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(The FASEB Journal. 2006;20:2476-2485.)
© 2006 FASEB

A paradigm for direct stress-induced mutation in prokaryotes

Josephine M. Kang1, Nicole M. Iovine and Martin J. Blaser

Departments of Microbiology and Medicine, New York University School of Medicine, and VA Medical Center, New York, New York, USA

1Correspondence: Department of Medicine, New York University School of Medicine, 550 First Ave., New York, NY 10016, USA. E-mail: kangm01{at}med.nyu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Environmental stresses may lead to selection for hypermutator bacterial cells, which have an increased chance of generating beneficial variants. With stress removal, cost of mutation exceeds the fitness advantage, selecting against hypermutators. Hypermutators arise through several mechanisms, including inactivation of mismatch repair genes (MMR) or induction of error-prone polymerases. Helicobacter pylori may provide an alternative mechanism of stress-induced mutagenesis, since it lacks the MMR genes and error-prone polymerases found in other bacterial species, and possesses an endogenously high mutation frequency. In this study, we expose H. pylori strains to reactive oxygen species and reactive nitrogen species, stressors found in their natural environment. These exposures directly resulted in elevated rates of spontaneous point mutation, deletion between direct repeats, and intergenomic recombination. We demonstrate that these effects are transient and do not involve selection for hypermutator strains. That H. pylori possesses direct repeats in regions where potential gene rearrangements can occur suggests a mechanism for targeted mutation in response to stress that avoids the deleterious fitness costs of fixed hypermutation. These studies provide a new paradigm for adaptation under increased selective pressures that may be present in other prokaryotes.—Kang, J. M., Iovine, N. M., Blaser, M. J. A paradigm for direct stress-induced mutation in prokaryotes.


Key Words: adaptation • repetitive DNA • reactive nitrogen species • ROS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACTERIA ARE CONFRONTED with stressful and changing environments, where the capacity for genetic adaptation is crucial to evolutionary success. Without mutation, adaptation would be impossible to achieve. Hypermutator bacteria have a greater likelihood of survival in a selective environment because they have an increased chance of generating beneficial mutations (1) . However, most mutations are deleterious or neutral, and an eventual loss of fitness due to constant accumulation of harmful mutations is the norm rather than the exception (1 , 2) . To optimize survival, organisms must maximize genomic integrity while minimizing loss of adaptive capacity. Thus, bacterial strains have fluctuating mutation rates, with greater representation of hypermutators in more stressful environments, where they possess the advantage (3 4 5) . A variety of stressors such as nutrient limitation, oxidative damage, UV irradiation, and antibiotic and acid exposure have been documented to result in increased mutation rates (4 5 6 7 8 9 10) .

The major molecular mechanisms through which stress leads to increased mutations include direct damage to DNA, induction of stress-inducible responses via depression of LexA and activation of sigma factors RpoS and RpoH (11 , 12) , and selection for preexisting hypermutator strains (i.e., mutSLH mutants) (13) . Damage to DNA can lead to point mutations and illegitimate recombination; however, since these events can hinder replication and become lethal, organisms have developed numerous pathways to repair DNA lesions. Stress can lead to global stress responses, resulting in the induction of error-prone [translesion synthesis (TLS)] polymerases and down-regulation of mismatch repair (MMR) genes (14 , 15) . In Escherichia coli, where TLS has been best studied, TLS polymerases PolII, PolIV, or PolV can bypass lesions that block replicative polymerase PolIII. Thus, although TLS facilitates survival, TLS polymerases have low fidelity and concomitantly introduce mutations into the genome at high frequency (16) . It has been hypothesized that TLS is an adaptive mechanism through which bacteria can transiently modulate their mutation rates, enhancing probability of survival in times of stress while minimizing fitness costs once the stress is removed (17) . Known as second-order selection, this adaptationist hypothesis has generated controversy by challenging neo-Darwinian theory, which states that mutation is independent of selection (18) . Activation of alternative E. coli sigma factor RpoS down-regulates MMR genes with the same net result: transient elevation in mutation rates (14) . A third mechanism for stress-induced adaptation is selection for preexisting hypermutator strains, which may comprise up to 1–5% of natural isolates of Escherichia coli and Salmonella typhimurium (19) . Hypermutator strains arising from loss of the MMR function have been found with increased frequency in numerous organisms including E. coli, Pseudomonas aeruginosa, and Neisseria meningitidis (7 , 19 20 21) .

In this study we focus on Helicobacter pylori, Gram-negative, microaerophilic bacteria with remarkably high levels of genetic variation (22 , 23) . H. pylori releases proinflammatory effectors that provoke host immune responses resulting in chronic inflammation, with release of reactive oxygen and nitrogen species. Yet H. pylori successfully colonizes this dynamic environment, persisting for decades in its human host without treatment and, in most cases, without pathological outcome (24 , 25) . H. pylori lacks homologs of TLS polymerases and is one of only a few known organisms to lack an MMR pathway (26 , 27) . H. pylori has no evidence of an SOS response, lacks the stress-induced sigma factors RpoS and RpoH, and possesses relatively few transcriptional regulators compared with other organisms (27 , 28) . Thus, H. pylori lacks two widely conserved mechanisms of stress-induced mutagenesis. Therefore, we sought to determine whether DNA damage-induced mutation, from reactive oxygen species (ROS) and reactive nitrogen species (RNS), plays a prominent role in affecting H. pylori genome stability and subsequent adaptation. We find that exposure to reactive nitrogen and oxygen species, stressors found in the natural environment of H. pylori, significantly increases point mutations, intergenomic recombination, and rearrangements between direct DNA repeats. In silico analyses demonstrate that these direct DNA repeats are especially concentrated in the H. pylori pathogenicity island and restriction modification (RM) systems, suggesting H. pylori may have evolved to "target" mutations to favorable genetic loci, particularly in times of stress. Such stress-induced hypermutation may be a fundamental mechanism for maintaining homeostasis in the gastric niche and provide an explanation for the high degree of genomic plasticity and persistent colonization of H. pylori. Our work suggests that H. pylori may be a model for stress-induced mutation in other prokaryotes as well.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Statistical analyses
Student’s t test was used, where appropriate, and P value < 0.05 was deemed statistically significant.

Bacterial strains, plasmids, and chemicals
The plasmids and H. pylori strains used in this study are listed in Supplemental Table 1 and in Table 1 , respectively. H. pylori strains were routinely grown at 37°C in 5% CO2 on trypticase soy agar (TSA) plates or on brucella agar (BA) plates with 10% newborn calf serum (NCS) supplemented with the appropriate antibiotic. The presence or absence of cagA and the genotype of vacA were determined by polymerase chain reaction (PCR), as described (29) . Sodium nitroprusside (SNP) and methyl viologen (MV) were obtained from Sigma-Aldrich (St. Louis, MO, USA); NOC-18 was obtained from Calbiochem (San Diego, CA, USA). SNP and NOC-18 are sources of RNS, and MV a source of ROS.


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  		Table 1. Point mutation rates of H. pylori strainsa

Determination of spontaneous mutation rates
Since a point mutation in rpoB confers resistance to rifampin in H. pylori and E. coli (30) , rifampin resistance was used as a marker for spontaneous mutation frequency. Nine colonies of each strain to be tested were picked and expanded on BB plates for H. pylori. Expansion of colonies was necessary in order to produce a sufficient number of cells to detect mutants. After 48 h growth at 37°C in 5% CO2, bacteria were suspended in 1 ml PBS and serially diluted onto TSA plates alone or to BA plates supplemented with rifampin (7.5 µg/ml). Plates were incubated for 5 days at 37°C in 5% CO2 and frequencies were determined by dividing the number of colony-forming units (CFU) on rifampin by total CFUs; mutation rates were calculated based on the Lea-Coulson algorithm (31) .

Recovery from DNA damage
To examine susceptibility to free radical damage, H. pylori cells were suspended in brucella broth supplemented with 10% NCS, aliquoted into eight tubes, and treated with either SNP (at 100 nM to 10 µM concentrations), NOC-18 (10 µM to 100 µM), MV (100 nM to 100 µM), or PBS alone (control). Liquid cultures were utilized instead of agar plates to ensure a uniform distribution of RNS and ROS in contact with the bacterial cells. Cells were grown with shaking for 24 h at 37°C in 5% CO2, then serially diluted onto TSA plates, plates incubated for 5 days at 37°C in 5% CO2, and survival rates were calculated for ≥3 separate assays for each strain examined.

Quantitation of NO formation by NOC-18 and SNP
The Griess reagent system (Promega, Madison, WI, USA) was used to quantitate levels of nitrite, which is a stable, NO derivative capable of exerting nitrosative stress (32) . Levels of nitrite in H. pylori cultures untreated or exposed to SNP and NOC-18 were determined spectrophotometrically at 530 nm immediately upon addition of reagents and after 24 h growth with shaking at 38°C in 5% CO2, as per the Griess reagent protocol (Supplemental Fig. 1 ).


Figure 1
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  		Figure 1. Susceptibility of H. pylori cells to reactive nitrogen species (RNS) and ROS. H. pylori strains JP26, 26695, and J99 were examined for susceptibility to RNS through exposure to SNP (A), NOC-18 (B), and to ROS through exposure to methyl viologen (C). A representative experiment from ≥3 trials is shown.

Assays to determine influence of RNS and ROS on spontaneous mutation frequencies
H. pylori strains to be tested were treated with SNP, NOC-18, or MV as above, grown for 24 h with shaking at 37°C in 5% CO2, and inoculated to BA plates supplemented with rifampin (7.5 µg/ml) or serially diluted onto TSA plates. Resulting colonies were counted after 5 days of incubation and spontaneous mutation frequencies were calculated. To determine whether rifampin-resistant mutants had a hypermutator phenotype, rifR colonies from the rifampin plates and rifS colonies from TSA plates as a control were streaked onto fresh plates, grown for 48 h at 37°C, 5% CO2, harvested into 1 ml PBS, and inoculated onto BA plates supplemented with metronidazole (7.5 µg/ml) or serially diluted onto TSA plates. A point mutation in rdxA confers H. pylori resistance to metronidazole (33) . Plates were incubated for 5 days at 37°C in 5% CO2 and spontaneous mutation frequencies were calculated by comparing growth on the metronidazole-containing (selective) and nonselective plates. A minimum of five frequencies were averaged for each data point; any outliers (jackpot cultures) were removed.

Construction of H. pylori mutants used in mating experiments
A plasmid containing a ureAB fragment, pAD1, was digested with SmaI and ligated to a chloramphenicol resistance cassette, cat, to create pAD1-Cat. H. pylori strain JP26 was subsequently transformed to chloramphenicol resistance with pAD1-Cat to create JP26 ureA::cat, and the chromosomal insertion of the cassette was verified using PCR. The construction of JP26 vacA::aphA has been described (34) .

Influence of RNS and ROS on mating frequencies
JP26 strains ureA::cat and vacA::aphA were harvested into PBS to achieve optical density (OD)600 = 1.0. From each suspension, 500 µl was added to 9 ml brucella broth supplemented with 10% newborn calf serum, aliquoted into eight tubes, and treated with SNP, NOC-18, MV, or mock-treated as a control. Cells were grown for 24 h with shaking at 37°C, 5% CO2, and inoculated onto BA plates supplemented with both kanamycin (m) (25 µg/ml) and chloramphenicol (com) (20 µg/ml) or serially diluted onto TSA plates. Resulting colonies were counted after 5 days of incubation and mating frequencies were calculated. As controls, unmixed cultures of JP26 ureA::cat and vacA::aphA were plated onto BA plates supplemented with Km and Cam; as expected, no colonies were detected in any case.

To determine whether RNS or ROS treatment results in a persistent elevation in transformation frequencies, H. pylori cells from the BA plates supplemented with Km and Cam or from the TSA plates were inoculated onto fresh plates without antibiotics, incubated for 48 h, harvested into PBS, and 25 µl was spotted onto a fresh TSA plate combined with 50 ng of donor DNA, and incubated for 18 h at 37°C in 5% CO2. Donor DNA was an 800 bp PCR product of H. pylori rpsL (A128G) from streptomycin-resistant (35) strain JP26. The transformation mixture was then harvested into 1 µl PBS and 100 µl of the appropriate serial dilutions was inoculated to either TSA or BA plates containing 10% NCS and 25 µg/µl streptomycin. After the plates were incubated for 5 days at 37°C in 5% CO2, the recombination frequency was calculated by comparing cfu on the streptomycin-containing (selective) and the nonselective plates.

Assay to determine intergenomic recombination frequencies of H. pylori mutants
To examine the permanence of the hyperrecombinant phenotype, we assessed the frequency of a second intergenomic recombination event in an H. pylori ureA::cat/vacA::aphA progeny strain (that had been selected based on its recombination of chromosomal markers) along with its parental (unselected control) strains. Donor DNA was an 800 bp PCR product of H. pylori rpsL with A128G from streptomycin-resistant (35) strain JP26. H. pylori strains JP26, JP26 ureA::cat and JP26 vacA::aphA were grown on TSA plates for 48 h, harvested into 1 ml of PBS, and 25 µl was spotted onto a fresh TSA plate; 50 ng of donor DNA was added and plates were incubated for 18 h at 37°C in 5% CO2. The transformation mixture was then harvested into 1 ml PBS, and 100 µl of the appropriate serial dilutions were plated onto either TSA or BA plates containing 10% NCS and 25 µg/µl streptomycin. The plates were incubated for 5 days at 37°C in 5% CO2, and the total recombination frequency was determined by dividing the number of StR colonies by the total number of colonies. As negative controls, H. pylori strains with no DNA added were examined in parallel in each experiment; no colonies were detected in any case.

Determining influence of RNS and ROS on frequency of deletion between direct DNA repeats
The construction and use of the deletion cassettes and H. pylori deletion strains have been described (34 , 36) . H. pylori strains JP26 vacA::0 and vacA::100 were treated with SNP, NOC-18, or MV as above, grown for 24 h with shaking at 37°C in 5% CO2, and inoculated onto BA plates supplemented with Km (25 µg/ml) or serially diluted onto TSA plates. Plates were incubated for 5 days at 37°C in 5% CO2 and deletion frequencies were calculated by comparing numbers of cfu on the kanamycin-containing (selective) and nonselective plates.

In silico analyses of potential intragenomic recombination hotspots
The repeat visualization program REPUTER (37) was used to identify potential intragenomic (deletion/duplication) recombinational hotspots in sequenced strains 26695 and J99, defined as paired repeat sequences ≥ 24 bp occurring at chromosomal loci ≤ 5 kb apart. For each potential recombination hotspot, the number of potential recombination events was identified through REPUTER. All ORFs associated with potential recombination hotspots were further classified based on known or putative function using the Entrez Protein database, www.ncbi.nlm.nih.gov/entrez, and the Comprehensive Microbial Resource on www.tigr.org.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
H. pylori strains have a narrow distribution of mutation rates
H. pylori lacks methyl-directed MMR (38) , the primary pathway for recognizing and repairing spontaneous point mutations, and it has been hypothesized that the absence of MMR contributes to higher spontaneous point mutation frequencies in H. pylori (38 , 39) . An earlier study of 29 H. pylori isolates revealed a >700-fold range in spontaneous point mutation frequencies (39) , suggesting that extreme variability exists between strains. Mutation frequencies reflect a ratio of mutants divided by the total number of bacteria at a given time and show large fluctuations depending on when during the culture the mutation occurred. Early events give rise to a larger clone of events, referred to as "jackpot cultures," whereas mutations arising later give rise to a correspondingly smaller clone of mutants. Thus, calculation of mutation rates are more reliable because the above variables are taken into account. We utilized an algorithm based on Lea-Coulson’s method (31) to calculate mutation rates for 10 H. pylori strains of varying genotypes from different parts of the world (Table 1) . We observe a fairly narrow 48-fold range in mutation rates (average 2x10–8), ~10-fold higher than such rates in E. coli (40) .

Exposure to RNS or ROS can elevate point mutation frequencies
To determine the effect of exposure to RNS and ROS on H. pylori cells, we first examined susceptibility of three wild-type (WT) strains to RNS, using SNP and NOC-18 as NO donors, or to ROS, using the superoxide donor MV (Fig. 1 A–C). The Griess reaction was used to confirm the release of RNS from SNP and NOC-18 (see Supplemental Fig. 1 ). We demonstrate that H. pylori cells are susceptible to SNP, NOC-18 and MV (paraquat) with dose-related killing (Fig. 1A-C ). No colonies were detected at SNP, NOC-18, or MV concentrations > 1 mM, indicating lethality. Since susceptibilities showed little variation among the three strains tested; strain JP26, which has been the subject of extensive studies of DNA repair, recombination, and variation (36 , 38) , was selected for further experimentation (38 , 41) . Next, we determined the influence of exposure to nonlethal levels of RNS or ROS on the generation of point mutations in JP26; both RNS and ROS exposures led to elevated point mutation frequencies (Fig. 2 A–C). Not surprisingly, exposure to NOC-18, which is highly lethal, increased point mutation frequencies (Fig. 2B ), but exposure to low levels of RNS and ROS, produced by MV and SNP, that mildly (<1 log) impair H. pylori survival also significantly elevated the point mutation frequency.


Figure 2
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  		Figure 2. Effect of exposure to reactive nitrogen and oxygen species on point mutation frequencies. Cells of H. pylori strain JP26 exposed to SNP (A), NOC-18 (B), or methyl viologen (C) were examined for point mutation frequencies based on spontaneous resistance to rifampin. Significant (P value<0.05) increases in point mutation frequencies (indicated by asterisks) were observed at concentrations of SNP (1 µM and 10 µM), NOC-18 (50 µM and 100 µM), and methyl viologen (10 µM and 100 µM). D) To determine whether RifR H. pylori colonies arose from preexisting hypermutator strains or were due to a transient mechanism, RifR and RifS colonies were selected from JP26 exposed to SNP (10 µM) or methyl viologen (10 µM), or neither (unexposed control). Point mutation frequencies in these strains were determined based on resistance to metronidazole. No significant differences in point mutation frequencies were observed between the RifR and RifS strains, suggesting that the mutations arose randomly in nonhypermutator strains.

DNA damage-induced point mutations do not arise from a hypermutator phenotype
In other bacteria, direct free radical damage to DNA bases creates mutagenic species such as 8-oxoguanine or abasic sites, leading to base mispairing; these stress-induced point mutations are one-time (transient) events (42 , 43) . TLS polymerases induced by DNA damage also transiently introduce point mutations into bacterial genomes with a longer effect due to gene induction (17) . Stress also may select for (nontransient) hypermutator strains that generate spontaneous point mutations at high frequency, often due to MMR pathway defects (5 , 7 , 19) . To determine the permanence of the hypermutation observed in the RNS- and ROS-exposed H. pylori strains, we isolated RifR and RifS colonies from the previous experiment (Fig. 2A-C ) and examined forward spontaneous mutation frequencies by inoculating the cells onto plates containing metronidazole. The lack of differences in point mutation frequencies between the RifR and RifS colonies (Fig. 2D ) provides evidence that the RifR colonies do not have a global hypermutator phenotype, as assayed at an independent locus (rdxA).

Exposure to RNS or ROS leads to increased mating frequencies
As naturally competent organisms, H. pylori cells can incorporate environmental DNA via recombination (44) . Mating between isogenic H. pylori strains with different antibiotic markers occurs at high frequency, resulting in doubly resistant progeny; this event can occur through free DNA uptake (44) , possibly from lysed cells, as well as by a separate DNase I-resistant mechanism requiring direct cell-to-cell contact (45) . Since intergenomic recombination is an important source of diversity in H. pylori, we next assessed the influence of RNS and ROS exposures. These exposures significantly elevated recombination (Fig. 3 A–C), with effects more pronounced for RNS than for ROS. RNS or ROS exposure at levels that only moderately impair survival (Fig. 1) resulted in a significantly increased genetic exchange.


Figure 3
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  		Figure 3. Effect of exposure to reactive nitrogen and oxygen species on intergenomic recombination frequencies. Mating experiments for H. pylori ureA::cat and vacA::aphA were conducted as described, with cells exposed to varying levels of RNS (A, SNP; B, NOC-18) or ROS (C, methyl viologen). Recombination (mating) frequencies significantly (*P<0.05) increased as RNS or ROS concentrations increased. D) Recombination frequencies of H. pylori cells in relation to a prior intergenomic recombination event. H. pylori parental and progeny cells after a mating were examined for their ability to be transformed by an 800 bp dsDNA product encoding StR. H. pylori WT strain JP26, parental strains JP26 ureA::cat and JP26 vacA::aphA, and a product of mating (H. pylori JP26 ureA::cat/vacA::aphA) were studied. The latter strains had been exposed to SNP (10 µM), MV (10 µM), or were unexposed (control) during mating experiments. No significant differences in ability to be transformed by the heterologous DNA encoding StR were detected among the strains tested.

To examine the permanence of the hyperrecombinant phenotype, we assessed the frequency of a second intergenomic recombination event in an H. pylori ureA::cat/vacA::aphA progeny strain, along with parental (control) strains. In these experiments, we used as donor DNA an 800 bp rpsL PCR product that confers resistance to streptomycin on recombination within the host cell. Prior exposure to RNS or ROS did not significantly affect recombination frequency (Fig. 3) . These studies provide evidence that the increased mating frequency observed upon exposure to RNS or ROS is due to direct DNA damage rather than to selection for a nontransient mutation that promotes recombination.

DNA damage results in increased deletions between direct repeats
In silico analyses of the H. pylori genome have revealed a large number of nonrandomly distributed direct DNA repeats that facilitate deletions or expansions of intervening segments, as verified experimentally (36 , 46 , 47) . Many of these repeats involve genes related to antigenic variation and/or immune evasion (Table 2 ), including those encoding the Bab adhesin, lipopolysaccharide synthesis, and CagY proteins. We evaluated the influence of RNS or ROS on H. pylori deletion frequencies involving direct repeats by utilizing a constructed cassette with 100 bp direct DNA repeats 900 bp apart (36) (Fig. 4 A). Low-level exposure to RNS or ROS exposure resulted in increased frequency of deletions between the direct repeats in a dose-dependent manner (Fig. 4B-D ). These results provide evidence that RNS or ROS exposure increases H. pylori intragenomic recombination plasticity as well.


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  		Table 2. H. pylori genes with direct DNA repeatsa


Figure 4
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  		Figure 4. Effect of exposure to reactive nitrogen and oxygen species on frequency of deletions involving direct DNA repeats. A schematic of the deletion cassette (34 , 36) used to assess frequency of intergenomic recombination between direct DNA repeats is shown (A). Insertion of the complete cassette into a host H. pylori strain confers resistance to chloramphenicol. The chloramphenicol cassette can subsequently be deleted by recombination between the two flanking identical repeat segments (IDS) DNA repeats, resulting in resistance (R) to kanamycin and susceptibility (S) to chloramphenicol. Cells of H. pylori strain JP26 vacA::100, containing 100 bp identical repeats, were exposed to RNS, using SNP (B), or NOC-18 (C) or ROS, using methyl viologen (D). Deletion frequencies were significantly (P value<0.05) increased at higher concentrations for SNP and methyl viologen.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been hypothesized that organisms possess a baseline rate of point mutations, ~10–9 to 10–10, suggesting that this is the level of fidelity that optimally balances fitness costs with evolutionary potential (40) . Our results for WT H. pylori strains document a higher rate of mutation, demonstrating that H. pylori is an exception to this rule. For H. pylori, the higher fitness costs associated with elevated point mutation rates may be worth the gain in adaptive capacity. Perhaps H. pylori has evolved a higher rate of mutation because the environment it colonizes is most similar to the selective environments that select for hypermutator E. coli, N. meningitidis, and P. aeruginosa (3 , 7 , 20) . H. pylori has limited opportunity for genetic exchange with other organisms; therefore, its high degree of genome plasticity may be evolutionarily advantageous by providing a broad range of variants on which selection can occur. This view is consistent with the hypothesis that H. pylori exists as an RNA virus-like quasispecies (48) in which isolates undergo constant genetic variation, resulting in a multitude of phenotypic variants. Through direct mutagenesis, reactive nitrogen species have been implicated to influence evolution and pathogenicity in certain RNA viruses (49 , 50) , similar to our current observations for H. pylori.

That H. pylori has a disproportionate number of direct repeats in genes related to antigenic variation and/or immune evasion (Table 2) , and that stress can directly induce genomic rearrangements between such repeats (Fig. 4) , suggests that repetitive DNA may represent an evolved strategy through which H. pylori generates targeted mutations under stressful conditions. An overabundance of repeats in stress response genes (51) is not unique to H. pylori, suggesting our results may be generalizable to other organisms. Intragenomic recombination involving cagA DNA repeat sequences leads to deletion (or addition) of tyrosine phosphorylation sites, altering host-bacterial interactions (47) . Similar alterations in other antigenic proteins can result in phenotypic variants with altered immunogenic potentials (52) , leading to variation in RNS/ROS levels. Stronger immune responses are associated with elevated inflammatory radicals, such as RNS and ROS (53 54 55) , which in turn may increase the generation of H. pylori variants, facilitating selection. In theory, high levels of ROS and RNS are detrimental to H. pylori colonization of the gastric mucosa; therefore, H. pylori variants that can evade the host immune response would be selected for. Thus, DNA-induced hypermutation may function as a rudimentary feedback mechanism for H. pylori, promoting long-term equilibrium between microbe and host (56) . H. pylori also has a higher concentration of repetitive DNA around RM genes. Previous studies have demonstrated that direct repeats can result in deletion of the HpyII RM system (46) , resulting in loss of function. Our results now demonstrate that increased exposure to DNA damage can increase frequency of rearrangements between such repeats, possibly influencing RM system expression as well. Although the role of RM systems in H. pylori has not been clearly delineated, studies in other organisms have shown RM systems may be involved in modulating gene expression (57) .

Adaptation to the human host is also mediated by homologous recombination in H. pylori. H. pylori strains deficient in homologous recombination fail to elicit a proper T helper (Th) 2 response in mice, necessary for persistent infection, and instead elicit Th1 responses, resulting in clearance within 70 days (58) . These results demonstrate the importance of recombination in facilitating H. pylori adaptation during colonization. That RNS and ROS exposure results in increased genetic exchange suggests that DNA damage-induced recombination facilitates rapid adaptation of H. pylori to its environment when generation of diversity is most crucial.

We find that direct DNA damage-induced hypermutation, as observed in H. pylori, is transient and essentially limited to the duration of stress. The effect of RNS and ROS on hypermutation may be more pronounced in H. pylori, which lacks MMR, since MMR proteins play a role in repairing various types of DNA lesions. An elevation in baseline mutation rates, though beneficial during adverse conditions, usually becomes detrimental to overall fitness once the stress has been removed (59) ; therefore, transiently increased mutation rates may be most advantageous since organisms do not become burdened with high mutation rates.

The majority of our experiments were performed in H. pylori strain JP26, which has been used extensively in other studies (34 , 36 , 38 , 41 , 60 , 61) . Since H. pylori is highly diverse, JP26 may not be representative of all H. pylori strains, and studies in multiple strains may be necessary before generalizations about variation within H. pylori can be made. However, previous studies using the deletion cassette in multiple H. pylori strains and our data for mutation rates suggest there is a relatively narrow range of intragenomic and spontaneous mutation events among strains, and our results suggest a similar overall trend of increased genomic plasticity may exist in response to DNA damage in H. pylori.

In conclusion, computational, experimental, and environmental studies have shown that stressful environments, where organisms are under constant selection, favor enrichment of hypermutating organisms, which have an increased probability of generating fitter variants. In this study, we demonstrate that H. pylori takes advantage of direct DNA-induced damage to manifest transient hypermutation phenotypes with elevated point mutations, deletions between repetitive DNA sequences, and intergenomic recombination. We hypothesize that such stress-induced hypermutation may represent a mechanism to maintain homeostasis in the gastric niche, where variation in surface antigen expression is met with corresponding changes in host immune responses and altered levels of RNS, ROS release from inflammatory cells. This model of direct DNA damage-induced mutagenesis is likely applicable to other prokaryotes and may represent a strategy to increase diversity in stringent environments.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Hannah Klein for assistance with the Lea-Coulson computations. Supported in part by RO1GM62370 and 5T32 GM07308 from the National Institutes of Health, by the Medical Research Service of the Department of Veterans Affairs, and by the Diane Belfer Program in Microbial Ecology.

Received for publication April 3, 2006. Accepted for publication July 11, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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