Molecular Microbiology (2015) ■
13272
Molecular Microbiology (2016) 99(5), 897–908 䊏
Escherichia coli ClbS is a colibactin resistance protein
Nadège Bossuet-Greif,1,2,3,4 Damien Dubois,1,2,3,4,5
Claude Petit,1,2,3,4,6 Sophie Tronnet,1,2,3,4
Patricia Martin,1,2,3,4,5 Richard Bonnet,7
Eric Oswald1,2,3,4,5 and
Jean-Philippe Nougayrède1,2,3,4*
1
INRA, USC 1360, Toulouse, France.
2
Inserm, UMR 1043, Toulouse, France.
3
CNRS, UMR 5282, Toulouse, France.
4
Université de Toulouse, UPS, Toulouse, France.
5
CHU Toulouse, Service de bactériologie-Hygiène,
Toulouse, France.
6
INP-ENVT ESC, Toulouse, France.
7
Université d’Auvergne, Inserm UMR 1071, INRA USC
2018, Clermont-Ferrand, France.
Summary
The genomic pks island codes for the biosynthetic
machinery that produces colibactin, a peptide-
polyketide metabolite. Colibactin is a genotoxin that
contributes to the virulence of extra-intestinal patho-
genic Escherichia coli and promotes colorectal
cancer. In this work, we examined whether the pks-
encoded clbS gene of unknown function could par-
ticipate in the self-protection of E. coli-producing
colibactin. A clbS mutant was not impaired in the
ability to inflict DNA damage in HeLa cells, but the
bacteria activated the SOS response and ceased to
replicate. This autotoxicity phenotype was markedly
enhanced in a clbS uvrB double mutant inactivated
for DNA repair by nucleotide excision but was sup-
pressed in a clbS clbA double mutant unable to
produce colibactin. In addition, ectopic expression of
clbS protected infected HeLa cells from colibactin.
Thus, ClbS is a resistance protein blocking the geno-
toxicity of colibactin both in the procaryotic and the
eucaryotic cells.
Introduction
The genomic pks (polyketide synthase) island present in
pathogenic but also commensal and probiotic Escherichia
coli of the phylogenetic group B2 encodes a biosynthetic
machinery allowing the production of colibactin, a geno-
toxic secondary metabolite (Nougayrède et al., 2006). This
Accepted 9 November, 2015. *For correspondence. E-mail jean-
philippe.nougayrede@toulouse.inra.fr; Tel. +33 (0)5 62 74 45 25; Fax
+33 (0)5 62 74 45 25.
© 2015 John Wiley & Sons Ltd
C 2015 John Wiley & Sons Ltd
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doi:10.1111/mmi.
doi: 10.1111/mmi.13272
First published online 1 December 2015
island is also found in certain strains of Enterobacter
aerogenes, Citrobacter koseri and Klebsiella pneumoniae
(Nougayrède et al., 2006; Johnson et al., 2008; Putze
et al., 2009; Lai et al., 2014). Epidemiological studies
suggest that the prevalence of pks+ E. coli is increasing in
the human microbiota in developed countries. There is a
shift in the E. coli population from the phylogenetic group A
to the newly dominant group B2, and up to 40% of B2 E. coli
strains host the pks island (Nougayrède et al., 2006;
Johnson et al., 2008; Putze et al., 2009; Payros et al.,
2014). Carriage of the pks island could provide a fitness
advantage for long-term persistence in the human gut
(Nowrouzian and Oswald, 2012). Colibactin is also a viru-
lence determinant of extraintestinal pathogenic E. coli, as
its production aggravates the lymphopenia and lethality in
mouse and neonatal rat systemic infection models (Martin
et al., 2013; Marcq et al., 2014; McCarthy et al., 2015).
Exposure of mammalian cells to live pks+ bacteria induces
DNA double strand breaks (DSB) in vitro and in vivo
(Nougayrède et al., 2006; Putze et al., 2009; Cuevas-
Ramos et al., 2010; Marcq et al., 2014). This DNA damage
leads to gene mutations, chromosomal instability and pre-
mature senescence that ultimately drive tumorigenesis
(Cuevas-Ramos et al., 2010; Secher et al., 2013;
Cougnoux et al., 2014). It was recently shown in different
mouse models that pks+ E. coli strains promote intestinal
tumor progression (Arthur et al., 2012; Cougnoux et al.,
2014; Dalmasso et al., 2014) and modulate gut homeosta-
sis (Olier et al., 2012; Payros et al., 2014; Secher et al.,
2015). Such pks+ B2 E. coli strains are overrepresented in
human colon cancer biopsies (Arthur et al., 2012; Buc
et al., 2013; Cougnoux et al., 2014). In Taiwan, more than
25% of K. pneumoniae isolates are pks+, two-third of which
belongs to the highly virulent K1 type causing pyogenic
liver abscesses that are associated with a higher risk of
colorectal cancer (Lai et al., 2014).
The 54 kb pks gene cluster is highly conserved in
Enterobacteriaceae with ∼100% identical nucleotide
sequences and preserved syntheny (Putze et al., 2009).
The chromosomal insertion at asn tRNA loci, the flanking
by direct repeats and by genes for integration and conju-
gation indicate it was shared recently by horizontal gene
transfer. The pks gene cluster is physically associated with
the High Pathogenicity Island (HPI) coding for the sidero-
phore yersiniabactin, another polyketide metabolite (Putze
et al., 2009; Martin et al., 2013). Similar biosynthetic gene
clusters were recently identified in the honeybee gut com-
mensal Frischella perrara (43–81% protein sequence
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identity with that in Enterobacteriaceae) and in the marine
sponge commensal Pseudovibrio sp. (28–65% protein
identity with that in F. perrara) (Bondarev et al., 2013; Engel
et al., 2014). It is not known whether the pks island in
Pseudovibrio induces genotoxicity, but despite genetic
divergence, infection with F. perrara induces DNA DSB in
HeLa cells. This conservation of function together with the
spread by horizontal transfer suggest that colibactin allows
similar interactions in distinct ecological communities.
The pks island clusters the clb genes encoding a hybrid
nonribosomal peptide synthase (NRPS)-polyketide syn-
thase (PKS) multi-modular assembly line related to that
synthesizing many microbial secondary metabolites of
polyketide and non-ribosomal peptide origin (Fischbach
and Walsh, 2006). These multimodular enzymes are acti-
vated by ClbA, a phosphopantetheinyl transferase that
primes the NRPS–PKS enzymes to allow ketide–peptide
chain elongation (Beld et al., 2014). The primed assembly
line utilizes malonyl-coA and amino acids, including the
unusual nonproteinogenic aminocyclopropane-carboxic
acid, to synthesize the non-ribosomal peptide-polyketide
hybrid metabolites (Bian et al., 2015; Brotherton et al.,
2015; Vizcaino and Crawford, 2015). These inactive
metabolites (called precolibactin) contain a prodrug motif
that is hydrolyzed to produce the active genotoxin colibac-
tin. This prodrug activation is achieved by ClbP, a
membrane-bound protein with a periplasmic D-amino
peptidase activity (Dubois et al., 2011; Cougnoux et al.,
2012; Brotherton and Balskus, 2013). The high instability
of the activated colibactin has precluded its purification
and characterization during a decade. The relatively
stable intermediate biosynthesis derailment products
were recently characterized, shedding light on the active
colibactin ‘warhead’, an unusual spirobicyclic ring system
and ultimate electrophilic species that could alkylate DNA
or proteins (Bian et al., 2015; Brotherton et al., 2015;
Vizcaino and Crawford, 2015).
Colibactin was recently shown to possibly induce cross-
links on purified DNA in vitro (Vizcaino and Crawford,
2015). This raises the question of colibactin autotoxicity
and the self-resistance mechanisms in the producing bac-
terium. The inactive prodrug assembly and activation by
ClbP cleavage during periplasmic export was previously
proposed as a resistance mechanism (Dubois et al., 2011;
Brotherton and Balskus, 2013). In a screen using activity-
based probes, Kunzmann and Sieber (2012) discovered
that the c2450 protein, a pks island-encoded protein with
previously unknown function (Nougayrède et al., 2006),
could bind a α-methylene-γ-butyrolactone probe with
intrinsic reactivity found in potent anticancer and antibac-
terial drugs. Homologue c2450 proteins are encoded on
the genomic islands of Pseudovibrio and Frischella (53%
and 64% amino-acid identity respectively), suggesting a
conserved function. In this work, we uncover the self-
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defense function of the E. coli c2450 protein, hereby
called ClbS. We observed that a clbS mutant E. coli is not
impaired for colibactin production but exhibits a marked
autotoxicity phenotype. In addition, clbS ectopic expres-
sion within HeLa cells confers protection against
colibactin-inflicted DNA DSB, demonstrating that ClbS is a
colibactin resistance protein.
Results
clbS is not required for genotoxicity of pks+ E. coli
To examine whether the clbS gene could play a role in the
genotoxin synthesis or trafficking, we quantified the geno-
toxicity of a wild-type pks and an isogenic clbS mutant E.
coli. The laboratory E. coli strain DH10B hosting the
BACpks, a clbS isogenic mutant or DH10B hosting the
empty BAC vector as a negative control, were grown to
reach the exponential growth phase (OD600 = 0.3), and
then inoculated to HeLa cells at various multiplicity of
infection (moi). After a 4 h infection, the HeLa cells were
further incubated for 4 h, and the host DSBs were demon-
strated by immunostaining S139-phosphorylated histone
H2AX (pH2AX) (Rogakou et al., 1998) (Fig. 1A). HeLa
cells exposed to the wild-type pks and clbS mutant strains
exhibited similar levels of pH2AX in relation to the moi
(Fig. 1B). However, when the bacteria were grown over-
night before infection, the clbS mutant strain displayed a
marked decrease of genotoxicity compared with the wild-
type pks and with the clbS mutant in exponential culture
(Fig. 1D–E). This defect could be fully reversed by reintro-
ducing a single copy of the clbS gene with its cognate
promoter region at the chromosomal attTn7 site (Fig. 1E).
The decreased genotoxicity of the clbS mutant grown
overnight was correlated with lower colony-forming unit
(CFU) counts in the cell culture medium at the end of the
infection (Fig. 1C and F). The genotoxicity of the clbS
mutant grown overnight could be restored to wild-type
levels with a 10-fold increase of the moi (Supplementary
Fig. S1), indicating that the decreased genotoxicity of the
clbS mutant grown overnight resulted from the reduced
number of infecting bacteria. Together, these results
confirm that clbS is not required for colibactin synthesis or
trafficking, but for the fitness of the bacteria producing the
genotoxin.
A clbS mutant exhibits decreased CFU numbers in the
stationary growth phase
To investigate the fitness of the clbS mutant, we deter-
mined its growth kinetics. The wild-type pks, clbS mutant
and complemented mutant strains were grown in LB
(inoculated with pre-cultures in the exponential phase)
and plated at indicated times (0–24 h) to determine the
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 ClbSClbS protects
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Fig. 1. Colibactin production and growth of a clbS mutant. E. coli DH10B hosting the BAC vector, or the BACpks (pks), the isogenic clbS
mutant or the clbS mutant complemented with the clbS gene inserted at the chromosomal attTn7 site, were pre-cultivated in the exponential
growth phase (panels A–C) or overnight to reach stationary phase (panels D–F) then inoculated to HeLa cells with given multiplicities of
infection (moi, number of bacteria per cell at the onset of infection). After a 4 h infection followed by 4 h post-incubation, host DNA double
strand breaks were quantified by staining S139-phosphorylated histone H2AX (pseudocolored green) relative to DNA (red) (panels A and D).
Panels B and E: Genotoxic indexes (phosphorylated-H2AX fold-induction relative to control cells). Panels C and F: Colony-forming units (CFU)
per milliliter in the interaction medium were determined after the 4 h infection.
This figure is available in colour online at wileyonlinelibrary.com.

numbers of CFUs. The clbS mutant showed an exponen-
tial growth phase closely similar to that of the wild-type
pks (Fig. 2). Similar CFU numbers were observed up to
the late exponential phase. In the stationary phase, in
contrast to the wild type that showed stable counts, the
clbS mutant exhibited a significant reduction of CFU
counts (Fig. 2). Chromosomal complementation of the
clbS mutant with the wild-type gene restored CFU levels
similar to that of the wild-type pks strain (Fig. 2). Similarly,
a plasmid-encoded clbS gene cloned with its cognate
promoter region abrogated the growth defect of the clbS
mutant (Fig. 3). It was previously observed that clb gene
transcription peaked in the late exponential to early sta-
tionary phase, suggesting that this transition phase sup-
ports maximal genotoxin production (Homburg et al.,
2007). We thus next investigated whether the decreased
numbers of CFU in the clbS mutant culture in the station-
ary phase was indeed associated to production of colibac-
tin. We constructed a clbS clbA double mutant in which
removal of the ClbA PPTase required for maturation of the
NRPS–PKS machinery completely abolishes production
of colibactin and a clbS clbP double mutant deficient for
the ClbP peptidase that cleaves precolibactin to release
the active genotoxin. The drop in CFU counts was not
observed in the clbS clbA double mutant (Fig. 3). Com-
plementation of the clbS clbA double mutant with a
plasmid-encoded clbA restored and even increased the
growth defect, possibly as a result of ClbA overexpression
and increased synthesis of colibactin (Fig. 3). Similarly,
the clbS clbP double mutant did not show reduced CFU
counts and was fully complemented by a plasmid-
encoded clbP gene (Fig. 3). Thus, the reduction of CFU in
the clbS mutant in the stationary growth phase was tightly
associated with production of active colibactin.
Autotoxicity of colibactin in a clbS mutant induces a
viable-non replicating state
In line with an autotoxicity of colibactin, we noted a distinct
colony morphology in the clbS mutant grown on agar

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Fig. 2. Decreased numbers of clbS mutant culturable cells in the
stationary growth phase. E. coli DH10B hosting the BACpks (pks),
the isogenic clbS mutant or the mutant complemented by
reinsertion of the clbS gene at the chromosomal attTn7 site (clbS
attTn7::clbS) were pre-grown in LB to reach exponential phase,
2 × 106 bacteria ml−1 were inoculated in LB and grown for indicated
times before plating on LB to determine colony-forming unit (CFU)
numbers. Each point represents the mean and standard error of
the mean (SEM) of three to six independent experiments. Two-way
ANOVA and Bonferroni post-test, ***P < 0.001.
plates. The clbS mutant colonies exhibited flattened or
depressed centers, giving the colonies a ringed ‘draughts-
man’ appearance. This phenotype was suppressed by
complementation (Supplementary Fig. 2). We thus next
Fig. 3. Decreased numbers of CFU in overnight cultures of clbS
but not in clbS clbA and clbS clbP mutants. E. coli DH10B hosting
the BACpks (pks), the clbS mutant, the clbS clbA and clbS clbP
double mutants, and the complemented mutants, were pre-grown in
LB to reach exponential phase, 106 bacteria ml−1 were inoculated in
LB and grown overnight before plating on LB to determine
colony-forming unit (CFU) numbers. The median and individual
results of independent experiments are shown. One-way ANOVA
and Bonferroni post-test, ***P < 0.001.
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investigated clbS mutant viability by assessing bacterial
membrane integrity with cell impermeant dyes. The wild-
type pks, clbS and clbS clbA mutants, the complemented
mutants and the vector control strain were grown over-
night in LB, then propidium iodide (PI) was added, and the
PI-uptake by membrane-compromised (i.e. dead) bacteria
was assessed by flow cytometry. In contrast to control
heat-killed bacteria, none of the strains tested was found
to take up the dye (Fig. 4A and Supplementary Table S1).
Similar results were obtained using the lower molecular
weight dye ethidium bromide (Supplementary Table S1).
We also monitored the bacteria metabolic state, by quan-
tification of cellular ATP levels using a luciferase-based
luminescence assay. All the overnight cultures of wild-type
pks, clbS and clbS clbA mutants, and the complemented
mutants showed similar levels of ATP (Fig. 4B). Thus,
clbS mutant bacteria producing colibactin exhibit reduced
numbers of CFU together with largely conserved mem-
brane integrity and metabolic activity, suggesting that
these bacteria remained viable but did not proliferate. We
thus monitored the replication of clbS mutant bacteria in a
fluorescence dilution assay. The wild-type pks and clbS
mutant strains transformed with a plasmid encoding an
IPTG-inducible gfp gene were grown overnight in the
presence of IPTG, further incubated in fresh LB without
inducer for 0–7 h, then the dilution of the preformed pool
of fluorescent GFP protein in dividing cells was monitored
by flow cytometry. Although fluorescence intensities of the
pks bacterial cell populations were gradually reduced as a
result of replication, the clbS mutant population remained
highly fluorescent with no dilution of GFP, indicating that it
was non-replicating (Fig. 4C). Thus, clbS mutant bacteria
producing colibactin remain viable but cease to replicate.
We next asked whether arrested clbS mutant cells could
be rescued by expression of the clbS gene cloned under
the control of an inducible promoter. The clbS mutant was
transformed with a gfp-clbS gene cloned on an IPTG
inducible expression vector. Following a culture without
IPTG up to the stationary phase to express the autotox-
icity phenotype, the bacteria were induced (or not) with
IPTG. Although the induced cells expressed GFP fluores-
cence (but not the control non-induced cells, not shown),
no significant increase of CFU levels was observed (Sup-
plementary Fig. 3). Thus, colibactin-induced stasis could
not be reversed following expression of ClbS.
The SOS-response is activated in a colibactin-producing
clbS mutant
Induction of a senescent-like state in which viable cells
are not able to form colonies has been described in E. coli
treated with a DNA-damaging antibiotic that activates the
co-ordinated cellular SOS response (Pennington and
Rosenberg, 2007; Dörr et al., 2009). We examined the
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induction of the SOS response in the clbS mutant, using
the plasmid pRecA-GFP coding for the recA promoter
controlling the gfp reporter gene. The wild-type pks, clbS
and clbS clbA mutants and vector control strains hosting
pRecA-GFP were grown overnight in LB and GFP fluo-
rescence was measured (Fig. 5). In contrast to positive
control bacteria treated with ciprofloxacin, wild-type pks or
Fig. 5. Induction of the SOS response in the clbS mutant
producing colibactin. DH10B pBACpks (pks), the clbS, clbA and
clbS clbA mutants, hosting the plasmid pRecA-GFP (coding for gfp
reporter gene under control of the DNA damage-inducible recA
promoter) were grown 17 h in LB broth and the GFP fluorescence
per OD600 units was measured with a fluorescence plate reader
and normalized to that in the DH10B pBAC vector control strain.
Individual results of four independent experiments and the median
are shown. As a DNA damage positive control, ciprofloxacin (cipro,
1 μg ml−1) was added to a DH10B pBAC vector overnight culture
and incubated 1 h at 37°C. One-way ANOVA with Bonferonni
multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001.
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ClbSClbS protects
protects fromfrom colibactin9015
colibactin
Fig. 4. The clbS mutant exhibits a viable but
non-replicating state. Panel A: DH10B hosting
the BAC vector, the BACpks (pks) and the
clbS mutant were grown overnight in LB, then
propidium iodide (PI) was added and the dye
uptake by membrane-compromised bacteria
was quantified by flow cytometry. Heat-killed
(95°C) bacteria were used as a positive
control. Percentages of various strains in the
positive gate (bar) are shown in
Supplementary Table S1. Panel B: The
bacteria were grown overnight in LB, then
bacterial ATP levels were determined with a
luminescence assay. Relative light units
(RLUs) per optical density units (OD) at
600 nm were recorded with a microplate
reader. The median and individual results of
three independent experiments are shown.
Panel C: DH10B pBACpks (pks) and the clbS
mutant hosting the plasmid pDIGi coding for
an IPTG-inducible GFP were grown overnight
in LB + IPTG, then without inducer for the
indicated times. The dilution of GFP
fluorescence (caused by bacterial replication;
Helaine et al., 2010) was analyzed by flow
cytometry.
clbA mutant cells did not show activation of the recA
promoter. Interestingly, the clbS mutant exhibited a sig-
nificant increase of fluorescence (Fig. 5). No increase of
fluorescence was observed in the clbS clbA double
mutant, showing that the SOS-response activation in the
clbS mutant was associated to colibactin production
(Fig. 5). Similar results were obtained with bacterial colo-
nies grown overnight on LB plates (Supplementary
Fig. 4). These results suggest that the clbS mutant pro-
ducing colibactin activates the SOS response following
self-inflicted DNA damage.
Mutating the nucleotide excision repair system strongly
enhances the autotoxicity of colibactin in a clbS
mutant background
The SOS induction can be driven by primary damage to
DNA such as covalent binding of adducts or cross-links,
and colibactin was recently proposed to cross-link DNA
(Vizcaino and Crawford, 2015). As these damages are
repaired mainly by the nucleotide excision repair uvrABC
repair system (Truglio et al., 2006), we reasoned that an
uvrB mutant would be highly sensitive to colibactin in a
clbS mutant background. We thus constructed an uvrB
single mutant and a clbS uvrB double mutant, and as a
control an uvrB mutant in the E. coli strain hosting the
BAC vector. Whereas the single uvrB mutants did not
exhibit an altered growth, the clbS uvrB double mutant
shown a marked reduction of CFU numbers compared
with that of the clbS single mutant (Fig. 6). Complemen-
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Fig. 6. Enhanced growth defect in a clbS uvrB double mutant. E.
coli DH10B hosting the BAC vector, BACpks (pks), the clbS and
uvrB single mutants, the clbS uvrB double mutants, and the double
mutant complemented with the empty vector plasmid (pASK) or the
plasmid coding for clbS or uvrB (pClbS or pUvrB) were pre-grown
in LB to reach exponential phase, 106 bacteria ml−1 were inoculated
in LB and grown 17 h before plating on LB to determine
colony-forming unit (CFU) numbers. The median and individual
results of independent cultures in five experiments are shown.
One-way ANOVA and Bonferroni post-test, ns: not significant,
**P < 0.01, ***P < 0.001.
tation of the clbS uvrB mutant with pClbS (but not with the
empty pASK75 vector) restored CFU levels similar to that
of the single uvrB mutant. Similarly, complementation with
the uvrB gene restored a growth level similar to that of the
single clbS mutant (Fig. 6).
Together these results support a model where ClbS is a
colibactin resistance protein that, coupled with the
prodrug assembly and efflux strategies, prevents self-
inflicted DNA damage.
Ectopic expression of clbS protects HeLa cells
from colibactin
To provide additional support to the hypothesis that ClbS
is a colibactin resistance protein, we tested whether its
expression within mammalian HeLa cells could protect
the host cell DNA against colibactin. To allow ectopic
expression and visualization of expressing eukaryotic
cells, the clbS gene was cloned in a mammalian expres-
sion vector as a translational fusion with the gfp gene. The
GFP-ClbS fusion protein was functional for complemen-
tation of the clbS mutant, as verified with a bacterial
expression vector (Supplementary Fig. 3). The mamma-
lian GFP-ClbS expression vector was then transfected in
HeLa cells, in parallel with the expression vector coding
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only for GFP as a control. Microscopic examination of the
transfected HeLa cells showed that both the GFP control
and GFP-ClbS proteins exhibited similar diffuse localiza-
tion in the host cell cytoplasm and nucleus and that
expression of GFP-ClbS did not induce signs of toxicity
(Fig. 7A). Following transfection with GFP or GFP-ClbS
expressing vectors, the HeLa cells were infected 4 h with
the wild-type pks and DNA DSBs were demonstrated by
staining pH2AX 4 h later (Fig. 7A). In HeLa cells that were
transfected with the GFP control plasmid, the cells
expressing or not GFP exhibited similar nuclear pH2AX
foci. In contrast, HeLa cells that were transfected with
GFP-ClbS showed a non-homogeneous pH2AX staining
pattern: GFP-ClbS positive cells exhibited less pH2AX
staining compared with that in nearby cells not expressing
GFP-ClbS (Fig. 7A, arrows). To quantify this effect, the
pH2AX signal in relation with GFP fluorescence was
recorded by flow cytometry (Fig. 7B). The GFP/pH2AX
scatter graphs showed that pH2AX levels did not change
with the GFP signal in pks-infected and GFP-transfected
cells. In GFP-ClbS transfected cells, however, pH2AX
levels diminished in conjunction with increased GFP-ClbS
expression (Fig. 7B). Following infection with wild-type
pks at various moi the numbers of cells with a pH2AX
signal above a defined threshold in GFP-ClbS expressing
cells were significantly reduced compared with that in
GFP control cells (Fig. 7C). In contrast, GFP-ClbS
expressing cells were not protected against the DNA-
damaging peptide-polyketide bleocin (Supplementary
Fig. 5). These results show that clbS gene expression in
mammalian cells confers a specific protection against the
genotoxicity of colibactin, confirming that ClbS is a coli-
bactin resistance protein.
Discussion
Drug-producing bacteria protect themselves against the
toxicity of their biosynthesized products by using multiple
resistance mechanisms in combination, including drug
sequestering proteins, which are typically encoded on the
biosynthesis gene cluster (Cundliffe and Demain, 2010).
We thus tested whether the pks-encoded ClbS protein,
which binds reactive α-methylene-γ-butyrolactones
(Kunzmann and Sieber, 2012), could participate in the
self-protection of E. coli producing colibactin. A clbS
mutant displayed an autotoxicity phenotype characterized
by a marked growth defect with a decrease of CFU
numbers in overnight cultures. This was linked to produc-
tion of colibactin, as this phenotype was expressed at the
transition between the exponential and stationary phases,
the time that supports maximal clb gene expression (and
supposedly colibactin production) (Homburg et al., 2007).
In addition, the growth defect was abrogated in a clbS
clbA double mutant inactivated for the ClbA PPTase that
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 ClbSClbS protects
protects fromfrom colibactin9037
colibactin

Fig. 7. clbS ectopic expression protects HeLa cells from colibactin. HeLa cells were transfected with plasmids coding for a GFP-ClbS fusion
protein or for GFP alone and 24 h later, infected 4 h with DH10B pBACpks (pks) or with a clbA mutant at given multiplicities of infection (moi,
number of bacteria per cell at the onset of infection). Host DNA double strand breaks were demonstrated 4 h later by staining phosphorylated
histone H2AX (pH2AX). Panel A: epifluorescence optical section (Apotome) images of pH2AX (red), GFP (green) and DNA (blue) in cells
expressing GFP or GFP-ClbS and infected with DH10B pBACpks (MOI = 50). Bar = 20 micrometer. The green arrow points to a GFP-ClbS
expressing cell with low pH2AX, and the red arrow a cell that does not express GFP-ClbS but shows high pH2AX. Panel B: HeLa cells were
transfected and infected as in panel A and analyzed by flow cytometry to generate density plots of GFP and pH2AX fluorescence. Panel C:
cell counts of H2AX positive and negative cells in the GFP and GFP-ClbS-expressing population, based on the quadrant gates shown in panel
B. The experiment was performed three times in duplicates, with similar results. Chi-square test, *P < 0.001.
This figure is available in colour online at wileyonlinelibrary.com.

primes the NRPS–PKS assembly line and therefore does
not produce (pre)colibactin. Complementation with a
plasmid-encoded clbA gene restored and even increased
the growth defect, suggesting that overexpression of the
activating PPTase resulted in increased colibactin synthe-
sis and enhanced autotoxicity. The growth defect was also
suppressed in a clbS clbP double mutant, inactivated for
the periplasmic ClbP peptidase that cleaves precolibactin
to generate mature active colibactin.
The altered growth of the clbS mutant was associated
with the activation of the SOS response, the bacterial
response to DNA damage. The severe growth defect
observed in the clbS uvrB double mutant, in which the
nucleotide excision repair UvrABC system is inactivated,
further indicates that colibactin inflicted DNA damage in
the absence of the self-defense ClbS protein. As uvrB
mutants are unable to repair cross-links as well as
adducts (Truglio et al., 2006), our results also suggest that
colibactin can produce such DNA damages in bacterio.
This is in line with the recent finding that colibactin might
cross-link DNA, based on the in vitro reactivity of a can-
didate purified precolibactin (Vizcaino and Crawford,
2015). More work is required to establish the mode of
action of the mature colibactin, the binding to DNA, the
nature of the DNA damage(s) inflicted and how it ulti-
mately results in DNA DSB and mutagenesis in the mam-
malian cell.
The clbS mutant producing colibactin appeared to stop
replication without showing significant cell death, as exam-
ined by fluorescence dilution, membrane impermeant dyes
uptake and levels of intracellular ATP. The growth arrest is
reminiscent of persistence, a phenotype where a subpopu-

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C 2015
V John Wiley
Wiley && Sons
Sons Ltd,
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Molecular Microbiology,
Microbiology 99, 897–908
8 N. N.
904 Bossuet-Greif et alet. al.■ 䊏
Bossuet-Greif
lation of non-growing bacteria escapes the lethal action of
antibiotics without acquiring heritable resistance (Balaban
et al., 2004). It was shown that persistence can be medi-
ated by the SOS response, providing a mechanism of
survival upon DNA-damage (Dörr et al., 2009). The SOS
response has also been shown to induce a prolonged
senescence-like state in E. coli cells sustaining spontane-
ous DNA damage (Pennington and Rosenberg, 2007).
Senescence is triggered in mammalian cells exposed to
colibactin (Secher et al., 2013; Cougnoux et al., 2014;
Dalmasso et al., 2014). Senescence is regarded in eukary-
otes as a programmed cellular response to DNA damage
that suppresses proliferation of potentially mutated cells. In
E. coli enduring colibactin-inflicted damage, induction of
SOS followed by replication arrest could represent a similar
mechanism to cease replication of genetically damaged
bacterial cells. The analogous responses of the clbS
mutant E. coli and the eukaryotic cells may also relate to
the type of DNA damage induced by colibactin. Indeed
DNA cross-links present a unique challenge for the bacte-
rial or eukaryotic cell to repair and a major senescence
inducer in mammalian cells (Grillari et al., 2007).
The drug-binding property of the ClbS protein
(Kunzmann and Sieber, 2012), the SOS-response activa-
tion, the autotoxicity phenotype and increased sensitivity
of a uvrB mutant in a clbS background, together with the
protection of HeLa cells expressing GFP-ClbS, support
the hypothesis that ClbS is a colibactin resistance protein.
We propose that ClbS can sequester, or modify colibactin
and thereby prevent self-inflicted DNA-damage. Such
resistance proteins are common in antibiotic-producing
microorganisms. For example, two resistance proteins
have been characterized in Streptomyces sp. producing
the peptide-polyketide antitumor antibiotic bleomycin, the
BlmA sequestering protein and the N-acetyl-transferase
BlmB that acetylates and inactivates bleomycin (Galm
et al., 2005). Kunzmann and Sieber (2012) could not
detect any esterase, peptidase or protease activity in the
ClbS protein, suggesting that ClbS would rather act as a
colibactin-sequestering protein. Our results also indicate
that ClbS protective function is specific to colibactin, as
ectopic expression of GFP-ClbS did not protect HeLa
cells against the DNA-cleaving activity of bleocin. In addi-
tion, the pClbS plasmid did not confer any resistance to a
variety of antibiotics, including the DNA-damaging drugs
bleocin, ciprofloxacin and mitomycin-C (data not shown).
We propose a putative model in which ClbS functions
as a backup for the prodrug assembly and efflux mecha-
nisms, to confer a robust protection system to the produc-
ing bacteria (Fig. 8). Following synthesis by the NRPS–
PKS enzymes, the inactive precolibactin equipped with an
N-terminal acylated D-asparagine is secreted to the peri-
plasm, possibly by the putative efflux pump ClbM. The
peptidase ClbP cleaves the prodrug to release the active
C 2015 John ©
V 2015& John
Fig. 8. Putative model of suicide avoidance in
colibactin-producing E. coli. The NRPS-PKS enzymes,
post-translationally modified by the ClbA PPTase, synthesize
colibactin as a prodrug. The multidrug and toxic compound
extrusion (MATE family) efflux pump ClbM secretes the prodrug
into the bacterial periplasm, where the peptidase ClbP cleaves the
prodrug to release the active colibactin ‘warhead’. ClbS could
sequester or modify colibactin and thereby prevent DNA-damage.
Ultimately, the SOS-response and DNA nucleotide excision repair
system stands as the last-line of defense.
colibactin ‘warhead’ in the periplasm, away from the
genomic DNA. This prodrug assembly strategy with cleav-
age of an acylated D-asparagine to produce active drugs
upon secretion is also found in other NRPS and hybrid
PKS-NRPS antibiotic biosynthesis pathways, such as
xenocoumacin, amicoumacin and zwittermicin (Reimer
et al., 2011). Bacterial resistance during the biosynthesis
of intracellular toxic compounds typically involves multiple
mechanisms in combination (Cundliffe and Demain,
2010). Indeed the producing bacteria have also to cope
with the previously exported drug that might reenter into
the producer, and with exogenous drug produced by
sibling bacteria. ClbS could thus function as a comple-
mentary protection component, where the prodrug activa-
tion mechanism is ineffective against active genotoxin
(re)entry. Microorganisms that synthesize DNA-damaging
drugs develop such complementary self-protection strat-
egies relying on drug sequestration or modification
together with efflux systems. For example, the antitumor
antibiotic mitomycin-C produced by Streptomyces laven-
dulae is synthesized as an inactive prodrug that is
sequestered by the small soluble resistance protein Mrd,
delivered to the Mct efflux pump, and deactivated by the
McrA resistance protein (Sheldon et al., 1997; 1999). Fol-
lowing efflux in the periplasm and cleavage by ClbP, the
Wiley Sons Wiley & Sons Ltd,
Ltd, Molecular Molecular 99,
Microbiology, Microbiology
897–908

colibactin warhead is further trafficked to the eukaryotic
cell through a yet unexplored pathway. The protection of
HeLa cells by ectopic expression of GFP-ClbS indicates
that the colibactin warhead likely enters the eukaryotic
cells to inflict DNA damage.
In conclusion, we have gained a novel insight into how
toxigenic E. coli protects itself against colibactin. Ulti-
mately, this knowledge could help to further decipher coli-
bactin mode of action and to design new means to
prevent colibactin-mediated carcinogenesis.
Experimental procedures
Bacterial strains, mutants and plasmids
Strains used in this study were Escherichia coli DH10B
hosting the BACpks (Nougayrède et al., 2006) and isogenic
mutants and complemented mutants that are listed in Sup-
plementary Table S2. Gene mutagenesis was performed by
using the lambda Red recombinase method (Datsenko and
Wanner, 2000), with primers used listed in Supplementary
Table S2. The double mutants were constructed sequentially.
The mutations and deletion of FRT cassettes were verified by
PCR using primers upstream and downstream of the target
genes. For clbA and clbP complementation, the mutants
were transformed with the previously described plasmids
pClbA (Cuevas-Ramos et al., 2010) and pClbP (Dubois et al.,
2011). To construct plasmid pClbS, the clbS gene (with
207 bp upstream sequence) was PCR-amplified using
primers CPTPNG9/10, digested by XhoI and XmaI, and
ligated into pASK75 (Skerra, 1994). The resulting plasmid
pClbS was verified by sequencing. The clbS chromosomal
complementation was constructed by mini-Tn7 insertion of
the clbS gene at the attTn7 site (Choi and Schweizer, 2006);
the clbS gene was subcloned (XmaI/XhoI) from pClbS into
pUC18R6K-mini-Tn7T-Gm and the resulting plasmid was
electroporated in the clbS mutant together with the pTNS2
helper plasmid. The insertion of the clbS gene at the attTn7
site was selected on LB medium supplemented with
15 μg ml−1 gentamicin and verified by PCR using the
PTn7R and PglmS-down-Ecoli primers. To construct plasmid
pEGFP-ClbS for the eukaryotic cell transfection experiments,
the clbS gene was PCR-amplified using primers
CPTPNG1/2; the PCR product was digested with XhoI/XmaI
and ligated in the pEGFP-C2 vector (Clontech). The con-
struction was verified by sequencing. Plasmid pGFP-ClbS
was constructed by PCR amplifying the GFP-ClbS gene
fusion from pEGFP-ClbS with primers CPTPNG2/11 and
ligating the PCR product into pSC-B-amp/kan vector under
control of the IPTG-inducible promoter (Stratagene). Plasmid
pUvrB was constructed by PCR amplifying uvrB from DH10B
with primers CPTPJPN34/35; the PCR product was digested
with XmaI/BamHI and ligated into pASK75.
Bacterial growth, enumeration and kinetics
Escherichia coli strains were routinely grown at 37°C with 240
r.p.m. shaking in 5 ml of Lennox L broth (LB, Invitrogen) in
closed 16 × 100 mm tubes (Kima), or on LB agar plates.
© Sons Ltd,
C 2015 John Wiley & Sons
V Ltd, Molecular
Molecular Microbiology,
Microbiology 99, 897–908
ClbSClbS protects
protects fromfrom colibactin905
colibactin 9
Carbenicillin (50 μg ml−1), kanamycin (50 μg ml−1), gen-
tamicin (15 μg ml−1) or chloramphenicol (25 μg ml−1) were
added to the medium as required. For enumeration and
kinetic experiments, each strain was pre-grown in LB from
a −80°C glycerol stock to reach exponential growth
(DO600 = 0.4) and then inoculated at 2 × 106 bacteria ml−1 in
5 ml LB and grown overnight (17 h) or for indicated times. For
CFU enumeration, the culture broth was serial-diluted in
PBS, plated on LB agar plates and incubated overnight at
37°C.
Eukaryotic cell culture, transfection and infection
HeLa cells (ATCC) were grown in Dulbecco’s modified
Eagle’s medium (DMEM glutamax, Invitrogen) supplemented
with 10% (vol/vol) Foetal Calf Serum (FCS; Eurobio) and 1%
(vol/vol) Non Essential Amino Acid (Invitrogen) at 37°C in a
5% CO2 atmosphere. Cells were confirmed mycoplasma-free
by PCR and maintained by serial passage. HeLa cells were
infected as described previously (Nougayrède et al., 2006).
Briefly, the day before transfection or infection, HeLa cells
were trypsinized and seeded in cell culture plates or slides.
The following day, cells were washed three times with HBSS
(Invitrogen), inoculated with a defined multiplicity of infection
(moi; number of bacteria per cell at the onset of infection) in
infection medium (DMEM with 25 mM Hepes, Invitrogen) and
incubated with the bacteria for 4 h at 37°C in a 5% CO2
atmosphere. Cells were then washed three times with HBSS
and incubated 4 h in cell culture medium supplemented with
200 microgram ml−1 gentamicin, and then processed for
analysis. For ectopic expression experiments, HeLa cells
were transfected with plasmids pEGFP-C2 or pEGFP-ClbS
(EndoFree plasmid preparation, Qiagen) using TurboFect
(ThermoScientific), following the manufacturer protocol.
Twenty-four hours later, cells were infected as above and
processed for fluorescence microscopy or flow cytometry.
Phospho-H2AX staining and quantification by
microscopy, flow cytometry and in-cell western
For the imaging and quantification of pH2AX and GFP, HeLa
cells were grown in 8-chamber slides or 6-wells plates, trans-
fected and infected as described above. As a control, cells
were treated 4 h with 120 microgram ml−1 bleocin (Calbio-
chem). The cells were fixed, and immunostained as
described previously (Nougayrède et al., 2006). The antibod-
ies used were rabbit monoclonal anti pH2AX (20E3, Cell
Signaling Technology) and TexasRed or AlloPhycoCyanin
(APC) conjugated secondary antibodies (Jackson). DNA was
labeled with DAPI. Optical section images were acquired
using an ApoTome microscope (Zeiss). The GFP and APC
fluorescence of individual cells was quantified with a FACS-
Calibur flow cytometer (Becton). FL1 and FL4 data of at least
50 000 events (gated on SSC/FSC) and 10 000 GFP-positive
cells were recorded and analyzed with FlowJo 7.6 software.
The in-cell western (ICW) procedure was performed as
described previously (Audebert et al., 2010; Martin et al.,
2013). Briefly, the cells were grown and infected in 96-wells
cell culture plate. Following fixation with formaldehyde, per-
meabilization and blocking, the cells were incubated over-
 Bossuet-Greifetetalal. . ■䊏
10 N.N.Bossuet-Greif
906
night at 4°C with anti pH2AX antibodies (20E3, Cell Signaling
Technology; 1:200). An infrared fluorescent (800 nm) second-
ary antibody (Rockland; 1:500) was used to detect pH2AX.
DNA was counterstained with RedDot2 (Biotium; 1:500).
DNA and pH2AX were visualized simultaneously using an
Odyssey Infrared Imaging Scanner (LI-COR Biosciences) at
680 nm (pseudocolored red) and 800 nm (pseudocolored
green). Relative fluorescent units for pH2AX per cell (as
determined by the 800 nm signal divided by the 700 nm
signal) were divided by untreated controls to determine the
genotoxic index.
Bacterial viability, ATP content and fluorescence
dilution assay
To examine the bacterial membrane integrity, 25 μg ml−1 of
propidium iodide (Sigma) or 30 μg ml−1 ethidium bromide
(Sigma) was added to 8 μl of 17 h LB culture diluted in 1 ml of
PBS and incubated in the dark during 20–30 min at room
temperature. Dead control bacteria were prepared by heating
at 95°C for 10 min. To monitor bacterial division by fluores-
cence dilution, bacteria hosting pDIGi were cultured 17 h in
LB 0.5 mM IPTG to induce production of GFP (Helaine et al.,
2010), centrifuged, resuspended in fresh LB medium without
the inducer and further incubated during 0 to 7 h. GFP fluo-
rescence in bacteria was measured by flow cytometry, with a
FACSCalibur (Becton). PI uptake and GFP fluorescence was
recorded in the FL2 and FL1 channels respectively, in at least
50 000 bacteria (gated on SSC/FSC) and analyzed with
FlowJo 7.6 software. Bacterial cell metabolic activity (ATP
content) following 17 h growth in LB was measured using the
BacTiter-Glo assay (Promega) following the manufacturer
recommendations. Luminescence and optical density at
600 nm (OD600) was recorded with a Tecan Infinite Pro
microplate reader.
SOS response monitoring
The activation of the SOS response pathway was monitored
with a GFP reporter under control of the recA promoter
(Aertsen et al., 2004). Bacteria hosting the pRecA-GFP
plasmid were grown 17 h in LB, then 100 μl samples were
transferred to a microplate and GFP fluorescence was meas-
ured with a Tecan Infinite M200 microplate reader. As a posi-
tive control, ciprofloxacin (1 μg ml−1) was added to a 16 h
culture of DH10B hosting the BAC vector and incubated for
1 h at 37°C. The OD600 was measured, and fluorescence at
505 nm (bandwidth 20 nm) was measured using an excita-
tion wavelength of 475 nm (bandwidth 9 nm). Fluorescence
was expressed per unit of OD600 (Aertsen et al., 2004).
Alternatively, the strains were grown overnight on LB plates,
isolated bacterial colonies were resuspended in 1 ml PBS
and GFP fluorescence was analyzed with a FACSCalibur
(Becton) flow cytometer, in at least 50 000 bacteria (gated on
SSC/FSC) and analyzed with FlowJo 7.6 software.
Statistical analyses
Statistical analyses were carried out using GraphPad Prism
5.0b. P values were calculated using one-way or two-way
C 2015 John ©
V 2015
ANOVA followed by Bonferroni post-tests. CFU/ml and
RLU/OD600 were log-transformed for the analyses. P
values < 0.05 were considered significant and are denoted by
*, P < 0.01 is denoted by ** and P < 0.001 by ***. For cell
counts by flow cytometry, a Chi-square test was used with the
number of events per region, a conservative value of
P < 0.001 was considered significant.
Acknowledgements
The authors have no conflict of interest to declare. We thank
Michèle Boury for the initial cloning of clbS, Sophie Allart,
Astrid Canivet and Fatima-Ezzahra L’Faqihi-Olive for techni-
cal assistance at the cellular imaging and flow cytometry
facilities of INSERM UMR1043, Toulouse. We thank David
Holden for the gift of pDIGi and Chris Michiels for the pRecA-
GFP plasmid. This work was supported by grants ANR-13-
BSV1-0028, ANR-13-BSV3-0015 and INCA-PLBIO13-123.
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