Prophage Induction Is Enhanced and Required for Renal
Disease and Lethality in an EHEC Mouse Model
Jessica S. Tyler1.¤a, Karen Beeri1.¤b, Jared L. Reynolds1¤c, Christopher J. Alteri1, Katherine G. Skinner1,
Jonathan H. Friedman2, Kathryn A. Eaton1, David I. Friedman1*
1Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, United States of America, 2Department of Mathwork, Mathworks, Natick,
Massachusetts, United States of America
Enterohemorrhagic Escherichia coli (EHEC), particularly serotype O157:H7, causes hemorrhagic colitis, hemolytic uremic
syndrome, and even death. In vitro studies showed that Shiga toxin 2 (Stx2), the primary virulence factor expressed by
EDL933 (an O157:H7 strain), is encoded by the 933W prophage. And the bacterial subpopulation in which the 933W
prophage is induced is the producer of Stx2. Using the germ-free mouse, we show the essential role 933W induction plays
in the virulence of EDL933 infection. An EDL933 derivative with a single mutation in its 933W prophage, resulting specifically
in that phage being uninducible, colonizes the intestines, but fails to cause any of the pathological changes seen with the
parent strain. Hence, induction of the 933W prophage is the primary event leading to disease from EDL933 infection. We
constructed a derivative of EDL933, SIVET, with a biosensor that specifically measures induction of the 933W prophage.
Using this biosensor to measure 933W induction in germ-free mice, we found an increase three logs greater than was
expected from in vitro results. Since the induced population produces and releases Stx2, this result indicates that an activity
in the intestine increases Stx2 production.
Citation: Tyler JS, Beeri K, Reynolds JL, Alteri CJ, Skinner KG, et al. (2013) Prophage Induction Is Enhanced and Required for Renal Disease and Lethality in an EHEC
Mouse Model. PLoS Pathog 9(3): e1003236. doi:10.1371/journal.ppat.1003236
Editor: Ralph R. Isberg, Tufts University School of Medicine, United States of America
Received November 16, 2012; Accepted January 22, 2013; Published March 28, 2013
Copyright: ? 2013 Tyler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by Public Health Service grant AI11459-10, the University of Michigan Endowment for the Basic Sciences, and the 599 NIAID,
NIH, Department of Health and Human Services, under Contract No. N01-AI-30058 from the National Institutes of Health. JST was supported in part by NIH
Training Grant GM008353. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤a Current address: Novartis Vaccines & Diagnostics, Inc. Cambridge, Massachusetts, United States of America
¤b Current address: Microbial and Environmental Genomics, J. Craig Venter Institute, San Diego, California, United States of America
¤c Current address: Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
. These authors contributed equally to this work.
Enterohemorrhagic E. coli (EHEC) has emerged as a serious
health threat with numerous outbreaks most commonly due to
contaminated beef, but also to contaminated vegetables and
water . Although EHEC strains , and another recently
identified pathogenic E. coli , encode a number of virulence
factors, the most serious sequelae of infection by these strains are
due to the acquisition and expression of genes encoding Shiga
In many EHEC strains these toxins are encoded in the genomes
of prophages of the l family (referred to as lambdoid phage) .
Two major classes of Shiga toxins, Stx1 and Stx2, have been
identified in EHEC strains . Although sharing the same activity,
they differ somewhat in sequence and Stx2 is associated with the
more severe sequelae in humans  and is the cause of disease in
animal models . These members of the AB5class of toxins bind
eukaryotic cells by attachment of the pentameric structure of the B
subunit to a glycoprotein receptor on the eukaryotic cell .
Retrograde transit through the endosomic pathway to the cytosol
results in the A subunit, a glycosidase, reaching the ribosomal
RNA . There, a specific adenine residue in the large ribosomal
subunit is cleaved, resulting in arrested protein synthesis that leads
to cellular intoxication . EHEC strains commonly isolated in
outbreaks are those of the O157:H7 serotype .
Members of the lambdoid family of temperate phages share a
common genome organization with prototypical l. Genes at the
same relative position on their respective genomes may differ in
sequence, but for the most part they share the same activity .
For example, the repressors and operators may differ in sequence
and specificity, but the different lambdoid phages have a common
structure and location for these genetic elements on their genomes
. Moreover, the lambdoid phages are mosaics with each phage
sharing a number of different genes with different members of the
family [11,13]. These conserved structure-function relationships
allowed for the relatively rapid determination of the role of the
phage in Stx expression .
When present, the stxA and B genes are located downstream of
PR9, the late phage promoter [15,16], and upstream of the phage
lysis genes (Fig. 1) [14,17]. In vitro and in vivo studies with the
O157:H7 strain 1:361 and its resident stx2-phage, w361, showed
that transcription from PR9 is required for Stx2 production . In
vitro studies with the E. coli strain K9675 (a derivative of the
nonpathogenic strain K37 lysogenized with the stx2-phage 933W)
showed that Stx2 expression requires prophage induction .
Hence, Stx2 expression, at least under these in vitro conditions,
PLOS Pathogens | www.plospathogens.org1March 2013 | Volume 9 | Issue 3 | e1003236
depends on the phage induction cascade. Prophage induction
explains why patient treatment with antibiotics that can act as
inducing agents, such as the quinolones, lead to higher Stx levels
 and exacerbate the disease .
The lambdoid phage regulatory cascade which leads to phage
production and cell lysis has been the subject of years of study with
l and to a lesser extent with other members of this family of
phages . Induction, which results in the initiation of the
regulatory cascade, is set in motion when the bacterium containing
the prophage (lysogen) sustains DNA damage and responds by
activation of the LexA regulon, leading to a cellular change in
gene expression termed the SOS response [23,24]. One member
of this regulon, RecA, increases in quantity and assumes an
activated form, RecA*, by interacting with single-stranded DNA
generated by DNA damage . RecA*, through its co-protease
activity, facilitates the autocleavage of phage repressor ,
allowing initiation of transcription from the early PL and PR
promoters (Fig. 1). Transcription from PLresults in expression of
N protein, which acts to modify RNA polymerase initiating
specifically at PL and PR to a form resistant to downstream
terminators . N-modified transcription from PRtranscends
downstream terminators resulting in Q expression. Q in turn
modifies transcription initiating at the late PR9 promoter to a
termination-resistant form allowing expression of downstream
genes , including stx A and B in stx-phages [14,17,18,28].
A l prophage fails to induce if the repressor gene (cI) has a
mutation that inhibits autocleavage [29,30]. These mutations,
called ind, change amino acid residues within the repressor that
participate in a serine protease activity that catalyzes autocleavage
We have previously suggested that the induced subpopulation is
responsible for Stx production and release . Lysogens with
most lambdoid prophages are stable with only an extremely small
fraction of the population, in the absence of an external inducing
agent, sustaining sufficient DNA damage to be induced, a
stochastic process referred to as ‘‘spontaneous induction’’ . It
has been suggested that collapse of the replisome in normally
growing bacteria caused by single-stranded breaks or noncoding
lesions may be an internal event responsible for spontaneous
induction . DNA damage-inducing agents change induction
from a stochastic to a deterministic process that activates RecA
and, in turn, repressor cleavage . Although recA mutants have
been used to study conditions where the prophage fails to be
induced and Stx is not expressed , such an experimental
approach suffers from the disadvantage of the pleiotropic effects
on bacterial physiology due to loss of RecA activity [34,35]. Using
a phage with an ind mutation avoids this problem by limiting the
failure of SOS control only to the prophage with the ind mutation.
Linkage of Stx expression to prophage induction raises the
question as to whether the intestinal environment increases Stx
levels by causing prophage induction. One way this could occur
would be by increasing DNA damage in the bacterium. In vitro
experiments showed a modest increase in Stx production by an
O157:H7 strain when bacteria were cultured with neutrophils
, which produce H2O2that can cause DNA damage leading to
an SOS response and prophage induction.
Here, we report experiments with the O157:H7 strain EDL933
and derivatives of EDL933 that carry a 933W prophage with a cI
ind mutation. Using a germ-free mouse model of disease, we show
that whereas the parent EDL933 with wild-type 933W prophage
produces high levels of Stx in vivo and causes severe disease that
can lead to death, a derivative isogenic except for a cI ind mutation
in the 933W prophage produces extremely low levels of Stx2 and
does not cause any observable disease. These results provide
compelling evidence that induction of the 933W prophage is a
major factor in pathogenesis of EDL933 and prophage induction
may play a role in the severity of infection by other O157:H7
strains. Using an EDL933 SIVET reporter strain, which survives
induction but undergoes a change in antibiotic resistances
following induction, we show that the intestinal environment
contributes to induction of the 933W prophage in EDL933.
Characterization of the 933W ind mutant in EDL933
Induction of lambdoid prophages, including that of 933W,
occurs when the repressor protein autocleaves in the presence of
activated RecA protein . Mutations, ind, in the cI gene result in
a noncleavable repressor and thus an uninducible prophage .
The strategy used to obtain the ind mutation in the cI gene of the
933W prophage in EDL933, a change of Lys codon 178
[suggested by John Little ], was based in part on the
procedure previously employed by our laboratory to construct
an identical point mutation in the cI gene of the 933W prophage in
strain K9675 . Sequencing confirmed that the cI gene in
EDL933 with the mutant 933W had only the designed nucleotide
substitution at codon 178. The mutation, named ind1, is a change
of the Lys codon to an Asn codon (K178N). This change interferes
with the autocatalytic serine protease activity of the CI repressor
, rendering the prophage uninducible. We will refer to the
derivative of EDL933 carrying the 933W prophage with the cIind1
mutation as EDL933cIind1 (Table 1). This strain carries the stx2
genes and differs from EDL933 only by the 933W cI mutation.
To assess the effectiveness of the ind1 mutation on prophage
induction, we treated EDL933 and EDL933cIind1 with mitomycin
C . At an appropriate concentration, this DNA damaging
agent activates the SOS response of most of the population
sufficiently to induce the prophage . Treatment of the
EDL933 parent with 2 mg/ml of mitomycin C led to full induction
of the culture; i.e., lysis was nearly complete (Fig. 2). Identical
treatment of EDL933cIind1 failed to cause lysis (Fig. 2). This result
confirms that the ind1 mutation blocks induction of 933W.
Additionally, it shows that the inducing agent does not cause
Infection with Enterohemorrhagic E. coli (EHEC), and more
recently with the Enteroaggregative E. coli strain O104:H4,
is a significant health risk, causing bloody diarrhea, kidney
failure, and even death. The virulence factor in these
bacteria responsible for the severe outcomes is Shiga toxin
(Stx). Genes encoding Stx are in the genome of bacterial
viruses (prophages) on the pathogenic E. coli chromo-
somes. The prophage remains quiescent until damage to
the bacterial chromosome occurs causing prophage gene
expression (called induction), which leads to production of
bacteriophages that are released into the environment.
Because stx expression is controlled by the phage
regulatory system, prophage induction leads additionally
to production and release of Stx. This study provides
conclusive evidence that in a mouse model of EHEC
infection, induction of the prophage carrying the stx genes
is specifically required for EHEC to cause disease and that
the intestinal environment adds to the induction and
therefore to the production of Stx. Similar events likely
regulate Stx production and release by the Stx encoding
phage in the O104:H4 strain. Controlling prophage
induction offers a means to control EHEC infection.
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any of the large number of defective prophage in EDL933  to
express lytic activity. This finding provides direct evidence that
induction of 933W is not only responsible for Stx2 production, as
shown below, but also for the lysis that releases Stx2 from the
Shiga toxin production
We used an ELISA to assess Stx2A levels; comparing levels in
EDL933 with those in the EDL933cIind1 derivative and the
nonpathogenic 933W lysogen K9675. In the absence of an
inducing agent, the parent EDL933 expresses ,40 times the level
of Stx2 expressed by EDL933 cIind1 mutation (Fig. 3). This result
provides compelling evidence that in culture a significant fraction
of Stx2 production derives from the subpopulation of EDL933 in
which the 933W prophage is induced. These results are only
partially consistent with our previous findings with strain K9675
. In that study we found that in the absence of an external
inducing agent, the level of Stx2A produced by K9675 was ,10
fold lower than the level produced under these conditions by
EDL933 with its wild-type 933W prophage. In the current study,
we confirmed these findings, showing that in the absence of an
external inducer (spontaneous induction) EDL933 produces ,10
times more Stx2A than K9675 (Fig. 3).
To rule out the possibility that the low Stx2A levels in the
nonpathogenic strain resulted from alteration of the prophage or
the host, we measured Stx2A production from another nonpatho-
genic K12 related strain, MC1000 , with a 933W prophage.
As above, we observed the lower level of Stx2A expression in the
non-pathogenic strain compared to EDL933 (data not shown).
Although comparison of spontaneous induction shows that
EDL933 produces ,10 fold higher levels of Stx2A than K9675,
the source of Stx2A for each is primarily that fraction of the
population in which the prophage is induced (this study and Tyler
et al. .
Figure 1. Diagrams illustrating genetic arrangements and constructs. I. The regulatory region of stx-carrying lambdoid phage and patterns
of regulated transcription (not drawn to scale) (A) Arrangement of relevant genes. (B) Operators and promoters. (C) Transcription terminators. (D)
Transcription patterns based on studies with l. Temporal order: Early transcripts from PLand PRending at indicator terminators, with ,40%
transcending tR1. Action of antitermination protein N allows synthesis of delayed early transcripts. Action of antitermination protein Q immediately
downstream of PR9 allows maximum synthesis of late transcripts that include the stx genes. II. Components of SIVET (not drawn to scale). Top: (E) The
early regulatory region of the 933W SIVET prophage. Deletion substitutions, N::spc and O-P::amp eliminate lethality by the induced prophage. The
tnpR gene encodes the cd resolvase and is transcribed from the prophage PRpromoter and under control of the 933W repressor. Hence, TnpR is not
expressed by the repressed lysogen, but is expressed when induction leads to autocleavage of the phage repressor. Bottom: (F) SIVET reporter
cassette with the cat gene interrupted with a kanR cassette flanked by resC sites (targets for TnpR resolvase). In this form the reporter cassette confers
KanR. (G) Action of TnpR removes the kanR cassette and one flanking resC site. This leaves the cat gene interrupted by one resC site on the bacterial
chromosome. By proper designing the position of the insertion site of the cassette in the cat gene of the reporter cassette as well as engineered small
nucleotide changes in the res sequence, excision of the kanR cassette leaves a functional cat gene even though a resC sequence remains within the
cat gene (confers CamR) . (H) The kanR cassette with the other resC site is excised as a non-replicating circle and lost by segregation. The thick
horizontal arrows in E represent patterns of transcription from early promoters following induction, loss of repression.
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Table 1. Strain and plasmid list.
Strain Name Relevant Information Source/Reference
K37 Nonpathogenic laboratory E. coli strain derived from NIH strain N99 
K9675 K37 (933W)
DY406 W3110 lcI857 D(cro-bioA) N-kil::cat-sacB 
EDL933 Isolated from ground beef, Serotype O157:H7[74,75]
K10595 K37 (933WcIind (K178N) 
DY378W3110 lcI857 D(cro-bioA) 
K9685DY406 (cat-sacB (CSB))Court lab
K10985EDL933 pKD46-ampR This work
K11078EDL933 (933W N::kan) pKD46-spcThis work
K11084 K11078 (cro::tnpR 168, OP::amp) This work
K1111411084 (CP-933V N-cII::cat-sacB) This work
K11115K1114 (CP-933V DN-cII) This work
K11161K1115 lacZ::catThis work
K11173 EDL933 SIVET 1: (cat::resC-tetR-resC::cat) This work
K11349EDL933 (933WcIind1)This work
K11604EDL933 SIVET 2: (cat::resC-kanR-resC::cat) This work
K11607 K11604 (933WcIind1 KanR)This work
K11608K11604 (933WcIind1 CamR) This work
Plasmid pKD46 (ampicillin R) 
PlasmidpKD46spcR (spectinomycin R) This work
Plasmid pKD46hygR (hygromycin R) This work
Figure 2. Induction and lysis following mytomycin C treatment. Aliquots of EDL933 and the cIind1 derivative untreated and treated with
2 mg/ml of mitomycin C were incubated with shaking at 37u. Ind: (+) EDL933, inducible, (2) EDL933cIind1 (K11349), not inducible. Mitomycin C: (+)
treated, (2) untreated. Vertical arrow indicates time mitomycin C added.
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Prophage induction and Stx2 production
To specifically assess the role of induction of the 933W
prophage in Stx2 production, we determined Stx2 levels following
treatment with mitomycin C (2 mg/ml). As shown in Fig. 3,
mitomycin C treatment resulted in a 100 to 200-fold increase in
Stx2 production by EDL933. Although K9675 produced signif-
icantly less Stx2 than EDL933 in the absence of an inducing agent,
it produced about the same levels of Stx2 following treatment with
mitomycin C as similarly treated EDL933.
The EDL933cIind1culture treated with mitomycin C produced
5- to 10-fold more Stx2 than the untreated culture. Although two
orders of magnitude lower than the Stx2 production reached by
EDL933 treated with mitomycin C, the increased levels we
observed with the treated EDL933cIind1 were reproducible. The
increase in Stx2 following mitomycin C treatment is consistent
with the observation of measurable levels of Stx2 produced by
EDL933cIind in the absence of an inducing agent. Either the
mutant repressor retains some ability to autocleave (leaky mutant)
in the EDL933 environment or there is an alternative route to
Stx2 expression. However, in either event the production of Stx2 is
extremely low in the presence of the cIind1 mutation.
Prophage induction and EDL933 pathogenicity
Results of clinical studies of children with EHEC infection show
that phage induction likely plays an important role in the disease;
e.g., those treated with antibiotics that elicit an SOS response may
experience more severe outcomes . In mice, treatment with
ciprofloxacin (an antibiotic that elicits the SOS response) also
results in greater in vivo expression of Stx, likely via prophage
Although suggestive, these findings are far from definitive. As
discussed, the SOS response has pleiotropic effects on bacterial
gene expression and does far more to affect cell physiology than
induce prophage [34,35]. Relevant to our studies, treatment of
EDL933 with the DNA damaging agent norfloxacin results in
changes in the expression of a number of prophage and non-
prophage genes in EDL933 . Because the only effect of the
ind1 mutation is to interfere with induction of the 933W prophage,
experiments with EDL933cIind1 allowed us to ask specifically how
significant induction of the 933W prophage is in causing the
pathology associated with EDL933 infection.
The germ-free mouse has proven an effective and practical
found that germ free mice infected with O157:H7 strains such as
EDL933 develop acute renal tubular necrosis and renal glomerular
thrombosis leading to renal failure and death. In the same study, we
also reported that a similar infection with a derivative of EDL933
isogenic except for a deletion of the stx2 genes does not result in any
of the pathogical changes seen with the wild-type parent strain.
Hence, in this animal model, all of the described pathological
changes result from the action of Stx2. For these reasons, we chose
the germ-free mouse to assess the role in the disease process of
induction specifically of the 933W prophage carried by EDL933.
Groups of 6 (3 female and 3 male) germ-free Swiss-Webster mice
were used in the experiments. They were infected with one of three
bacteria, EDL933 or either of two isogenic strains that differed by
having the Dstx::cat deletion substitution or the cIind1 point
mutation. For all strains tested, each animal was challenged with
106cfu administered orally. All three groups of mice were equally
colonized over the seven days ofthe experiment in whichbacteria in
the feces were measured (,1010cfu/g). As expected from our
previous work, all 6 mice infected with the wild-type EDL933
parent strain became moribund prior to the scheduled time mice
were euthanized at three weeks. All mice infected with the Dstx::cat
deletion derivative showed no signs of disease. Like the latter group,
miceinfected with EDL933cIind1showed no signs of disease (Fig. 4).
Figure 4A shows a Kaplan-Meier survival curve of mice
inoculated with the three strains. All 6 mice given EDL933 became
moribund or died prior to 21 days after inoculation. At necropsy,
these mice were dehydrated and thin, and their ceca were distended
with fluid contents. Mice in this group had moderate-severe acute
renal tubular necrosis (Fig. 4B), failed to gain weight as indicated by
Figure 3. Expression of Stx. Levels of Stx were determined as outlined in Materials and Methods. Mitomycin C was used at a concentration of
2 mg/ml. Strains: EDL933, EDL933cIind1 (K11349), and K37-933W (K9675). Error bars indicate standard deviations. P values (mc=mitomycin C treated):
(1) EDL9336EDL933 (mc)=6.661027; (2) EDL9336K11349=0.0056; (3) K113496K11349 (mc)=0.018; (4) EDL9336K9675=0.0069; (5) K96756K9675
(mc)=3.561026; (6) EDL933 (mc)6K9675 (mc)=0.00044.
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significantly lower body weights at necropsy (Fig. 4C), and all and
dilute urine (Fig. 4D), indicating renal failure. Histologically, renal
disease was characterized by necrosis of renal tubules and
occasional glomerular fibrin thrombi (Fig. 4E). Mice in the other
two groups did not show any signs of disease, and had normal renal
morphology Fig. 4F). As noted above, cecal colonization was similar
in all three groups of mice ruling out poor colonization as an
explanation for the failure of EDL933cIind1 to cause disease.
As discussed, in vitro EDL933cIind1produces measurable levels
of Stx2, raising the question of whether it produces measurable
levels of Stx2 in the infected mouse. Although there was wide
variation, we found low but measurable levels of Stx2 in the feces
of some of the mice infected with EDL933cIind1, 0–300 ng/ml of
feces. Much higher levels of Stx2, with considerable variation,
were found in the feces of mice infected with EDL933,
652964432 ng/ml of feces (P=0.0039).
Effect of in vivo environment on prophage induction
Based on the RIVET (recombinase based in vivo expression
technology) [45,46], we developed SIVET (selectable in vivo
Figure 4. Disease in mice infected with EDL933. A: Kaplan-Meier survival curve; B: Acute tubular necrosis score; C: Body weight at necropsy; D:
Urine specific gravity; E: Hematoxylin and Eosin stained section of kidney from a mouse inoculated with EDL933. Many tubules are necrotic and
contain cellular debris (arrows). A few glomeruli contain fibrin thrombi (arrowheads). F. Mouse infected with the EDL933cIind1 negative mutant.
Tubules and glomeruli are normal. Bars=50 microns. * Significantly different from mice infected with Ind- or Stx- mutants, p,0.001. Slides were
scored blind without knowledge of their source. Animals were euthanized and tissues removed for examination; at 10 days post infection with
EDL933 (day at which they became moribund) and at 21 days post infection with EDL933cIind1I (did not become moribund).
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expression technology), with the aim of determining if there is any
effect on prophage induction when bacteria are in the intestine.
Studies with the first generation SIVET, constructed in the
nonpathogenic E. coli strain MC1000, established this reporter
system as a valid method for measuring prophage induction .
Here we report construction of a second generation SIVET
through modification of EDL933 (see Materials and Methods for
details). Figure 1-II outlines the essential features of the SIVET
system. Briefly, the 933W and 933V prophages in EDL933 were
genetically altered so that functions lethal to the bacterial host 
are not expressed upon induction and the bacterium therefore
survives challenge with an inducing agent. The tnpR gene from the
cd transposon  was cloned downstream of the 933W early PR
promoter distal to the cro gene. Thus, following induction of the
933W prophage transcription initiating at the phage promoter PR
results in production of the TnpR resolvase that, in turn, acts at
another site on the bacterial chromosome to excise a kanR cassette
that interrupts a cat gene. This recombination serves two purposes,
establishes a functional cat gene and removes the kanR cassette,
conferring CamR. Hence, upon induction of the altered 933W
prophage there is an irreversible and inheritable change of the host
bacterium from KanR/CamS to KanS/CamR. The fraction of
the total bacterial count that is CamR provides a measurement of
the number of bacteria in which the prophage was induced.
That this change is due to prophage induction is shown by the
results of the following experiments. First, treatment of the SIVET
strain with mitomycin C, known to cause prophage induction ,
results in an increase of ,1000 fold in CamR colonies and a
reduction of ,1000 fold of KanR colonies (Fig. 5). Second,
treatment of a cIind1 mutant derivative of the SIVET strain
(K11607) under exactly the same conditions used with the SIVET
parent failed to cause any measurable change in the levels of
KanR or CamR bacteria (Fig. 5).
In the following in vitro and in vivo experiments, the ratio of
CamR/KanR SIVET was standardized to simplify the presenta-
tion using what will be referred to as the ‘‘Induction Index’’. This
function is calculated as the log10 of (CamR/KanR output)/
(CamR/KanR input) (for details see Materials and Methods).
Because of the way the Induction Index is calculated, the starting
point in the graphs, the input, is equal to log10(1) or 0. This allows
changes in induction to be monitored by observing movement of
the Index away from 0.
The only way we see the ratio deviate, beyond expected scatter,
from 0 on the Induction Index, is if one of the two populations
increases more than the other either by a growth advantage or by
addition of newly generated derivatives. To rule out alteration in
the induction index due to a growth advantage of one or the other
marked strain, we used two SIVET derivatives; one, K11607,
locked in the KanR form by virtue of the cIind1mutation and the
other, K11608, a derivative of K11607 which is isogenic except for
the excision of the KanR cassette and thus is locked in the CamR
form. The CamR/KanR ratio (calculated employing the formula
used to generate the Induction Index) following coinfection with
the locked in CamR and KanR derivatives hovers around 0
(Fig. 6A). Since there is no growth advantage to either form, any
positive increase in the CamR/KanR Induction Index of the
parental SIVET would have to be explained as addition by
conversion from the KanR population to the CamR population, a
direct consequence of induction of the 933W prophage in the
As discussed above, a small fraction of a population of lysogens
growing in the absence of an added inducing agent undergo
induction, a process called spontaneous induction . To
determine whether spontaneous induction of the SIVET prophage
adds to the population of CamR bacteria, we measured the
CamR/KanR ratio, determined as the Induction Index, over the
course of a large number of doublings in vitro in two different ways
(Fig. 6). In both approaches, the SIVET strain was serially
passaged in vitro for a number of generations in LB medium and
the CamR and KanR populations periodically measured by viable
counts. In one set of experiments, the SIVET bacteria were grown
to stationary phase and diluted 10-fold for the next passage
(Fig. 6B) while in the other, the bacteria were kept in log phase and
diluted from an OD600of ,1.0 to an OD600of 0.1 for the next
passage (Fig. 6C). Both protocols yielded similar experimental
results; the Induction Index remained relatively constant over
many doublings, hovering around 0. These results lead us to
conclude that spontaneous induction does not significantly affect
Figure 5. SIVET induction by treatment with mitomycin C. SIVET strains EDL933 (K11604) and EDL933cIind1 (K11607) were treated as
described in the Materials and Methods. Bars show colony forming units with and without mitomycin C treatment; Left (ind+) EDL933 and right
(ind2) EDL933cIind1. Error bars represent standard error of the mean.
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the CamR/KanR ratio. We consider these results further in the
Induction in the mouse intestine
To determine if the intestine environment contributes to
prophage induction and thus Stx production, we employed the
ELD933 SIVET strain using the infection protocol as described
above. Each mouse was orally infected with ,106SIVET
bacterium. Because the 933W prophage was mutationally
disarmed (see Materials and Methods for details) and thus does
not produce Stx2, as expected, mice infected with EDL933 SIVET
did not show signs of disease. Feces were isolated each day for
seven days and bacterial counts were determined by plating on LB
agar plates containing kanamycin or chloramphenicol. The total
EDL933 SIVET count remained relatively constant over the
course of the experiment, ,108CFU/g of feces, although slightly
decreasing by the seventh day (data not shown). The Induction
Indexes over the 7 days presented in Fig. 7 were compiled from
results of three independent experiments, each comprised of five
By day seven the Induction Index has increased by over three
logs. The study was terminated at day 7, when the onset of severe
disease caused by EDL933 usually occurs .
To determine if the change in the CamR/KanR Induction
Index during in vivo growth of SIVET reflects a difference in
viability of the two forms of the SIVET, we employed the SIVET
pair K11607 and K11608. These derivatives, as discussed above,
are locked in either the KanR or CamR form. Mice were co-
infected with K11607 and K11608 and followed essentially as
Figure 6. Measurement of in vitro prophage induction. SIVET
strains were grown in LB broth and serially diluted into fresh LB.
Cultures were grown to desired densities and concentrations of CamR
and KanR bacteria in cultures measured at indicated points in growth
cycle. Multiple rounds of dilutions and growth allowed for indicated
rounds of duplication. Where appropriate, low dilutions ensured that
any CamR bacteria would be carried over during dilutions. Graphs show
ratios of CamR/KanR on a log scale (calculated using the Induction
Index formula) over the indicated number of generations. A. SIVET
cIind1strains K11607 (locked in KanR form) and K11608 (CamR) were
grown together in LB to stationary phase at which time bacterial counts
were determined and dilutions were made for next round of growth.
Error bars represent standard error of the mean. B. Cultures of K11604
were grown to stationary phase at which time bacterial counts were
determined and dilutions were made for next round of growth. C.
Cultures of K11604 were grown to late log phase at which time
dilutions were made for next round of growth. Aliquots were removed
at mid-log phase for determination of bacterial counts.
Figure 7. Measurement of in vivo prophage induction. Germ-
free mice were infected with 106bacteria. At the indicated times feces
were collected, suspended in diluent, and dilutions plated on LB plates,
with kanamycin (30 mg/ml), or chloramphenicol (9–10 mg/ml). Graphs
show ratios of CamR/KanR on a log scale (calculated using the Induction
Index formula) over the indicated number of days. (N) SIVET strain
K11604. (#) K11604 derivatives with 933W cIind mutation; K11607
(locked in KanR) and K11608 (CamR) were co-inoculated. Error bars
represent standard error of the mean.
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described for the in vivo SIVET study outlined above. Examina-
tion of fecal samples showed that the ratio of CamR/KanR
(calculated using Induction Index formula) did not significantly
change over the course of 7 days (Fig. 7); i.e., neither form of
SIVET has a growth advantage during in vivo growth. Hence, the
null hypothesis stands and we conclude that the increase of the
CamR/KanR Induction Index observed during growth in the
mouse intestine results from prophage induction.
Based on this collection of data, we conclude that there is
significant induction of the 933W prophage in the germ free
mouse intestine. Since Stx2 production is directly linked to 933W
induction, it follows that the intestine, through action of a yet to be
identified factor(s), stimulates Stx2 production through induction
of the 933W prophage.
With the information gained from sequencing numerous
bacterial genomes, it has become apparent that virulence factors
are commonly located in genomes of prophage [50,51].
Introduction of a new function, such as a virulence factor, to
a bacterium by a prophage is referred to as lysogenic conversion.
Although Stx2 is an example of a phage-encoded toxin whose
expression is controlled by the phage regulatory cascade, many
other phage-encoded toxins are expressed independently of
prophage regulatory functions. This is true for the classic toxin
of Corynebacterium diphtheriae  and of cholerae toxin (CTX),
which is encoded in the genome of the CTXW prophage .
Expression of CTX is controlled by a complex circuitry of
proteins encoded by regulatory genes located outside of the
prophage genome . Observations like these led to the idea
that phage, like other mobile elements, serve as agents that can
transfer genetic information from one bacterium to another
. However, at least in the case of stx-phages, the phage
serves a wider role, not only being the source of transfer, but
also the regulator of expression from the transferred virulence
The construction of a derivative of EDL933 with the ind1
mutation in the 933W prophage coupled with an animal model
that mimics, to a large degree, the human disease, has allowed us
to specifically assess the contribution of induction of the 933W
prophage to the disease process. Like its EDL933 parent,
EDL933cIind1 effectively colonizes the host intestines. However,
unlike the parental strain, the ind1 strain fails to elicit any of the
hallmarks of an EHEC infection; e.g., physical signs of illness,
renal disease, and death. That EDL933cIind1 colonizes the host
intestine is consistent with our previously reported findings
showing that a derivative of EDL933 with a deletion-substitution
of the stx2 genes colonized as well as the parent strain with a
functional stx2 gene . This observation is contrary to the
findings of Robinson et al. , who reported that colonization
was reduced if the O157:H7 strain did not express Stx2. As we
have suggested previously , this difference may reflect our use of
germ-free mice, while Robinson et al. used mice with normal
Our results provide evidence that the major pathogenic effect of
EDL933 results from induction of the 933W prophage. Hence, the
phage regulatory cascade plays a central role in the pathogenesis of
this O157:H7 strain and likely many others. Since repressor auto-
cleavage requires activated RecA protein, which, in turn, is a
product of the SOS response, it is primarily that subpopulation of
bacteria, with a sufficiently vigorous SOS response that induces
the 933W prophage and results in the production and release of
Our observation that Stx2 production and disease in the mouse
are directly related to induction of the 933W prophage raises the
question as to whether there is a factor(s) in the intestines that
increases the SOS response resulting in increased prophage
induction beyond that expected from results of in vitro experi-
ments. Such a role was found for a factor in human pharyngeal
cells that induces a group A Streptococcus prophage . And a
small but significant level of induction of Stx was observed when
an EHEC strain was co-cultured with human neutrophils . In
a similar manner, a factor(s) in the intestines that induces an SOS
response might increase the levels of Stx produced by a population
of infecting EHEC. Such a factor(s) could be a product of the host
(e.g., neutrophils). Not considered here is the possible importance
of the interaction between the microbiota and the mammalian
intestine in the SOS response and resulting Stx2 production .
Our studies with germ free mice show that even in the absence of
the normal microbiota there is sufficient prophage induction to
produce and release levels of Stx capable of causing renal disease
Constructed regulatory networks as biosensors have wide
biological applications . The studies reported here demon-
strate the utility of the comparatively simple SIVET regulatory
network as a tool for identifying conditions where prophage
induction is enhanced. First, treatment in vitro of SIVET with the
inducing agent mitomycin C results in overwhelming conversion
of KanR to CamR (Fig. 5), confirming that SIVET responds to
inducing agents as designed. Second, the experiments with the
933W cIind SIVET derivatives showed that the increase in CamR
relative to KanR colonies observed during in vitro and in vivo
growth is not due to a growth advantage of the CamR variants
(Figs. 6 and 7). Third, no significant change in the ratio of CamR
to KanR was observed over a large number of doublings during
continuous in vitro growth of SIVET in the absence of an inducing
agent (Fig. 6). This observation held true whether cultures prior to
dilution were allowed to grow to stationary phase or were
maintained in log phase. In each case dilutions were at a
sufficiently high level to ensure that CamR bacteria were carried
over during each dilution. It might be expected that CamR
bacteria contributed de novo by induction should add to the
growing population, resulting in an increase in the CamR/KanR
ratio. However, the in vitro experiments failed to show an increase
in the Induction Index over a large number of doublings (we
discuss this apparent paradoxical finding in detail below). Based on
these results, we conclude that spontaneous induction (induction in
the absence of a known inducing agent) of the 933W prophage
fails to lead to a measurable increase in conversion of SIVET from
KanR to CamR. Hence, SIVET is not sufficiently sensitive to
distinguish no induction from low levels of induction.
By eliminating obvious alternative explanations and showing
that mitomycin C treatment results in an increase in the SIVET
CamR/KanR ratio, these results confirm that SIVET can be used
to identify the presence of inducing agents. Moreover, the failure
to observe changes in the Induction Index over many rounds of
doubling during in vitro growth, in the absence of an extrinsic
inducing agent, indicates that even small measurable increases in
the Induction Index should provide evidence of an extrinsic
In the light of this background information, the .3 log increase
in the Induction Index observed in SIVET isolated from feces
(Fig. 7) over the seven days following the initial infection provides
evidence for action of an inducing factor in the mouse intestinal
tract. We suggest three alternative, but not mutually exclusive,
scenarios to explain this increase in the rate of induction: 1) a
substantial portion of the bacteria reach a section of the intestinal
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tract that contains resident inducing activity; 2) the infection
causes an increase in the amount and/or activity of a resident
inducing activity; or 3) infection attracts an inducing activity or a
cell (e.g., neutrophils) producing an activity. Since Stx2 produc-
tion, in large measure, is directly related to phage induction (Fig. 3),
the intestinal environment likely contributes to the severity of the
Although we failed to observe any significant change in the
Induction Index over many generations of in vitro growth, an
increase in the Induction Index over time might be expected
because spontaneous prophage induction  should result in
TnpR expression and, at some level, conversion of KanR to
CamR bacteria. This, in turn, would add to the total of CamR
population over the number produced by replication of preexisting
CamR population resulting in an increase in the CamR/KanR
We used mathematical modeling to gain a quantitative
understanding of what the expected Induction Index over time
would look like if all of the spontaneously induced KanR bacteria
were able to contribute immediately to the CamR bacterial
population. Based on a starting Induction Index of 0, the model
adds the newly produced CamR bacterium at each division to the
growing preexisting CamR population, predicting an increase in
the Induction Index over time as shown in Fig. S1. If we assume a
doubling every hour over the seven days of in vivo growth, the
model predicts the Induction Index would increase a little over one
log and, even assuming a doubling time of 20 minutes, the Index
would increase by slightly over two logs, both substantially less
than the over three logs observed in the in vivo SIVET
The counterbalancing actions that we see as potentially
reducing the contribution of spontaneous induction might make
to the CamR population, include: 1) as discussed above, SIVET
may not be sufficiently sensitive to distinguish no induction from
low induction; 2) there may be a delay in initiation of growth
following recovery from the consequences of DNA damage that
caused the induction [60,61]; i.e., a phenotypic lag (graphed in
Fig. S1); 3) removal of the KanR cassette may occur in only one of
the multiple bacterial chromosomes  resulting in segregation of
both CamR and KanR derivatives from a single induced KanR
bacterium and thus resulting in no change in the CamR/KanR
ratio ; and 4) there may be sufficient DNA damage in some of the
bacteria to block further growth, compromising survival of those
bacteria. This subpopulation would be part of the induced pool
that although theoretically adding to the CamR population would
not be alive to do so. Although collectively these actions could
explain our results, we are far from having a definitive answer as to
how the Induction Index maintains this steady state. Nor can we
explain how the ratio of CamR/KanR colonies reaches a steady
state that is maintained for many generations. However, failure of
SIVET to identify low level induction (spontaneous), but identify
high level induction, as with mitomycin C, indicates measurements
by SIVET are likely to be an under representation.
Materials and Methods
All animal protocols were approved by the University Com-
mittee on Use and Care of Animals at the University of Michigan
Medical School. The University of Michigan is fully accredited by
the Association for Assessment and Accreditation of Laboratory
Animal Care, International (AAALAC, Intl) and the animal care
and use program conforms to the standards of ‘‘The Guide for the
Care and Use of Laboratory Animals’’ (published by the NRC).
Bacteria, phage, and plasmids
See Table 1.
Primers and oligonucleotides
See Table 2.
LB, 10 g tryptone, 5 g yeast extract, 5 g NaCl/liter of H2O. For
LB plates 10 g of agar was included. LB sucrose plates are LB
plates without NaCl and made 10% in sucrose. Antibiotics were
added at the following concentrations; spectinomycin 80 mg/ml,
ampicillin 100 mg/ml (plasmids) 25 mg/ml (chromosomal), kana-
mycin 30 mg/ml, hygromycin 200 mg/ml, and chloramphenicol
9–10 mg/ml. TB plates, 10 g tryptone, 2.5 g NaCl, and 10 g agar/
liter of H2O.
All of our constructs were engineered using the l Red
recombination system, colloquially referred to as recombineering
. The l Red functions were supplied in either of two ways:
transiently by a heat pulse freeing a l promoter on a truncated l
prophage from control by a Ts repressor (cI857) so that the
downstream red genes could be transcribed, using DY378  or
from pKD46 and derivatives of that plasmid carrying cloned l red
genes by adding arabinose to the growth medium to activate an
Ara-regulated promoter . Single-stranded oligonucleotides or
double-stranded PCR products of varying lengths having ,40
nucleotides of flanking sequences with homologies to the target
regions were introduced by electroporation into bacteria express-
ing l Red functions. The expressed Red functions recombine the
introduced DNAs with the target site. In the absence of a
selectable marker, a two-step procedure was used: a cat-sacB (CSB)
cassette  was inserted by recombineering and the recombinant
selected by resistance to chloramphenicol. This cassette was then
exchanged by recombineering with the designed DNA product
using as selection resistance to sucrose and confirming by
screening for CamS. DNA sequencing by the University of
Michigan Sequencing Core Facility confirmed structure of
Construction of EDL933 with a 933W ind1 prophage
Recombineering was used to cross the designed mutation from a
single stranded oligonucleotide to the chromosome of strain
K10985. The oligonucleotide contained a single nucleotide change
that resulted in a replacement of Lys codon 178 (AAG) with an
Asn codon (AAC). The following is the sequence of the DNA
oligonucleotide (oligo #2) with the mutant nucleotide capitalized:
tatgacagagatggagaataccaatttacaagcattaacca-39. The pairing of the
oligonucleotide with its complementary chromosomal DNA strand
forms a C-C mismatch at the position of the nucleotide change.
This mispairing is not repaired by the mismatch repair system
. In the absence of mismatch repair there is a significant
increase in isolation of bacteria with the designed nucleotide
change . K10985, an EDL933 derivative with the pKD46
plasmid , was prepared for electroporation essentially as
described by Murphy and Campellone . Following electropo-
ration, bacteria were resuspended in 10 ml of LB broth and grown
at 37u. After ,5 hrs of growth, dilutions of the bacteria were
placed on LB plates and incubated overnight at 37u. The following
day colonies were picked and stabbed to an LB plate and a TB
plate that was layered with a lawn of K37, a strain that supports
growth of 933W. Plates were incubated at 37u for two hours and
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the seeded plate was UV irradiated (1.6 Joules/M2/S for
30 seconds). Following overnight incubation at 37u, a zone of
lysis in the lawn showed phage had been synthesized by an
induced prophage. Two clones out of 160 tested showed no zones
of lysis. These derivatives failed to lyse following treatment with
mitomycin C and subsequent DNA sequencing showed that
although they both had the cIind mutation, only one, EDL933-
cIind1, had no other changes and was selected for further study.
A similar strategy was used to construct an EDL933 SIVET ind
mutant, K11607, that was KanR. A CamR derivative, K11608,
isogenic except for the loss of the KanR cassette and thus
converted to CamR, was constructed from K11607 using a
plasmid, pJLTnpRhygro, which supplied the TnpR resolvase.
Overnight cultures were diluted and grown to early log phase in
LB. The cultures were divided into two aliquots; one grown
untreated and the other treated with 2 mg/ml mitomycin. Cultures
were grown for 3–4 hours, based on time of lysis for the
mitomycin C treated culture. Uninduced cultures were diluted
every 30 minutes to maintain them in logarithmic growth.
Cultures were sonicated 36 for 10 seconds at amplitude of 30%
to obtain total cell lysis. Lysates were passed through 0.22 mm filter
and concentrated using Amicon Ultra-4 (Millipore). Stx2A levels
in supernatants were measured using an enzyme-linked immuno-
absorbent assay (ELISA) following a previously published proce-
dure  using anti-Stx2A monoclonal and anti-Stx2 polyclonal
antisera. Results were determined as ng Stx2A/mg total protein.
Germ-free Swiss-Webster mice of both sexes were raised in the
University of Michigan Laboratory of Animal Medicine germ free
colony, housed in soft-sided bubble isolators, and fed autoclaved
water and laboratory chow ad libitum. Inoculations, monitoring of
animals, and sample collections were performed as previously
described . In brief, mice were inoculated orally with ,106cfu
of LB-cultured bacteria. Each group of inoculated animals
contained 3 male and 3 female mice between 5 and 6 weeks of
age. Throughout the experiment and at necropsy, feces or cecal
contents were collected for quantitative EHEC culture. Gram stain
and aerobic and anaerobic culture were used to demonstrate the
absence of microorganisms other than EHEC. Mice remained
Table 2. Primers and oligonucleotides.
# Name OligonucleotidesRole
359 catsacB (inactivate CP933V)59–TTCTCGCTGTGTTGGCTTGCTGTAGCTTGCTTGTGCCAGTTACTTAGATATTGGCCTTGG–39
439 catsacB (inactivate CP933V)59–TGCCGTACCTTTGGATTCTTTCCAGACAATGGTTACACTGTCCATATGCACAGATG–39
559 cat (synthesize
639 cat (synthesize
759 933WOR (synthesize
59 cat in
39 cat in
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sterile (except for the infecting EHEC strain) throughout the
course of the experiment.
Mice inoculated with EDL933Dstx:cat or EDL933cIind1 showed
no signs of disease and were euthanized 3 weeks after inoculation.
All of the mice inoculated with EDL933 became moribund prior
to the scheduled necropsy date, and these mice were necropsied
when they became moribund, between 10 and 18 days after
inoculation (see Results). All animal experiments were conducted
with the approval of the University of Michigan Animal Care and
At necropsy, cecal contents were cultured to determine bacterial
colonization density. Quantitative counts were determined using
LB agar plates containing appropriate antibiotics. Stx concentra-
tion in cecal contents was measured using a commercial kit
(Premier) as previously described . For histologic examination,
right and left kidney were immersion-fixed in formalin, embedded
in paraffin, cut in 5 micron sections, and stained with hematoxylin
and eosin (Fig. 4). Kidney sections were scored by a single
pathologist without knowledge of the source of the section. For
quantitation, a midline section of the right renal cortex was
examined in its entirety, and the number of 2006 fields with
tubular or glomerular lesions was recorded. Acute tubular necrosis
was subjectively scored as mild, moderate, or severe.
For the SIVET experiment, animals were similarly infected with
,106cfu of LB-cultured bacteria. Because of the deletion-
substitutions in the 933W prophage, the SIVET strain does not
express significant levels of Stx2. Details of the experiment
procedure have been discussed above. In these experiments
colony counts were obtained using LB plates containing either
kanamycin (30 mg/ml) or cloramphenicol (9 mg/ml).
Statistics: Quantitative data were analyzed by Mann-Whitney U
test. Multiple groups were compared by ANOVA and Fisher’s
Least Significant Difference.
Construction of EDL933 SIVET strain
The design of SIVET  is based on Camilli and colleague’s
‘‘Recombinase-based Reporter of Transcription (RIVET) system’’
[45,46]. However, SIVET differs from RIVET in providing a
selection for cells in which the assayed transcription occurred
(Fig. 1-II). The first generation of EDL933 SIVET, was
constructed similarly to the original K12 SIVET strain [47,70]
using recombineering , with a SpcR (this laboratory) derivative
of pKD46  supplying the l Red functions. The 933W
prophage was inactivated by elimination of genes controlling two
critical components of phage growth, transcription and replica-
tion. The N gene, encoding a transcription regulator, was deleted
and replaced with a KanR cassette. The O and P genes, encoding
proteins involved in initiation of DNA replication , were
replaced with the tnpR gene and ampR cassette. This was
accomplished using a PCR product containing the ampR cassette
and the sequence encoding the 168 variation of the cd resolvase,
tnpR-168,  with flanking sequences having homology to the
933W cro and ren genes (Fig. 1-I). These changes generated strain
K11084 that, even though having a defective 933W prophage, is
unable to survive treatment with an inducing concentration of
mitomycin C. The cryptic prophage CP933V in EDL933,
although defective, has nearly a complete lambdoid phage genome
, leading us to suspect that its induction might be responsible
for this sensitivity to mitomycin C. Therefore, we deleted the
control region of CP933V rendering that prophage uninducible;
the deletion included the putative repressor (cI) gene with
immediate surrounding putative promoters, operators, genes,
and relevant associated genetic material in a two-step process. A
cat-sacB (CSB) cassette  with flanking ends having appropriate
homologies to CP933V (primers 3 and 4, template K9685) was
recombined into the targeted region, extending from N to cII
(Fig. 1-I) in CP933V, generating strain K11114. The CSB inserted
in CP933V was then replaced with a single-stranded DNA
oligomer (oligo #1) , generating strain K11115. This strain
survives the inducing levels of mitomycin C used in our studies.
Addition of the reporter cassette in a two-step procedure
completed the construction of the EDL933 SIVET strain. First,
K11161 was constructed by crossing a cat cassette (primers: 9 and
10, K10373 template) into the lacZ gene of K11115 providing
homology for the next step. Second, K11173 was constructed by
crossing the cat::resC-tetR-resC::cat cassette (primers7 and 8, K10449
template) into the inserted cat gene in K11161 with selection for
This first EDL933 SIVET construct had to be modified because
its constitutive expression of TetR made the bacteria sensitive to
the in vivo environment. We therefore made the following changes
using recombineering, l Red functions were supplied by a
hygromycin resistant derivative of pKD46 (pKD46hygR). The
KanR cassette in the N gene was replaced by a spcR cassette and
the selective tetR cassette in the cat::resC::tet::resC::cat reporter was
replaced by a kanR cassette yielding the cat::resC-kan-resC::cat
reporter. To complete the process, the strain was cured of
pKD46hygR yielding K11604, the SIVET strain used in the
experiments reported here.
Mitomycin C induction
The method used to obtain the results shown in figure 5 was
essentially those outlined in Livny and Friedman . Briefly,
SIVET strain was grown ,108/ml in LB, made 2 mg/ml in
mitomycin C, grown for 2 hrs, washed and resuspended in LB,
grown for 4 hours, and dilutions of bacteria were plated on
This metric provides a log10scale readout that allows for a
simplified comparison of results of different SIVET experiments.
The calculations compare the ratio of CamR/KanR colonies at
any given time relative to the starting ratio of CamR/KanR
colonies. It is calculated as log10[(CamR titer/KanR titer at any
time after start of experiment)/(CamR titer/KanR titer at start of
experiment)]. It follows that the Induction Index at the start would
obviously be 0; i.e., log101 (starting ratio/starting ratio).
Effect of induction of cIind1 mutant on lysis
Overnight cultures of O157:H7 and the cIind1 derivative grown
in LB broth were diluted 1:100 in LB and grown to early log
phase. Each were divided into two aliquots, one untreated and the
other treated with 2 mg/ml of mitomycin C. Samples, 200 ml, were
placed in a 96 well plate and grown at 37u with OD600read at
30 minute intervals in the SpectraMax 250 (Micro Devices).
To determine what would be expected if the contribution to the
CamR population by spontaneous induction during growth of the
SIVET population were unimpeded, we used mathematical
modeling to predict what such an unimpeded expansion would
look like. The change in the ratio of CamR/KanR, due to
spontaneous induction during bacterial growth is calculated and
displayed as an Induction Index over a range of population
doublings reaching 1000. This allows comparison with the results
obtained by experimentation. The CamR bacteria resulting from
Mathematical modeling of Induction Index.
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the conversion of KanR bacteria are added to the growing CamR
culture which is determined by the standard exponential growth
equation taking into consideration, as we have shown, that the two
populations grow at the same rate. The model is further expanded
to take into account possible effects of phenotypic lag, the period
following induction needed for the newly converted CamR
bacteria to recover from the damage inflicted during the resulting
SOS response. The top line shows the expected increase in the
ratio CamR/KanR populations due to spontaneous induction,
assuming the conversion was free of any factors impeding the
conversion number (see text for detailed discussion of possible
factors). The lines below show a range of possible delays due to
phenotypic lag prior to the first doubling. The starting ratio is set
to 1, or an Induction Index of 0 on the log scale. Other factors
possibly reducing the actual conversion number were not
considered in the modeling.
describing the mathematical modeling of Induction Index.
Supplementary methods. Experimental procedures
The authors thank John Little for identification of the Lys codon changed
to construct the cIind mutant and valuable discussion. Victor DiRita and N.
Cary Engleberg are thanked for helpful suggestions in preparation of the
manuscript. Celeste Thorpe and David Acheson are thanked for anti-Stx2
serum. Celeste Thorpe is thanked for purified Stx2. An anonymous
reviewer is recognized for pointing out that constitutive expression of TetR
could make SIVET sensitive to the in vivo environment. Simione Marino is
thanked for help with calculation. We thank Sara Poe and Chriss Vowles
for the germ free mouse studies, and Sara Smith for developing the in vivo
Conceived and designed the experiments: DIF CJA KAE JHF JST KB.
Performed the experiments: DIF CJA JST KB KGS KAE JLR. Analyzed
the data: DIF KAE. Contributed reagents/materials/analysis tools: JST
DIF JHF. Wrote the paper: DIF.
1. Melton-Celsa A, Mohawk K, Teel L, O’Brien AD (2012) Pathogenesis of shiga-
toxin producing Escherichia coli. In: Mantis NJ, editor. Ricin and Shiga Toxins
Berlin, Germany: Springer-Verlag. pp. 67–103.
2. Kaper JB (1998) Enterhemorrhagic Escherichia coli. Curr Opin Microbiol 1: 103–
3. Muniesa M, Hammerl JA, Hertwig S, Appel B, Brussow H (2012) Shiga toxin-
producing Escherichia coli O104:H4: a new challenge for microbiology. Appl
Environ Microbiol 78: 4065–4073.
4. Tyler JS, Livny J, Friedman DI (2005) Lambdoid Phages and Shiga Toxin. In:
Waldor MK, Friedman DI, Adhya SL, editors. Phages; Their role in
Pathogenesis and Biotechnology. Washington, D.C.: ASM Press. pp. 131–164.
5. O’Brien AD, Tesh VL, Donohue-Rolfe A, Jackson MP, Olsnes S, et al. (1992)
Shiga toxin: biochemistry, genetics, mode of action, and role in pathogenesis.
Curr Top Microbiol Immunol 180: 65–94.
6. Tarr PI, Gordon CA, Chandler WL (2005) Shiga-toxin-producing Escherichia coli
and haemolytic uraemic syndrome. Lancet 365: 1073–1086.
7. Eaton KA, Friedman DI, Francis GJ, Tyler JS, Young VB, et al. (2008)
Pathogenesis of Renal Disease Due to Enterohemorrhagic Escherichia coli in
Germ-Free Mice. Infect Immun 76: 3054–3063.
8. Jacewicz M, Clausen H, Nudelman E, Donohue-Rolfe A, Keusch GT (1986)
Pathogenesis of shigella diarrhea. XI. Isolation of a shigella toxin-binding
glycolipid from rabbit jejunum and HeLa cells and its identification as
globotriaosylceramide. J Exp Med 163: 1391–1404.
9. Sandvig K, Grimmer S, Lauvrak SU, Torgersen ML, Skretting G, et al. (2002)
Pathways followed by ricin and Shiga toxin into cells. Histochem Cell Biol 117:
10. Reisbig R, Olsnes S, Eiklid K (1981) The cytotoxic activity of Shigella toxin.
Evidence for catalytic inactivation of the 60S ribosomal subunit. J Biol Chem
11. Campbell A (1994) Comparative molecular biology of lambdoid phages. Ann
Rev Microbiol 48: 193–222.
12. Degnan PH, Michalowski CB, Babic AC, Cordes MH, Little JW (2007)
Conservation and diversity in the immunity regions of wild phages with the
immunity specificity of phage lambda. Mol Microbiol 64: 232–244.
13. Botstein D (1980) A theory of modular evolution for bacteriophages. Annals of
the New York Academy of Sciences 354: 484–490.
14. Neely MN, Friedman DI (1998) Functional and genetic analysis of regulatory
regions of coliphage H-19B: location of shiga-like toxin and lysis genes suggest a
role for phage functions in toxin release. Mol Microbiol 28: 1255–1267.
15. Roberts JW (1993) RNA and protein elements of E. coli and lambda
transcription antitermination complexes. Cell 72: 653–655.
16. Waldor MK, Friedman DI (2005) Phage regulatory circuits and virulence gene
expression. Curr Opin Microbiol 8: 459–465.
17. Plunkett G, 3rd, Rose DJ, Durfee TJ, Blattner FR (1999) Sequence of Shiga
toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-
gene product. J Bacteriol 181: 1767–1778.
18. Wagner PL, Neely MN, Zhang X, Acheson DW, Waldor MK, et al. (2001) Role
for a phage promoter in Shiga toxin 2 expression from a pathogenic Escherichia
coli strain. J Bacteriol 183: 2081–2085.
19. Tyler JS, Mills MJ, Friedman DI (2004) The operator and early promoter region
of the Shiga toxin type 2-encoding bacteriophage 933W and control of toxin
expression. J Bacteriol 186: 7670–7679.
20. Zhang X, McDaniel AD, Wolf LE, Keusch GT, Waldor MK, et al. (2000)
Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin
production, and death in mice. J Infect Dis 181: 664–670.
21. Wong CS, Jelacic S, Habeeb RL, Watkins SL, Tarr PI (2000) The risk of the
hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7
infections. N Engl J Med 342: 1930–1936.
22. Friedman DI, Court DL (2001) Bacteriophage lambda: alive and well and still
doing its thing. Curr Opin Microbiol 4: 201–207.
23. Little JW (1996) The SOS Regulatory System. In: Lin ECC, Lynch AS, editors.
Regulation of Gene Expression in Escherichia coli. Austin TX: R.G. Landes. pp.
24. Sutton MD, Smith BT, Godoy VG, Walker GC (2000) The SOS response:
recent insights into umuDC-dependent mutagenesis and DNA damage
tolerance. Annu Rev Genet 34: 479–497.
25. Little JW (2005) Lysogeny, prophage induction, and lysogenic conversion. In:
Waldor MK, Friedman DI, Adhya SL, editors. Phages: Their role in bacterial
pathogenesis and biotechnology. Washington, D.C.: ASM Press. pp. 37–54.
26. Friedman DI, Court DL (2006) Regulation of lambda gene expression by
transcription termination and antitermination. In: Calendar R, editor. The
Bacteriophages. Oxford: Oxford Press. pp. 83–103.
27. Roberts JW, Yarnell W, Bartlett E, Guo J, Marr M, et al. (1998) Antitermination
by bacteriophage lambda Q protein. Cold Spring Harb Symp Quant Biol
28. Karch H, Schmidt H, Janetzki-Mittmann C, Scheef J, Kroger M (1999) Shiga
toxins even when different are encoded at identical positions in the genomes of
related temperate bacteriophages. Mol Gen Genet 262: 600–607.
29. Roberts JW, Devoret R (1983) Lysogenic Induction. In: Hendrix RW, Roberts
JW, Stahl FW, Weisberg RA, editors. Lambda II. Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory. pp. 123–144.
30. Gimble FS, Sauer RT (1985) Mutations in bacteriophage lambda repressor that
prevent RecA-mediated cleavage. J Bacteriol 162: 147–154.
31. Little JW (2006) Gene regulatory circuitry of phage lambda. In: Calendar R,
editor. The Bacteriophages. New York, New York: Oxford University Press. pp.
32. Little JW (1984) Autodigestion of lexA and phage lambda repressors. Proc Natl
Acad Sci USA 81: 1375–1379.
33. Fuchs S, Muhldorfer I, Donohue-Rolfe A, Kerenyi M, Emody L, et al. (1999)
Influence of RecA on in vivo virulence and Shiga toxin 2 production in
Escherichia coli pathogens. Micro Path 27: 13–23.
34. Clark AJ (1973) Recombination deficient mutants of E. coli and other bacteria.
Annu Rev Genet 7: 67–86.
35. Little JW, Mount DW (1982) The SOS regulatory system of Escherichia coli. Cell
36. Wagner PL, Acheson DW, Waldor MK (2001) Human neutrophils and their
products induce Shiga toxin production by enterohemorrhagic Escherichia coli.
Infect Immun 69: 1934–1937.
37. Slilaty SN, Little JW (1987) Lysine-156 and serine-119 are required for LexA
repressor cleavage: a possible mechanism. Proc Natl Acad Sci USA 84: 3987–
38. Lin LL, Little JW (1988) Isolation and characterization of noncleavable (Ind-)
mutants of the LexA repressor of Escherichia coli K-12. J Bacteriol 170: 2163–
Prophage Induction and EHEC Infection
PLOS Pathogens | www.plospathogens.org13 March 2013 | Volume 9 | Issue 3 | e1003236
39. Iyer VN, Szybalski W (1963) A Molecular Mechanism of Mitomycin Action:
Linking of Complementary DNA Strands. Proc Natl Acad Sci U S A 50: 355–
40. Otsuji N, Sekiguchi M, Iijima T, Takagi Y (1959) Induction of phage formation
in the lysogenic Escherichia coli K-12 by mitomycin C. Nature 184: 1079–1080.
41. Perna NT, Plunkett Gr, Burland V, Mau B, Glasner JD, et al. (2001) Genome
sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409: 529–533.
42. Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA
fusion and cloning in Escherichia coli. J Mol Biol 138: 179–207.
43. Isogai E, Isogai H, Hayashi S, Kubota T, Kimura K, et al. (2000) Effect of
antibiotics, levofloxacin and fosfomycin, on a mouse model with Escherichia coli
O157 infection. Microbiol Immunol 44: 89–95.
44. Herold S, Siebert J, Huber A, Schmidt H (2005) Global expression of prophage
genes in Escherichia coli O157:H7 strain EDL933 in response to norfloxacin.
Antimicrob Agents Chemother 49: 931–944.
45. Camilli A, Beattie DT, Mekalanos JJ (1994) Use of genetic recombination as a
reporter of gene expression. Proc Natl Acad Sci USA 91: 2634–2638.
46. Lee SH, Hava DL, Waldor MK, Camilli A (1999) Regulation and temporal
expression patterns of Vibrio cholerae virulence genes during infection. Cell 99:
47. Livny J, Friedman DI (2004) Characterizing spontaneous induction of Stx
encoding phages using a selectable reporter system. Mol Microbiol 51: 1691–
48. Eisen HA, Fuerst CR, Siminovitch L, Thomas R, Lambert L, et al. (1966)
Genetics and physiology of defective lysogeny in K12 (l): studies of early
mutants. Virology 30: 224–241.
49. Grindley ND (1983) Transposition of Tn3 and related transposons. Cell 32: 3–5.
50. Waldor MK, Friedman DI, Adhya SL (2005) Phages; Their role in bacterial
pathogenesis and biotechnology. Washington, D.C.: ASM Press.
51. Brussow H, Canchaya C, Hardt WD (2004) Phages and the evolution of
bacterial pathogens: from genomic rearrangements to lysogenic conversion.
Microbiol Mol Biol Rev 68: 560–602.
52. Johnson EA (2005) Bacteriophages encoding botulism and diphtheria toxins. In:
Waldor MK, Friedman DI, Adhya SL, editors. Phage; Their Role in Bacterial
Pathogenesis and Biotechnology. Washington, D.C.: ASM Press.
53. Waldor MK, Mekalanos JJ (1996) Lysogenic conversion by a filamentous phage
encoding cholera toxin. Science 272: 1910–1914.
54. Matson JS, Withey JH, DiRita VJ (2007) Regulatory networks controlling Vibrio
cholerae virulence gene expression. Infect Immun 75: 5542–5549.
55. Frost LS, Leplae R, Summers AO, Toussaint A (2005) Mobile genetic elements:
the agents of open source evolution. Nat Rev Microbiol 3: 722–732.
56. Robinson CM, Sinclair JF, Smith MJ, O’Brien AD (2006) Shiga toxin of
enterohemorrhagic Escherichia coli type O157:H7 promotes intestinal coloniza-
tion. Proc Natl Acad Sci U S A 103: 9667–9672.
57. Broudy TB, Pancholi V, Fischetti VA (2001) Induction of lysogenic
bacteriophage and phage-associated toxin from group A Streptococci during
coculture with human pharyngeal cells. Infect Immun 69: 1440–1443.
58. de Sablet T, Chassard C, Bernalier-Donadille A, Vareille M, Gobert AP, et al.
(2009) Human microbiota-secreted factors inhibit shiga toxin synthesis by
enterohemorrhagic Escherichia coli O157:H7. Infect Immun 77: 783–790.
59. Khalil AS, Collins JJ (2010) Synthetic biology: applications come of age. Nat Rev
Genet 11: 367–379.
60. Friedman N, Vardi S, Ronen M, Alon U, Stavans J (2005) Precise temporal
modulation in the response of the SOS DNA repair network in individual
bacteria. PLoS Biol 3: e238.
61. Little JW (1983) The SOS regulatory system: control of its state by the level of
RecA protease. J Mol Biol 167: 791–808.
62. Sawitzke JA, Costantino N, Li XT, Thomason LC, Bubunenko M, et al. (2011)
Probing cellular processes with oligo-mediated recombination and using the
knowledge gained to optimize recombineering. J Mol Biol 407: 45–59.
63. Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using
homologous recombination. Annu Rev Genet 36: 361–388.
64. Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, et al. (2000) An efficient
recombination system for chromosome engineering in Escherichia coli. Proc Natl
Acad Sci USA 97: 5978–5983.
65. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes
in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97: 6640–
66. Lahue RS, Au KG, Modrich P (1989) DNA mismatch correction in a defined
system. Science 245: 160–164.
67. Costantino N, Court DL (2003) Enhanced levels of lambda Red-mediated
recombinants in mismatch repair mutants. Proc Natl Acad Sci U S A 100:
68. Murphy KC, Campellone KG (2003) Lambda Red-mediated recombinogenic
engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol Biol
69. Wagner PL, Livny J, Neely MN, Acheson DW, Friedman DI, et al. (2002)
Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli.
Mol Microbiol 44: 957–970.
70. Livny J, LaRock CN, Friedman DI (2009) Identification and isolation of lysogens
with induced prophage. In: Cloakie MRJ, Kropinski AM, editors. Bacterio-
phages, Methods and Protocls. New York, NY: Humana Press. pp. 253–265.
71. Ellis HM, Yu D, DiTizio T, Court DL (2001) High efficiency mutagenesis,
repair, and engineering of chromosomal DNA using single-stranded oligonu-
cleotides. Proc Natl Acad Sci USA 98: 6742–6746.
72. Gottesman ME, Yarmolinsky MB (1968) Integration-negative Mutants of
Bacteriophage Lambda. J Mol Biol 31: 487–505.
73. Datta S, Costantino N, Court DL (2006) A set of recombineering plasmids for
gram-negative bacteria. Gene 379: 109–115.
74. O’Brien AD, Newland JW, Miller SF, Holmes RK, Smith HW, et al. (1984)
Shiga-like toxin-converting phages from Escherichia coli strains that cause
hemorrhagic colitis or infantile diarrhea. Science 226: 694–696.
75. Riley LW, Remis RS, Helgerson SD, McGee HB, Wells JG, et al. (1983)
Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med
76. Livny J (2003) Characterizing the role of the lambdoid prophage H-19B in the
production and release of Shiga toxin. Ann Arbor, Michigan: Ph.D. Thesis,
University of Michigan.
Prophage Induction and EHEC Infection
PLOS Pathogens | www.plospathogens.org14March 2013 | Volume 9 | Issue 3 | e1003236