth defect, but its survival is greatly reduced under oxidative stress conditions. The latter conditions most likely closely resemble the in vivo stress conditions, at least in later stages of infection. Consistent with this idea, very little fragmented DNA was observed in either strain (wild type or recN mutant) when cells were cultured under normal microaerobic conditions. However, after oxidative stress treatment, a significantly higher proportion of the DNA was fragmented in the recN mutant cells than in the wild-type cells (Fig. 4). This strain comparison provided evidence that RecN functions in DNA recombinational repair. For the DNA fragmentation assays, we tested two oxidative stress exposure conditions: (i) exposure to air for 6 h, followed by a DNA fragmentation assay; and (ii) further incubation (after exposure to air for 6 h) under microaerobic conditions for 4 h (allowing time for DNA repair), followed by DNA fragmentation analysis. The same results were obtained for the two conditions, indicating that recombinational repair was functional during the exposure to air.

Repair of damaged DNA is known to be important for bacterial survival and pathogenesis (3, 28, 44). For H. pylori, it was documented that loss of DNA repair functions, such as EndoIII, RuvC, MutS, or MutY, resulted in a reduced ability to colonize the host stomach (8, 25, 31, 49). Our data demonstrated that H. pylori cells with recN disrupted were less able to colonize hosts than wild-type cells (Fig. 5). These results suggested that there were significant DSBs in the H. pylori DNA in the host environment and that the recN mutant strain was unable to efficiently repair them. Another interpretation is that DNA recombination may provide an alternative mechanism for gene regulation (e.g., immune modulation) that is essential for the long-term survival of H. pylori within the stomach (25). For example, while the wild-type H. pylori strain elicited a T helper 2 response in mice and established a persistent infection, a ruvC mutant elicited a T helper 1-mediated immune response that was more effective in eradicating H. pylori (34). Thus, an H. pylori ruvC mutant displayed decreased colonization ability in a relatively short time period (within 3 weeks) after infection, and this strain was completely eliminated from the murine gastric mucosa over a longer period (5 weeks after inoculation) (25). In contrast to RuvC, which is a major component of the DNA recombination machinery, RecN is a protein specific for repair of DSBs by linking DSB recognition and DNA recombination initiation. In our mouse colonization assay, the recN mutant cells were not completely eradicated 5 weeks after infection, although the colonization level of this strain was significantly lower than that of the wild-type strain (Table 3). We therefore propose that the attenuated ability of the recN mutant to colonize mouse stomachs was mainly due to its failure to repair DSBs through a DNA recombinational repair pathway.


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ACKNOWLEDGMENTS
 
This work was mainly supported by NIH grant R01DK06285201.

We thank Sue Maier for her expertise and assistance with mouse colonization assays.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, 815 Biological Sciences Building, University of Georgia, Athens, GA 30602. Phone: (706) 542-2323. Fax: (706) 542-2674. E-mail: rmaier{at}uga.edu Back

{triangledown} Published ahead of print on 22 October 2007. Back

Editor: A. Camilli


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Infection and Immunity, January 2008, p. 153-160, Vol. 76, No. 1
0019-9567/08/$08.00+0     doi:10.1128/IAI.00791-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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