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RESEARCH LETTER Genetic dissection of Helicobacter pylori AddAB role in homologous recombination Stéphanie Marsin1, Anne Lopes2, Aurélie Mathieu1, Eléa Dizet1, Emilie Orillard1, Raphaël Guérois2 & J. Pablo Radicella1 1 CEA, Institut de Radiobiologie Cellulaire et Moléculaire, UMR217 CNRS/CEA, Fontenay aux Roses, France; and 2Laboratoire de Biologie Structurale et Radiobiologie, CEA, IBiTecS, URA 2096, SB2SM, Gif sur Yvette, France Correspondence: J. Pablo Radicella, CEA, Institut de Radiobiologie Cellulaire et Moléculaire, UMR217 CNRS/CEA, 18 route du Panorama, F-92265 Fontenay aux Roses, France. Tel.: 133 1 46 54 88 57; fax: 133 1 46 54 88 59; e-mail: [email protected] Received 27 April 2010; revised 16 June 2010; accepted 14 July 2010. Final version published online 16 August 2010. DOI:10.1111/j.1574-6968.2010.02077.x Editor: Arnoud van Vliet MICROBIOLOGY LETTERS Keywords genetic variability; recombination; Helicobacter pylori; AddAB; RecO; RecR. Abstract Helicobacter pylori infects the stomach of about half of the world’s human population, frequently causing chronic inflammation at the origin of several gastric pathologies. One of the most remarkable characteristics of the species is its remarkable genomic plasticity in which homologous recombination (HR) plays a critical role. Here, we analyzed the role of the H. pylori homologue of the AddAB recombination protein. Bioinformatics analysis of the proteins unveils the similarities and differences of the H. pylori AddAB complex with respect to the RecBCD and AddAB complexes from Escherichia coli and Bacillus subtilis, respectively. Helicobacter pylori mutants lacking functional addB or/and addA show the same level of sensitivity to DNA-damaging agents such as UV or irradiation and of deficiency in intrachromosomal RecA-dependent HR. Epistasis analyses of both DNA repair and HR phenotypes, using double and triple recombination mutants, demonstrate that, in H. pylori, AddAB and RecOR complexes define two separate presynaptic pathways with little functional overlap. However, neither of these complexes participates in the RecA-dependent process of transformation of these naturally competent bacteria. Introduction The pathogen Helicobacter pylori colonizes the stomach mucosa of about half of the human population, frequently resulting in chronic gastritis, which can lead to peptic ulcers and, in a small fraction of cases, to cancer. Adaptation of H. pylori to the changing gastric environment within a host, or to new hosts, suggests an enhanced ability of this pathogen to change. Indeed, H. pylori is one of the most genetically diverse bacterial species. At the origin of such diversity are both mutations and recombination events (Suerbaum & Josenhans, 2007). Incorporation of DNA sequences by homologous recombination (HR) into the H. pylori chromosome, facilitated by the natural competence of this species, is crucial for horizontal gene transfer between unrelated strains colonizing the same host (Kersulyte et al., 1999). This process is believed to be the cause of its panmictic population structure (Suerbaum et al., 1998). Analysis of the genomic sequences has also underlined the 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c importance of intragenomic chromosomal rearrangements mediated by HR (Israel et al., 2001; Aras et al., 2003). In Escherichia coli, two major DNA recombination initiation (presynaptic) pathways coexist and are complementary: the RecFOR and the RecBCD pathways. The RecFOR pathway is essential for the postreplication repair of gaps and for the restart of replication following UV damage. However, none of the recF, recO and recR mutants show a decrease in HR following conjugation or transduction (Howard-Flanders & Bardwell, 1981; Kuzminov, 1999; Ivancic-Bace et al., 2003). We recently reported the presence in H. pylori of a functional RecO orthologue sharing o 15% identity with previously characterized homologues (Marsin et al., 2008). The RecBCD pathway is needed for the repair of doublestrand (ds) breaks and to resolve regressed forks. Consistently, E. coli mutants with null mutations in recB or recC genes have reduced viability and resistance to DNA-damaging agents such as ionizing radiation (IR). recBC mutants are also deficient in HR following conjugation or FEMS Microbiol Lett 311 (2010) 44–50 45 Helicobacter pylori recombination pathways transduction, whereas recD mutants display a hyper-recombination phenotype in these assays (Kuzminov, 1999). The RecBCD trimer is an ATP-dependent double-strand (ds) and single-strand (ss) exonuclease and a helicase. A functional analogue of the RecBCD complex, Bacillus subtilis AddAB, has been characterized genetically and biochemically (Kooistra et al., 1988; Chedin & Kowalczykowski, 2002). The add mutants are less sensitive to UV radiation compared with E. coli recBC mutants, and recombination during transformation is almost unaffected (Petit, 2005). Bacillus subtilis AddAB complex has associated both ATPdependent helicase and nuclease activities and loads to DNA at ds ends. The complex, either RecBCD or AddAB, binds a dsDNA end and initiates unwinding and degradation of both strands of DNA (Chedin & Kowalczykowski, 2002). Upon interaction with the host-specific sequence w (8 nt in E. coli and 5 nt in B. subtilis), the mediator complex generates a 3 0 -end ssDNA on which it loads RecA. This nucleoprotein filament proceeds to the synapsis step of recombination, searching for homology and invading a homologous dsDNA. In H. pylori, only a remote homologue of AddA (RecB), but not of AddB, had been predicted by sequence analysis (Tomb et al., 1997; Alm et al., 1999). It was recently shown that the H. pylori addA product is functional (Amundsen et al., 2008; Marsin et al., 2008; Wang & Maier, 2009). Indeed, it protects the genome from ds breaks, promotes intrachromosomal HR (Amundsen et al., 2008; Marsin et al., 2008) and contributes to the stomach colonization efficiency in mouse infection models (Amundsen et al., 2008; Wang & Maier, 2009). The works cited above explored the effect of inactivation of single HR genes. However, little is known regarding the overlapping functions of the two presynaptic pathways and the relative contributions of each gene to the genetic variability of H. pylori. Here, besides modeling the AddAB complex structure, we investigated using a genetics approach the in vivo roles of the H. pylori addA and addB gene products during recombinational repair, exogenous DNA incorporation and intrachromosomal recombination. Furthermore, using double or triple mutants in HR genes, we determined the different HR initiation pathways involved in these events and their relative contributions. profile–profile comparison (Soding, 2005). The sequence identities shared by RecB and RecC from E. coli with AddA and AddB are, respectively, 17% and 11%. It is known that below 30% identity, alignment errors are frequent. Therefore, several regions were further optimized manually in order to generate sequence alignments consistent with the structural topology and constraints imposed to the AddAB complex structure. Particularly, we manually adjusted the positions of insertions and deletions in order to ensure that burial positions are kept hydrophobic and that the secondary structures are minimally broken by insertions. These optimized alignments were then used as starting points for generating models with MODELLER. The quality of the resulting models was assessed using VERIFY3D (Luthy et al., 1992) or PROSA2003 (Wiederstein & Sippl, 2007). The alignments between the sequences and the template profiles were then iteratively refined in order to reduce the alignment errors pinpointed by the evaluation scores. Helicobacter pylori strains and growth conditions All H. pylori strains used were in the 26695 background (Tomb et al., 1997) and are listed in Supporting Information, Table S1. Plate cultures were grown at 37 1C under microaerobic conditions on a blood agar base medium supplemented with an antibiotic mix and 10% defibrillated horse blood (BAB). Plates were incubated from 24 h up to 5 days depending on the experiment or the strains involved. To generate the corresponding mutant derivatives, the gene of interest cloned into pILL570 was disrupted, leaving the 5 0 and 3 0 ends (300 bp) of the gene, by a cassette carrying a nonpolar kanamycin (Kn), an apramycin (Apr) or a chloramphenicol (Cm) resistance gene (Marsin et al., 2008). DNA was introduced into H. pylori strains by natural transformation and selection after 3–5 days of growth on 20 mg mL1 Kn, 12.5 mg mL1 Apr or 8 mg mL1 Cm. Allelic replacement was verified by PCR. Double or triple mutant strains were obtained by plasmid or genomic DNA transformation of single mutant or by mixing two mutant strains together before plating the mix on double or triple selection. Experiments were performed on a minimum of two mutants obtained independently for each construction. Sensitivity assays Materials and methods Generation of the structural models Models of both AddA and AddB were generated with MODELLER 9v5 (Sali et al., 2003) using as template the X-ray structure of the RecBCD complex in E. coli (PDB code: 1w36). Initial alignments between AddA and RecB and between AddB and RecC were obtained from the HHsearch FEMS Microbiol Lett 311 (2010) 44–50 For UV sensitivity assays, bacterial cell suspensions were serially diluted and 10 mL of each dilution was spotted on BAB plates. Cells were irradiated with 0, 15, 30, 45 and 60 J of 264-nm UV light delivering 1 J m2 s1. Gamma irradiation was performed using a 137Cs source delivering 30 Gy min1. Survival was determined as the number of cells forming colonies on plates after a given irradiation divided by the number of colonies from nonirradiated cells. 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 46 The intrachromosomal recombination substrate in the rdxA locus was described previously (Marsin et al., 2008). For insertion of the substrate into the recR gene, the Kndu<Apra structure was amplified by PCR from plasmid pTZ954Kndu-Apra. The PCR fragment was then inserted into the intact Kn gene present in recR by transformation of the LR122 strain. Integration of the recombination substrate into the chromosome was verified by PCR. Mutants in the rec genes were obtained by transformation of these strains with the corresponding plasmid as described above. Deletion assay Strains to be tested were grown on BAB plates containing apramycin (12.5 mg mL1). When they reached the exponential step (24 h), 25 mL of resuspended cells (2.5 105 cells) were spotted on BAB plates. After 24 h at 37 1C, appropriate dilutions were plated on BAB with and without 20 mg mL1 Kn and incubated for 3–5 days. The recombination rates and their SDs were calculated from 15–42 independent experiments using the method of the median (Lea & Coulson, 1949). P-values were calculated using the Mann–Whitney U-test. Natural transformation assay Two hundred nanograms of genomic DNA from strain LR133 (StrR) was mixed with 15 mL of resuspended exponentially growing cells (2.5 105 cells). Mixes were spotted on BAB plates. After 24 h at 37 1C, dilutions of the resuspended spots were plated on BAB with and without the appropriate antibiotic (50 mg mL1 Str) and incubated for 3–5 days. Transformation frequency was calculated as the number of resistant colonies per recipient CFU. P-values were calculated using the Mann–Whitney U-test. Results and discussion Amundsen et al. (2008) used the AddB nuclease motif ‘GRIDRID’ to identify the HP1089 as the H. pylori AddB orthologue. By complementing H. pylori single mutants or analyzing the AddAB activities in E. coli cells or extracts, they showed the importance of the helicase and nucleases activities in the AddAB complex (Amundsen et al., 2009). Based on a bioinformatic methodology similar to that used for the detection of the RecO orthologue (Marsin et al., 2008), we also converged on HP1089 as the orthologue of AddB. A remarkable feature of the HP1089 protein is that its length (778 residues) is only two-thirds that of E. coli RecC or B. subtilis AddB (spanning 1122 residues and 1166 residues, respectively). Such a large difference in the H. pylori sequence length prompted us to model the 3D structure of 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c the AddAB pylori proteins based on the RecBC template structures so as to map the major differences. These models and the resulting alignments provided as Supporting Information lend useful insights into the regions that remained conserved in all three species and can serve as a guide map to design mutants for further structure–function investigations. The major conclusion is that the nearly 400 residues deleted between H. pylori and B. subtilis concern in priority the 5 0 channel as if the active nuclease domain in HpAddB was sufficient for the function of the enzyme. In contrast, the architecture of the 3 0 channel in H. pylori enzyme is not drastically perturbed. Bacillus subtilis AddB appears as a hybrid system between RecC and HpAddB in which the nuclease domain is active and the 5 0 channel architecture has been slightly remodeled with respect to the E. coli enzyme. To define the roles of the addA and addB genes, we generated mutant strains combining the inactivation of either addA or addB with that of one or two other genes involved in recombination (Table S1). As we have described for the addA mutant (Marsin et al., 2008), growth was clearly impaired in an addB strain compared with that of the parental strain (Fig. 1), while no differences in cell size or filamentation were detected by microscopic observation. Strains impaired for AddA display a modest sensitivity to UV irradiation, intermediate between the recO and the wild type (Fig. 2a and Amundsen et al., 2008; Marsin et al., 2008). The addB single mutant showed exactly the same low UV 4 26695 3.5 addA addB 3 2.5 OD600 nm Construction of the intragenomic recombination substrate S. Marsin et al. 2 1.5 1 0.5 0 0 4 8 12 16 Time (hours) 20 24 Fig. 1. addA and addB mutants show growth impairment. Strains were grown in a liquid medium and OD600 nm taken at the indicated times. Growth curves displayed are representative of those obtained in three independent experiments. FEMS Microbiol Lett 311 (2010) 44–50 47 Helicobacter pylori recombination pathways 100 100 26695 10 addB addA addA addB Cell survival (%) 1 0.1 recO 0.01 26695 10 1 Cell survival (%) (a) 0.1 0.01 addA addB recA addA addB recO 0.001 0.001 addB recA recO addB recO addB recA recA recA 0.0001 0.0001 0.00001 0.00001 0 20 40 60 0 UV dose (J m–2) (b) 20 40 60 UV dose (J m–2) 100 100 26695 26695 10 10 1 recA addA addA addB addB recO addB 0.1 0.01 0 100 200 γ -Dose (Gy) 300 Cell survival (%) Cell survival (%) recO 1 recA addB recA addA addB recO addB recA recO addA addB recA 0.1 0.01 0 100 200 γ -Dose (Gy) 300 Fig. 2. UV and g irradiation sensitivities of Helicobacter pylori recombination mutant strains. Average from four to six experiments are shown. (a) UV light sensitivity. (b) IR sensitivity. sensitivity as the addA one. Furthermore, after UV irradiation, the double addA addB mutant behaved as the single mutants, confirming that both genes are involved in the same pathway (Fig. 2a). When the inactivation of addB was combined with that of recO, strains were much more sensitive to UV. Indeed, a double addB recO was as sensitive to UV as a recA. Similar results were obtained using a recRdisrupted strain instead of the recO mutant (data not shown). These results confirm that AddAB and RecOR act on distinct repair pathways. All triple mutants involving FEMS Microbiol Lett 311 (2010) 44–50 mutations in both pathways and recA inactivation presented sensitivities equivalent to that of the recA mutant. This result, together with the additive effect of RecO(R) and AddB(A) deficiencies, shows that in the case of UV-damaged DNA, RecA-mediated repair can be initiated through two nonoverlapping pathways defined by the RecOR and the AddAB complexes. Moreover, it can be concluded that no other mediator besides AddAB or RecOR participates in the RecA-dependent repair of UV DNA damage. However, we cannot rule out the possibility that, depending on the nature 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c 48 S. Marsin et al. of the damage, they could partially complement each other. Unlike what was shown for E. coli (Lloyd et al., 1988), the inactivation of RecOR in H. pylori has a more dramatic effect on UV survival than the inactivation of AddAB (RecBCD in E. coli). A different picture emerges from the analysis of the sensitivity to IR. Similar to addA (Marsin et al., 2008), the single addB mutant is extremely sensitive to IR. Inactivating both genes, addA and addB, resulted in the same sensitivity as that of the single mutants (Fig. 2b). These results confirm that AddA and AddB act together in the repair of IR-induced DNA damage. Inactivation of the AddAB complex made the strain as sensitive as a recA mutant and its combination with a recO mutation did not increase the sensitivity, strongly suggesting that in H. pylori, all recombinational repair of IRinduced lesions, mostly ds breaks, is mediated by AddAB. These results show that in H. pylori, in contrast to the E. coli model, RecOR cannot act as a backup of AddAB in RecAmediated ds break repair. To evaluate the involvement of the different initiation pathways of RecA-mediated HR, we used an intrachromosomal substrate consisting of direct repeats separated by a gene conferring apramycin resistance (Marsin et al., 2008). Deletion of the intervening sequence by recombination between the repeats yields a functional kanamycin-resistance gene. With this construct, 90% of the deletion events occurring spontaneously are dependent on a functional RecA (Table 1 and Marsin et al., 2008). As shown in Table 2, inactivation of addB resulted in a 40% reduction in recombination rates. This value is comparable to the one obtained in the single addA mutant (Marsin et al., 2008), suggesting that AddA and AddB are epistatic. In order to evaluate the relative contributions of the two pathways to intrachromosomal recombination, we introduced the recombination substrate into the recR gene, disrupting it (recR<KDA). The recombination rate in this case is slightly higher (Table 1) than the one obtained when the substrate was located in rdx (Table 1) probably due to sequence context. Inactivation of recO did not affect the rate obtained in the single recR mutant, again confirming the notion that recO and recR are likely to act as a complex in H. pylori. Conversely, the inactivation of addB reduced the rate of intrachromosomal recombination of the recR mutant by an additional 60% (Table 1). This result indicates that during spontaneous recombination of direct chromosomal repeats, both RecOR- and AddAB-dependent presynaptic pathways can act, but they do so in an additive way. It is tempting to speculate that the initial event, i.e. the formation of a gap or a ds break, will determine which presynaptic complex initiates recombination. During natural transformation, H. pylori can integrate exogenous DNA into its chromosome by HR. This process is dependent on a functional RecA (Schmitt et al., 1995); however, in strain 26695, the absence of either HR initiation complexes does not impair the integration process (Amundsen et al., 2008; Marsin et al., 2008). Consistently, Table 2 shows that disruption of addB did not reduce the frequency of transformation with chromosomal DNA carrying a mutation conferring resistance to streptomycin. Moreover, similar to what we have reported for the addA mutant, the transformation frequency in the addB mutant was fivefold higher than that in the wild-type strain. The double addAB mutant also had an elevated transformation frequency (Table 2), indicating that the AddAB complex might act as a suppressor of transformation. This adds the AddAB complex to RecG (Kang et al., 2004), UvrD (Kang & Blaser, 2006) and MutS2 (Pinto et al., 2005) in the list of DNA metabolism proteins suppressing transformation in H. pylori. While inactivation of RexAB, the functional homologue of AddAB in Streptococcus pneumoniae, did not significantly affect chromosomal transformation (Halpern et al., 2004), no data are available on mutants defective in the other presynaptic pathway. In the other transformation model system, B. subtilis, although inactivation of either AddAB or RecFOR had modest effects on chromosomal Table 1. Repeat deletion rates in recombination mutants Strain genotype (a) Single mutants (rdx<KDA) wt recA recR addB (b) Double mutants (recR<KDA) recR<KDA recR<KDA recO recR<KDA addB recR<KDA recA n Recombination rate ( 104)w Relative value P-value (MWU) 42 28 26 27 0.260 ( 0.007) 0.027 ( 0.005) 0.180 ( 0.006) 0.160 ( 0.006) 1 0.1 0.7 0.6 o 2 106 0.1 o 1 103 32 15 17 36 0.23 ( 0.006) 0.18 ( 0.008) 0.09 ( 0.006) 0.03 ( 0.003) 1 0.8 0.4 0.1 0.313 o 2 103 o 1 106 Number of independent determinations. w Recombination rates were determined as described in Materials and methods. Values correspond to the average and SD. 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c FEMS Microbiol Lett 311 (2010) 44–50 49 Helicobacter pylori recombination pathways Table 2. Transformation frequencies in recombination mutants Streptomycin resistance integration (genomic DNA) Strain genotype n Recombinant frequency ( 104)w Relative value P-value (MWU) wt recA recO recR addA addB addA recR addA recO addA addB addB recR addB recO addA recR recO addA addB recO 27 20 13 14 11 10 14 6 4 2 6 10 4 0.48 ( 0.51) o 0.0001 1.44 ( 1.93) 2.33 ( 3.31) 4.25 ( 2.23) 2.60 ( 2.49) 2.86 ( 2.62) 2.62 ( 3.38) 1.40 ( 0.86) 3.90 ( 1.03) 11.1 ( 3.85) 4.81 ( 3.66) 5.55 ( 1.17) 1 ND 3 4.9 8.9 5.4 6.0 5.5 2.9 8.1 23.1 10.0 11.6 1.3 101 1.8 101 o 1 108 o 2 105 o 1 105 o 0.1 o 0.05 o 5 103 o 1 105 o 5 105 o 5 105 Number of independent determinations. w Recombinant frequencies were calculated as the number of Strr colonies per recipient CFU. transformation capacity, strains defective in both presynaptic complexes have transformation frequencies more than 10-fold lower than that in the wild-type strain (Alonso et al., 1988). Table 2 shows that in H. pylori, all combinations resulting in the inactivation of both presynaptic pathways not only did not diminish the transformation capacity but also led to a significant increase in transformation frequencies. The dispensability of both mediator complexes indicates the existence of a specialized RecA-nucleation machinery for transformation. A possible explanation for the AddAB suppression of transformation is that the complex might exert its nuclease activity on some intermediate DNA substrate. In conclusion, the experiments described in this work using double or triple HR mutants show that H. pylori has two distinct functional presynaptic pathways for HR, defined by the RecOR and AddAB complexes. For recombinational repair, unlike what is found for E. coli, these two initiation pathways have little overlap in their substrate specificity, reflecting the lack of backup functions normally found in this pathogen. In the case of intrachromosomal recombination, although they both seem to contribute to a similar degree, they cannot compensate for each other, again suggesting differences in their substrates. We finally show that unlike in B. subtilis, neither of the two pathways can mediate the incorporation of exogenous DNA into the chromosome during natural transformation. Acknowledgements This work was supported by grants from the Agence Nationale de la Recherche (ANR-09-BLAN-0271-01 to J.P.R. and R.G.), the CEA, the CNRS and predoctoral fellowships from the CEA (to A.M. and E.O.) and the FEMS Microbiol Lett 311 (2010) 44–50 Association pour la Recherche contre le Cancer (to A.M.). We thank Agnès Labigne, Hilde de Reuse and members of their laboratories for sharing plasmids and strains. References Alm RA, Ling LS, Moir DT et al. (1999) Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397: 176–180. 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Nucleic Acids Res 35: W407–W410. Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Strategy used for the identification of HP1089 as Helicobacter pylori addB remote homologue. Appendix S2. Generation of a structural model for Helicobacter pylori and Bacillus subtilis AddAB complexes. Appendix S3. Comparative analysis of the structural models. Table S1. Helicobacter pylori strains used in this work. Fig. S1. Model of the AddAB complex of Helicobacter pylori (b) compared with the RecBCD X-ray complex (PDB: 1W36) (a) used as template of the comparative modelling. Fig. S2. Deletions in AddA highlighted by black secondary structures in the optimized alignment between RecB of Escherichia coli and AddA of Helicobacter pylori and Bacillus subtilis. Fig. S3. Deletions in AddB highlighted by black secondary structures in the optimized alignment between RecC of Escherichia coli and AddB of Helicobacter pylori and Bacillus subtilis. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. FEMS Microbiol Lett 311 (2010) 44–50