Download Genetic dissection of Helicobacter pylori AddAB role in homologous

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
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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.
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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
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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
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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.
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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.
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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