1

SOS RESPONSE AND THE MECHANISM OF ADAPTIVE TOLERANCE IN

Escherichia coli

A dissertation presented by

Tobias Dörr

to

The Department of Biology

In partial fulfillment of the requirements for the degree of

Doctor of Philosophy

in the field of

Biology

Northeastern University

Boston, Massachusetts

September 2010

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©2010

Tobias Dörr

ALL RIGHTS RESERVED

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SOS RESPONSE AND THE MECHANISM OF ADAPTIVE TOLERANCE IN

Escherichia coli

By Tobias Dörr

ABSTRACT OF DISSERTATION

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy in Biology

in the Graduate School of Arts and Sciences of

Northeastern University, September 2010

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ABSTRACT

Bacteria produce persisters, a small subpopulation of cells that neither grow nor

die in the presence of antibiotics. Persisters are tolerant against exposure to multiple

antibiotics and they likely contribute to the relapse of bacterial infections after antibiotic

therapy. The mechanism of persister formation is unknown, although several studies

have pointed towards redundancy in persister formation mechanisms and the possible

involvement of chromosomal toxin-antitoxin modules.

While studying the genetic requirements for Escherichia coli persister survival

after exposure to the DNA damaging antibiotic ciprofloxacin, we found that persister

formation was an adaptive response to the antibiotic. Survivors to ciprofloxacin

exhibited low levels of SOS induction and their survival depended largely on the SOS-

inducible small toxic peptide TisB. Ectopic overproduction of TisB decreased proton

motive force and induced growth arrest and multidrug tolerance. Further, synthesized

TisB peptide formed an anion-selective pore in an artificial lipid bilayer system. These

results suggest that TisB acts as an uncoupler of oxidative phosphorylation after

induction of the SOS response.

These results challenge the common view of persisters as a metabolically

inactive entity and show that persistence is in part an inducible response specific to a

certain stress.

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ACKNOWLEDGEMENTS

Countless people inside and outside the lab have made the past few years highly

enjoyable and stimulating and I wish to thank all of them.

First and foremost I would of course like to thank Kim Lewis, my advisor, for

giving me the opportunity to work in his lab. His patience, guidance and leadership style

have been an inspiration for my personal as well as professional development and all of

his book recommendations were excellent. Along the same lines I would like to extend a

big hvala to Marin Vulić for his scientific guidance, for commiserating and for Croatian

proverbs. Special thanks to Larry Mulcahy for his help with sorting and figuring out what

the best band in the world is.

Thank you to Sonja Hansen, Mike Lafleur, Iris Keren, Alyssa Theodore, Pooja

Balani, Tony D’Onofrio, Katya Gavrish, Eric Stewart, Janet Manson, Ron Ortenberg,

Gabriele Casadei and all other lab members for being awesome friends and colleagues

and for making this a fantastic place to work at.

An important thank you goes out to my committee members Drs. Veronica

Godoy, Slava Epstein and Abraham L. Sonenshein.

Outside the lab, I wish to thank my wife, Binu Shrestha for her love and support. I

am thankful to Riverhouse inhabitants past and present, especially Smita Das for helpful

comments. Importantly, thank you to my parents for their continuing support for my

endeavors.

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Table of contents

ABSTRACT 4

ACKNOWLEDGEMENTS 5

TABLE OF CONTENTS 6

LIST OF FIGURES 7

INTRODUCTION 9

CHAPTER 1 SOS RESPONSE INDUCES PERSISTENCE TO FLUOROQUINOLONES

IN ESCHERICHIA COLI 18

CHAPTER 2 CIPROFLOXACIN CAUSES PERSISTER FORMATION BY INDUCING

THE TISB TOXIN IN ESCHERICHIA COLI 52

CHAPTER 3 TISB MECHANISM OF ACTION 84

DISCUSSION 105

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List of Figures

Figure 1. Survival of the wild type and the mutants deficient in recombination and/or

SOS induction after ciprofloxacin challenge........................................................... 46

Figure 2. SOS induction and persister level during ciprofloxacin challenge in exponential

growth phase. ........................................................................................................ 47

Figure 3. Fraction of cells undergoing strong SOS induction during ciprofloxacin

challenge................................................................................................................ 48

Figure 4. Ciprofloxacin-induced persistence. ................................................................ 49

Figure 5. Mitomycin C-induced persistence. ................................................................. 50

Figure 6. Growth phase and persister formation. .......................................................... 51

Figure 7. Survival of the tisAB/istR mutants after ciprofloxacin exposure and

complementation of the phenoype ......................................................................... 76

Figure 8. Schematic of the tisAB/IstR locus. ................................................................. 77

Figure 9. Induction of LexA-controlled promoters by ciprofloxacin. ............................... 78

Figure 10. Adaptive ciprofloxacin tolerance in E. coli. ................................................... 79

Figure 11. TisB overproduction and antibiotic tolerance. .............................................. 80

Figure 12. TisB-dependent persister formation in SOS response mutants.................... 81

Figure 13. Model of ciprofloxacin-induced persister formation. ..................................... 82

Figure 14. Survival in antibiotics after overexpression of tisB from O157:H7 Sakai...... 98

Figure 15. Survival of strains carrying translational TisB – mcherry fusions in

ciprofloxacin. .......................................................................................................... 99

Figure 16. Overexpression of tisB in respiratory chain mutants. ................................. 100

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Figure 17. TisB overproduction under anaerobic conditions in different carbon sources.

............................................................................................................................. 101

Figure 18. Effects of cccp on multidrug tolerance ....................................................... 102

Figure 19. Proton motive force after TisB overproduction ........................................... 103

Figure 20. TisB-dependent conductance of a black membrane lipid bilayer. .............. 104

Figure 21. Model of stochasticity in TisB-mediated persister formation ...................... 113

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INTRODUCTION

Cell populations produce persisters, a small fraction of cells impervious to the

lethal effects of antibiotics. Persisters have been observed in bacteria (Lewis, 2010),

yeast (LaFleur et al., 2006) and cancer cells (Sharma et al.,2010).

Bacterial persisters were discovered by Joseph Bigger in 1944, who noticed that

cultures of Staphylococcus aureus could not be sterilized by the beta lactam antibiotic

Penicillin (Bigger, 1944). Bigger described the phenomenon and established that the

survivors were not resistant mutants but rather they were phenotypic variants of the wild

type arising at a low frequency (between 1 in 100 and 1 in 10,000 cells).

First clues towards a mechanism of persister formation came 40 years later from

Harrison Moyed and his coworkers, who isolated mutants that survived extended

treatment with the beta lactam antibiotic ampicillin. Mutants with an increase in

minimum inhibitory concentration (MIC) were discarded in order to exclude resistant

strains. One of the high-persister mutations was mapped to the hipA gene, the toxin part

of the toxin-antitoxin pair hipBA (Moyed & Bertrand, 1983, Black et al., 1994, Moyed &

Broderick, 1986). This hipA7 allele is a gain of function mutation increasing the

frequency of persisters to ampicillin and an unrelated class of antibiotics,

fluoroquinolones, by 10- to 100-fold (Scherrer & Moyed, 1988, Korch et al., 2003, Korch

& Hill, 2006). A knockout of hipA or hipBA, however, had no phenotype (Moyed &

Broderick, 1986, Hansen et al., 2008), leaving the role of the wild type allele in persister

formation unknown.

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Interest in persisters was renewed when Spoering et al. showed that these cells

were responsible for the enigmatic resistance of biofilm populations to killing by

antibiotics (Spoering & Lewis, 2001). Biofilm populations do not induce lifestyle-specific

antibiotic resistance mechanisms but they rather contain a large proportion of persister

cells, similar to stationary phase planktonic cultures. It was persisters that survived

antibiotic exposure and, due to the biofilm matrix, the host immune response (Vuong et

al., 2004). These persisters likely contributed to the common relapse of biofilm

infections after antibiotic therapy (Lewis, 2007).

The revival of interest in this phenomenon also led to a revival of mechanistic

studies. Keren et al. confirmed Bigger’s results (Keren et al., 2004a) and showed that

persisters were formed exclusively in mid-exponential to stationary phase.

Consequently, persisters could be eradicated by keeping a bacterial culture in early

exponential phase through constant dilution. Using an Escherichia coli strain carrying

the high persister allele hipA7, Keren et al. conducted transcriptomic analysis on cells

surviving extended ampicillin treatment and found that persisters had upregulated

conspicuously many chromosomal toxin-antitoxin modules (including relBEF, dinJ,

mazEF) as well as sulA, a septation inhibitor and rmf, the stationary phase ribosome

modulation factor (Keren et al., 2004b). It was subsequently revealed that

overexpression of the chromosomal toxins led to a multidrug tolerant, persister-like state

(Keren et al., 2004b, Shah et al., 2006), providing an attractive mechanism for persister

formation. Knockouts of TA modules, however, still had no phenotype, which led to the

hypothesis that persister formation might be mechanistically redundant (Lewis, 2007).

Escherichia coli contains at least 10 mRNA-interferases (yafQ, relE, mazF, ygiT, yafO,

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chpB, higB, hicA, yoeB, yhaV)(Gerdes et al., 2005, Christensen-Dalsgaard et al., 2010 )

and at least 14 other toxin antitoxin modules including several small, toxic peptides with

antisense RNA antitoxins (type 1 TA modules) (Fozo et al., 2008, Alix & Blanc-Potard,

2009, Fozo et al., 2010). A similar redundancy of TA modules has been reported for

most other free-living microbes (Pandey & Gerdes, 2005).

Shah et al. confirmed and extended these transcriptome results by using wild

type E. coli and a transcriptional fusion of an rRNA promoter with an unstable variant of

green fluorescent protein (GFP) (Shah et al., 2006). The rRNA promoter activity is

strictly coupled with the growth rate (Gourse et al., 1996) and is thus shut down in non-

growing cells. The unstable GFP has a half-life of ca. 30 minutes (Andersen et al.,

1998). Thus, exponentially growing cells were bright green and cells that stopped

growing quickly lost green fluorescence. Using FACS, it was shown that dim cells that

accumulated in a growing population were enriched for ofloxacin-tolerant persisters.

Transcriptome analysis of these cells revealed again an upregulation of toxin-antitoxin

modules (chp, dinJ, relE, dinJ/yafQ, yefM/yoeB, ygiUT) but also some stationary phase

genes (bolA, katE, hdeA) and glycerol metabolism genes (glpD, glpQ). Yet again, no

single knockouts or any attempted knockout combinations of genes upregulated in

persisters (including a strain with 8 TA module knockouts, (unpublished)) produced a

strain with reduced persister levels, reinforcing the redundancy hypothesis. Some other

studies, however, found persister phenotypes for diverse TA module knockouts and

overexpression in different conditions such as dinJ/yafQ (Harrison et al., 2009), hokA

and ygiUT (Kim & Wood, 2010). Although the observed effects were usually modest for

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single knockouts, these results strengthen the notion of involvement of TA modules in

persister formation and offer an explanation for redundancy.

A screen selecting for inducers of high persistence upon multicopy

overexpression from native promoters revealed glpD, coding for glycerol 3 phosphate

dehydrogenase, as a potential persister gene (Spoering et al., 2006). Overexpression of

glpD led to an increase in persister levels while deletion caused a decrease. This,

together with the fact that glpD had been shown to be upregulated in persisters

suggests the involvement of glycerol metabolism as at least one part of persister

formation. The role this pathway plays, however, remains unclear. Interestingly, no

toxin-antitoxin modules were found in this screen.

Redundancy in persister formation was further supported by results from screens

using an ordered, nearly complete E. coli knockout library (Hansen et al., 2008) and a

Pseudomonas aeruginosa transposon insertion library (De Groote et al., 2009) – in

neither case was a single knockout strain identified, which completely lacked persisters.

In summary, four different attempts of identifying the genetic basis of persistence

were only partially consistent: persister formation seemed to be related to toxin-antitoxin

expression and to glycerol metabolism.

Apart from mechanistic details, an important question was the role of persisters

in a population context. Balaban et al. and Shah et al. showed that persisters were pre-

exisiting in a population before addition of the antibiotic (Shah et al., 2006, Balaban et

al., 2004) and Wiuff et al. showed that persisters were multidrug tolerant (Wiuff et al.,

2005). Therefore it seemed that persisters represented a simple form of altruism, where

the individual persister bacterium forfeits propagation while constituting a kind of “life

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insurance” for regrowth of the whole population following a catastrophic event such as

exposure to a bactericidal antibiotic. Indeed, mathematical models have shown that

persistence can assure population survival in frequently changing environments

(Kussell et al., 2005) which most bacteria are subjected to.

Thus emerged the prevailing view of the persister population as an entity

comprised of multidrug tolerant, dormant cells. In time - dependent killing experiments,

however, different antibiotics and different concentrations of the same antibiotics

resulted in different persister plateaus indicating heterogeneity even within the persister

population (unpublished data). Most importantly, ampicillin, which kills only growing

cells, consistently revealed a lower persister plateau than ciprofloxacin, which kills

growing and non-growing cells. Strictly viewing persisters in a monolithic way is

inconsistent with this phenomenon unless one would suggest the highly unlikely

existence of exponentially growing cells that are tolerant to ciprofloxacin.

A more likely explanation is that persistence can be induced by the

fluouroquinolone antibiotic. To test this hypothesis, we studied the genetic requirements

for persister formation in response to ciprofloxacin.

We found that persister formation can indeed be an adaptive process induced by

an antibiotic and that this process was dependent on the SOS response and was mainly

caused by the DNA- damage inducible TisB toxin.

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CHAPTER 1 SOS response induces persistence to

fluoroquinolones in Escherichia coli

Published in the December 2009 issue of PloS Genetics

Tobias Dörr, Kim Lewis and Marin Vulić

Antimicrobial Discovery Center, Department of Biology, Northeastern University,

Boston, MA 02115

ABSTRACT

Bacteria can survive antibiotic treatment without acquiring heritable antibiotic resistance.

We investigated persistence to the fluoroquinolone, ciprofloxacin, in Escherichia coli.

Our data show that a majority of persisters to ciprofloxacin were formed upon exposure

to the antibiotic, in a manner dependent on the SOS gene network. These findings

reveal an active and inducible mechanism of persister formation mediated by the SOS

response, challenging the prevailing view that persisters are pre-existing and formed

purely by stochastic means. SOS induced persistence is a novel mechanism by which

cells can counteract DNA damage and promote survival to fluoroquinolones. This

unique survival mechanism may be an important factor influencing the outcome of

antibiotic therapy in vivo.

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AUTHOR SUMMARY

The frequent failure of antibiotic treatments is an acute public health problem.

Bacteria can escape the lethal action of antibiotics by a mutation in the cell’s DNA which

leads to antibiotic resistance. Alternatively, they can enter a distinct, temporary

physiological state in which the antibiotics do not affect them. This phenomenon,

referred to as persistence, is different from resistance because there is no genetic

modification and because it is transient. Persisters are believed to form stochastically

prior to antibiotic treatment.

The presence of persister cells in bacterial biofilms contributes to the difficulty in

treating biofilm related infections. Biofilms are implicated in many chronic infections,

such as middle-ear infections, gingivitis, endocarditis, infections in cystic fibrosis etc.

Biofilms are also a major cause of infections in leg prostheses, heart valves, catheters

and contact lenses.

In this study we investigated the persistence of Escherichia coli to one of the most

widely used antibiotics, ciprofloxacin. We show that the majority of persister cells are

formed in response to this antibiotic, contrary to the prevailing view of persister

formation. Ciprofloxacin kills bacteria by damaging their DNA and DNA damage

activates a SOS gene network, the result of which is the production of various repair

proteins. We uncovered a novel part of this network that leads to the formation of

tolerant persister cells. The induced tolerance as a side effect of antibiotic treatment is

an effective bacterial survival strategy and is likely to contribute to recalcitrance of

infections.

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INTRODUCTION

Persistence is the ability of a subpopulation of susceptible bacteria to survive

lethal doses of antibiotics. It is a transient and non-hereditary phenotype unlike

resistance, which is due to genetic modification. The transient nature of persistence

makes it inherently difficult to study therefore the underlying molecular mechanisms are

still poorly understood.

Persisters are thought to be slow growing, non-growing or dormant cells, which

escape the lethal action of antibiotics because their drug targets are inactivated due to

the physiological state. In an Escherichia coli high-persistence mutant, persisters to

ampicillin were shown to be non-growing prior to the addition of the antibiotic [1]. In

addition, a fraction of non-growing cells was isolated from untreated exponentially

growing E. coli and was shown to be enriched in persisters to ofloxacin [2]. These

studies demonstrated that persisters can form independently of antibiotics. The switch

from growing to non-growing state or dormancy, is thought to be a purely stochastic

process [1,3,4].

Both genetic and phenotypic variability can have important consequences on

bacterial survival of antibiotic treatment. One of the most prescribed broad spectrum

antibiotics today are the fluoroquinolones (FQ), which target gyrase and topoisomerase.

These essential enzymes regulate supercoiling of genomic DNA during replication and

transcription [5,6]. FQs prevent ligation reactions of gyrase and topoisomerase resulting

in double-strand breaks (DSB) [7]. DSBs are potentially lethal DNA lesions that occur

under physiological conditions through collapse of stalled replication forks, overlapping

repair tracts, spontaneous breakage of DNA, and other mechanisms. E. coli efficiently

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repairs DSBs through a series of reactions carried out by enzymes participating in

homologous recombination and replication [8]. Processing of DSBs leads to the

induction of the SOS response. SOS is a complex network composed of more than 40

genes [9,10]. Many of these genes are essential for efficient repair of various DNA

lesions, including DSBs [11,12].

Even though fluoroquinolones are potent bactericidal antibiotics they cannot

sterilize a bacterial culture. The bulk of the population rapidly dies in response to

fluoroquinolones but a small fraction persists. According to one model, persisters might

survive if gyrase and topoisomerase are inactivated due to cellular dormancy [3].

Dormant cells might be expected to form stochastically during growth of a culture, prior

to the antibiotic exposure [2,13-15].

Alternatively, the persister state might be inducible in a cell subpopulation by

exposure to the antibiotic, not stochastic and pre-existing. This could be because either

dormancy is inducible, or persisters might be active and have more efficient drug efflux

or more efficient repair of DSBs due to the stochastic overexpression of the genes

involved in those pathways or due to the physiological events leading to the activation of

the same pathways.

In order to distinguish between these possibilities, we measured numbers of

persisters to the fluoroquinolone ciprofloxacin in various genetic backgrounds with

altered capacity for SOS induction and DSB repair.

The majority of persisters were found to be formed upon exposure to the

antibiotic and formation was dependent on the SOS DNA damage response. Contrary

to the current view, a majority of surviving persisters to ciprofloxacin are not pre-

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existing, but induced by this antibiotic.

RESULTS

Fluoroquinolones (FQ) induce DSBs by interfering with the action of gyrase and

topoisomerase [16]. The cellular response to DSBs primarily consists of induction of the

SOS-regulon and ultimately in repair through recombination [17,18].

According to the prevailing model [3], persisters are dormant and are formed

stochastically prior to the addition of antibiotic. This suggests that persisters would not

experience DSBs, would not induce an adaptive response to that type of lesion, and

therefore would not need repair functions to survive. In order to test these predictions,

we wanted to determine whether persisters experience DSBs and induce SOS.

We measured the persister levels in different genetic backgrounds diagnostic of

specific molecular events linked with DSBs and SOS induction. The surviving fraction of

a wild-type culture treated with ciprofloxacin produces a typical biphasic pattern (Fig. 1

A). This reflects the rapid killing of the bulk of the cells, and a surviving persister

subpopulation.

We examined some of the well-known DNA-repair pathways in order to probe

their possible role in formation of persisters. RecA and RecBC are essential for repair of

DSBs in E. coli [19]. In strains lacking RecA and RecBC, DSBs are lethal. As expected,

the bulk of cells is more rapidly killed in both recA and recB backgrounds, compared to

the wild type, presumably because DSBs could not be repaired (Fig. 1 A). However, the

persister fraction was also greatly reduced (40-fold in recA, 35- to 103-fold in recB). In

recB, persisters were extremely rare or entirely absent after 6 hours of incubation. This

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shows that the persisters experience DSBs and hence depend on the repair functions.

RecA and RecB functions are essential not only for DSB-repair but for SOS

induction following processing of DSBs as well [18,20-22], so in order to test whether

persisters induce SOS we constructed strains unable to induce SOS but proficient for

homologous recombination; one carrying a non-inducible SOS-repressor (lexA3) [23]

and the other a mutant RecA able to function as a recombinase but unable to induce

cleavage of LexA (recA430) [24]. In both backgrounds the bulk of cells dies more rapidly

than in the wild type, confirming that SOS is efficiently induced following exposure to

ciprofloxacin and contributes to the survival (Fig. 1 B). Interestingly, the persister level is

decreased 43-fold and by 6 hours it is as low as in recA background. This shows that

the persistence to ciprofloxacin is largely dependent on a functional SOS response.

XerCD site-specific recombinase resolves chromosome dimers at a dif site [25-

27]. Chromosome dimers are formed by an odd number of recombination events. The

absence of xerCD function does not affect the proficiency for SOS induction, but is

lethal in cells in which chromosme dimers have formed. In xerC and xerD mutants the

persister level is reduced (7- and 9-fold, respectively, taking into account the 3-fold

reduction in viability of xerC and xerD mutants compared to the wild-type), suggesting

that most persisters have undergone at least one successful recombination event, most

likely repairing a ciprofloxacin-induced DSB (Fig. 1 C).

Taken together these results show that the formation of the majority of persisters

in the presence of ciprofloxacin is dependent on the SOS-response. They also suggest

that this antibiotic-tolerant state is induced, rather than pre-existing. The formal

possibility that an SOS controlled function is essential for reaching or exiting a pre-

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existing multidrug-tolerant state can be ruled out because tolerance to ampicillin and

streptomycin were not affected in recA, recB or lexA3 strains (Fig. 1 D). We cannot rule

out the possibility that spontaneous SOS induction was required for creating a pre-

existing ciprofloxacin-tolerant state.

A strain lacking the SOS-inducible RecN protein is also SOS proficient but

partially deficient in DSB repair. recN mutant exhibited increased sensitivity of the bulk

whereas persistence was largely unaffected (Fig. 1 F).

The entire population exposed to ciprofloxacin is expected to induce SOS yet

only a fraction survives. SOS is a gradual response where the strength of induction

reflects the extent and the persistence of the damage [28,29]. Upon addition of

ciprofloxacin the number and the chromosomal location of DSBs will vary across the

population depending on the activity and position of gyrase and topoisomerase

molecules in any given cell. The resulting distribution of DSBs is expected to translate

into a gradient of SOS induction. Therefore, it is possible that a specific level of SOS

induction is required for persistence. If this is the case, persister levels are expected

to change along with ciprofloxacin concentration and the overall level of SOS

induction.

We measured both the persister level and the induction of β-galactosidase under

the control of an SOS-inducible recA promoter [30] as a population average of SOS

induction for cultures exposed to increasing concentration of ciprofloxacin. Indeed, as

shown in Fig. 2, increased concentration of ciprofloxacin led to an increased average

SOS induction (Fig. 2 A) and decreased persister level (Fig. 2 B).

A strain constitutively expressing SOS functions (lexA300(Def)), also led to a 20-fold

25

increase in persister level compared to the wild type (Fig. 1 E).

In order to examine a difference in SOS induction between persisters and the

bulk at the single cell level, we followed a cI-cro gal reporter strain after addition of

ciprofloxacin [31]. In this strain the cleavage of λ repressor CI leads to a heritable

genetic switch rendering a cell gal+. gal

+ cells can be detected as red colonies on

MacConkey galactose plates. Unlike LexA, which undergoes auto-cleavage early in

SOS induction, CI cleavage occurs only if there is a high level of DNA damage and

activated RecA [32]. Therefore, the cI-cro system reports conditions of only strong SOS

induction. Following the addition of ciprofloxacin (> 0.5 µg/ml) the proportion of cells

giving rise to gal+ colonies increases, peaking at around 20 minutes and declines

thereafter (Fig. 3). This timing means that the massive amount of DNA damage occurs

readily leading to a strong SOS induction. Cells undergoing strong SOS induction are

able to withstand and repair the damage, if the ciprofloxacin is removed by plating.

However, upon extended exposure gal+ cells become fewer (Fig. 3), indicating that

additional damage occurs and eventually becomes lethal. The persister subpopulation

consisted almost entirely of gal-cells (Fig. 3) showing that persisters were not SOS-

induced prior to ciprofloxacin treatment (at least not highly induced) and also that they

did not experience high level of DNA damage nor strong SOS induction even in the

presence of the antibiotic. Because the persister level is greatly reduced in strains

unable to induce SOS (lexA3, recA430, Fig. 1 B) we conclude that persisters undergo

weak SOS induction. This is in contrast to the bulk of cells, probably because fewer

DSBs occur in eventual persisters. Increased sensitivity of the bulk and minimally

affected persistence in a recN strain also supports this conclusion (Fig. 1 F). SOS-

26

inducible RecN protein promotes efficient repair of DSBs. While it is dispensable for the

repair of a single break, it is essential for the repair of simultaneous multiple DSBs [33].

Next we exposed cells treated with a range of ciprofloxacin concentrations to a

higher dose (1 µg/ml) of the same antibiotic (Fig. 4). Control cultures were exposed to

1 µg/ml of ciprofloxacin for the duration of the experiment. The persister fraction

surviving exposure to 1 µg/ml was 10- to 40-fold higher in the cultures pretreated with

a low concentration of ciprofloxacin (0.05-0.2 µg/ml), compared to the control (Fig. 4

B). A dramatic, 1200-fold increase was found in cultures pretreated with sub-MIC

(minimal inhibitory concentration) concentration of ciprofloxacin (Fig. 4 B; compare full

bar at 0.03 µg/ml and the second dashed bar of the control). This shows that many of

the persisters are formed upon ciprofloxacin treatment rather than pre-existing. If they

were preexisting, the fraction surviving the exposure to the high concentration of

ciprofloxacin (1 µg/ml) would be the same regardless of the pretreatment.

It was important to learn whether SOS induction caused by treatments other than

FQ is able to induce persistence to ciprofloxacin. In order to test this we measured

persistence to ciprofloxacin in cells exposed to mitomycin C. Mitomycin C interacts with

DNA by intercalation and adduct formation, resulting in inter-strand crosslinks [34]. The

cellular response is a potent SOS-induction dependent on RecFOR pathway [35]. We

exposed exponentially growing cells to a sub-MIC concentration of mitomycin C and

compared the persister levels at two different time points during the treatment. The

results in Fig. 5 show a 180-fold increase in persistence to ciprofloxacin in the culture

treated with mitomycin C for 4 versus 2 hours, confirming the link between SOS

induction and persistence to FQs, regardless of the nature of the SOS inducing

27

treatment.

Persister levels are very low in early exponential phase and are maximal in

stationary phase [14]. We treated aliquots of growing cultures with ciprofloxacin at

different time points in order to determine the persister levels between these two

extremes and establish the role of growth phase in SOS-induced persistence. Fig. 6

shows an exponential increase in persister levels when cell densities reach around

5 x107 CFU/ml in both the wild-type and the strain unable to induce SOS (lexA3).

We conclude that the SOS-induced persisters make up the majority of persisters to

ciprofloxacin regardless of the growth phase.

DISCUSSION

The processes leading to genetic variability in bacteria, mutagenesis and

recombination, have been studied extensively [36-38] and their role in evolution of

bacterial antibiotic resistance by generating and disseminating mutations is well

established [39-46]. On the other hand, processes leading to phenotypic variability,

which is also an important factor influencing bacterial ability to survive antibiotic

treatments [47,48] have only recently become a subject of systematic investigation.

In contrast to the well-understood mechanisms of bacterial resistance to antibiotics,

molecular mechanism(s) of persistence have so far remained elusive. The current

model of persistence assumes that persisters are non-growing or dormant cells,

formed by stochastic process(es) independently of any physiological responses

normally elicited by antibiotics [1,3,4]. Studies involving persistence to two different

28

classes of antibiotics, a β-lactam ampicillin [1] and a fluoroquinolone ofloxacin [2] are

consistent with this model, which was therefore presumed to hold universally.

Here we show a mechanism of persister formation triggered by DNA damage

inflicted by the fluoroquinolone ciprofloxacin. Formation of persisters in response to

DNA damage reveals a deterministic component in this bistability phenomenon.

Bistability is the stochastic production of two phenotypically distinct cell types within a

clonal population of genetically identical kin cells. Bistability is observed in sporulation,

competence, and motility [49-51]. In all cases studied, there is both a stochastic and

deterministic component of bistability. Previous studies have shown that persisters can

form stochastically, prior to the addition of antibiotics [1,14]. The present findings show

that persister formation can also be induced by an antibiotic, through an active process.

This sheds an entirely new light on the problem of antibiotic tolerance and its role in

infectious processes.

We also show that mutants defective in persistence to ciprofloxacin have normal

persister levels to amipicillin and streptomycin (Fig. 1 D), therefore it is still possible

that persistence to β-lactams is purely stochastic and not inducible. These results

suggest that there are different mechanisms of persistence to different antibiotics.

Ciprofloxacin induces DSBs in cells with active gyrase and/or topoisomerase,

which in turn leads to the activation of the general DNA damage stress response, the

SOS gene network. Our results show that the majority of persisters to ciprofloxacin

are dependent on a functional SOS response.

DSBs and other SOS-inducing lesions occur under physiological conditions so at

any given time there is a fraction of a bacterial population undergoing a certain degree

29

of SOS induction [52,53]. However, we demonstrate that the SOS-dependent persister

state is induced upon exposure to ciprofloxacin. Manipulating the extent of SOS

induction by different antibiotic concentrations or by sequential exposure to a higher

dose dramatically affects persister levels (Fig. 2, Fig. 4). This would not be the case if

the persisters were pre-existing in the population. If they were, the bulk would be killed

by any bactericidal concentration of antibiotic, revealing the same pre-existing persister

population. In addition, increasing the basal level of expression of the SOS regulon by

genetic manipulation (Fig. 1 E) or by induction with different treatment (Fig. 5) also

leads to an increase in persister level.

Essentially all actively growing cells exposed to ciprofloxacin induce SOS, but not

all become persisters, suggesting that a specific level of SOS induction is required for

persister formation. SOS is a gradual response and depending on the nature of the

inducer, its concentration and the time of the exposure, different sets of genes are

induced [10,54,55]. Our data indicate that persister formation requires a functional SOS

response but a high level of induction is not required (Fig. 1 B, Fig. 3). Persister

formation also depends on functional DSB repair (Fig. 1 A) but does not need RecN

(Fig. 1 F), a function important for the repair of multiple DSBs [33]. This implies that

persisters are cells that experienced few DSBs upon ciprofloxacin addition and

underwent weak SOS induction.

Consistent with this, constitutive, full expression of the SOS regulon (equivalent

to high induction) does not lead to the tolerance of the entire population, but to an

increased level of surviving persisters (Fig. 1 E). Even in a lexA(Def) mutant, expression

levels of SOS genes appear to fall short of being truly uniform throughout the population

30

[52,53]. Persisters could be the cells that express a certain SOS function at a specific

high or low level. Additionally, other regulatory pathways could allow a persister

formation function to be expressed only in certain cells after induction. Turning on the

SOS response constitutively would increase the number of cells being able to express

this function.

Persister levels are known to change with the growth phase [14]. It is low in early

exponential phase and attains its highest level in stationary phase. An exponential

increase in persister levels begins when the cell density reaches around 5 x107 CFU/ml

(Fig. 6). The persister shoot up at similar cell density has been observed in other

studies under different antibiotic and growth conditions [1,14]. A cell density of 5 x107

CFU/ml coincides with the point at which the balanced growth of the culture ceases and

a slowdown of growth rate is observed, even though the population as a whole still

increases exponentially [56].

The extent of the DNA damage caused by ciprofloxacin would be expected to

reflect the activity levels of gyrase and topoisomerase. These enzymes are active

during replication and transcription [6,57]; therefore their maximal activity would occur in

rapidly growing and replicating cells and would be lowest in the non-growing state of

stationary phase. Lending support to this, transcription of gyrA and gyrB coding for

gyrase subunits is at the peak in the early exponential phase and the lowest in the

stationary growth phase [58,59]. It follows that ciprofloxacin would inflict maximal

damage, the irreparable chromosome fragmentation, in the exponentially growing cells

and fewer DSBs in the cells that slow down when the medium cannot support steady-

state growth [60]. Indeed, no cells survive treatment to ofloxacin, another FQ, when the

31

culture is kept at low density in constant exponential growth by repeated subculturing

[14], in other words no persisters are formed in that growth phase. On the other hand,

the surviving fraction increases dramatically between the end of true exponential growth

and stationary phase (Fig. 6, [14]). During that time the growth rate of the population

decreases from its maximum to zero, but because not all cells stop growing at the same

time the heterogeneity of growth rates across the population is expected within that time

frame. Those cells lacking steady state equilibrium might be the ones which experience

few DSBs, weak SOS-induction and enter the tolerant state. Consistent with this, the

difference in persister level in SOS proficient and deficient strains is minimal in early

exponential phase, whereas it increases after the cessation of steady state growth (Fig.

6).

Conditions for unrestricted growth are rarely met in natural environments, and

most bacteria are in a state of slow or no-growth [61-63]. However, physical and

chemical agents capable of causing DNA damage are ubiquitous, therefore the SOS-

induced persister state is probably quite common. Furthermore, in conditions of slow

growth and frequent or lasting presence of DNA damaging agents, damage

prevention would likely be advantageous over continuous active repair. The induction

of the persister state in response to DNA damage seems like such a strategy - the

avoidance of the damage build up as opposed to the costly repair.

SOS is induced in aging colony biofilms of E. coli [64] and in intracellular biofilms

formed by uropathogenic E. coli during cystitis [65]. Biofilms are notoriously hard to

eradicate even with bactericidal fluoroquinolones, and this enhanced ‘resistance’ could

in fact reflect the SOS-induced tolerance.

32

Virtually all natural isolates of E. coli and many other bacteria are lysogens and

many prophages are DNA-damage inducible [66-69]. Induction of λ prophage in E. coli

is a late SOS function. In that light, SOS-induced tolerance could have evolved as a life-

saving strategy preventing prophage induction upon DNA damage frequently

encountered.

There are at least 43 genes in the E. coli genome negatively regulated by LexA

[9,10]. Many encode proteins participating in repair by homologous recombination

and/or translesion synthesis and about one third are of unknown function. Among those

are several genes encoding toxin-antitoxin modules that are attractive candidates for

persistence genes, as the overexpression of some toxins has been shown to induce a

dormant-like state [13,70]. Indeed, in a parallel study we identified an SOS inducible

toxin/antitoxin module, tisAB, as a function needed for persister formation (Dšrr T., Vulić

M., Lewis K., submitted). However we cannot exclude that other LexA-regulated genes

also contribute to SOS-induced tolerance.

SOS has been shown to induce formation of a senescence-like state in which

cells are viable but unable to form colonies [53]. Here we show SOS-dependant

formation of persister cells. Both states could be formed through the common

mechanism, such as expression of SOS-regulated toxins. In that case the strength of

SOS induction and hence the toxin expression levels would determine which of

these two states a cell reaches.

In conclusion, we have discovered an active, regulated mechanism of persister

formation, which is part of the SOS response. SOS has been known to contribute to the

survival of antibiotic treatments by increasing the frequency of resistant mutants through

33

its mutagenic activities [44,71]. Here we show a novel function of this response, the

induction of a tolerant state. SOS-induced persistence having an immediate impact on

bacterial survival is likely an important factor influencing the outcome of antibiotic

treatment.

MATERIALS AND METHODS

Bacterial strains

Bacterial strains are listed in Table 1. Wild-type E. coli K-12 MG1655 was used as the

parental strain. Different alleles were moved into the parental background by P1

transduction [72]. The kanamycin resistance cassette from the alleles originated from

KEIO collection [73] was cured when needed by expressing the FLP recombinase

from the helper plasmid pCP20 according to the protocol in [74].

Persistence assay

Experiments were conducted at 37¡C in Mueller Hinton Broth (MHB) supplemented with

10 mg/L MgSO4 and 20 mg/L CaCl2 according to NCCLS (National Committee for

Clinical Laboratory Standards) guidelines for susceptibility testing and 0.1 M

HEPES/KOH pH 7.2. Persistence was measured by determining survival upon

exposure to 0.1 µg/ml ciprofloxacin (unless indicated otherwise), 100 µg/ml ampicillin

and 25 µg/ml streptomycin during time indicated on corresponding graph axes. All

antibiotics were purchased from Sigma. Prior to the addition of antibiotic overnight

34

cultures were diluted 100-fold in 3 ml of fresh medium in 17- by 100-mm polypropylene

tubes and incubated for 1.5 hrs with shaking, typically reaching ~ 2 x 108 CFU/ml. For

determination of CFU counts, cells were washed in 1% NaCl solution, serially diluted

and plated on LB (Luria-Bertani medium) agar plates supplemented with 20 mM

MgSO4. Persister fraction, reflected as a plateau in CFU counts, was calculated as an

average of CFU counts at 3- and 6-hour time points. In lexA3 and recA430 strains the

CFU counts stabilize later than in the wild-type and in that case the CFU counts at 6-

hour time point were used as a representative of the persister fraction.

Measurements of SOS induction

For plate assays using the CI-cro-gal construct, overnight cultures grown in LB medium

at 37¡C were diluted 1:200 in 15 ml of fresh medium and incubated in 125 ml flasks for

1.75 hrs at 37¡C with shaking. Ciprofloxacin was added and aliquots of the culture were

taken at different time points, washed in 1% NaCl solution, serially diluted and plated on

LB agar plates supplemented with 20 mM MgSO4 for total CFU counts and on

MacConkey agar plates supplemented with 1% galactose in order to determine the

fraction of gal+ cells. For β-galactosidase activity measurement, overnight cultures

grown in supplemented MHB medium (see above) at 37¡C were diluted 1:100 in 3 ml of

fresh medium in 17- by 100-mm polypropylene tubes and incubated for 1.75 hrs at 37¡C

with shaking.

Ciprofloxacin was added and after 15 minutes an aliquot of culture was taken and

recA::lacZ expression was measured as described in [72].

35

Mitomycin C treatment and persistence to ciprofloxacin

Overnight cultures in supplemented MHB medium were diluted 1:1000 in 15 ml of fresh

medium and incubated in 125 ml flasks for 1 hr at 37¡C with shaking after which 0.25

µg/ml of mitomycin C (Sigma) was added to the cultures. This concentration did not

inhibit the growth of the culture. After 2 hrs the total CFU counts were determined by

dilution and plating and an aliquot of the culture was taken out and exposed to 0.3 µg/ml

of ciprofloxacin for 3 hrs. The number of survivors was determined by plating on LB

agar plates supplemented with 20 mM MgSO4 after washing in 1% NaCl solution. The

same procedure was repeated after 4 hours of exposure to mitomycin C.

Kinetics of persister formation

Overnight cultures in MHB medium were diluted 1000-fold in 100 ml of fresh medium in

500 ml flasks and incubated at 37¡C with shaking. At defined time intervals the cultures

were serially diluted and plated on LB agar for determination of total CFU counts. In

the same time 1 ml aliquots were transferred into 2 ml eppendorf tubes and 0.1 µg/ml

ciprofloxacin was added. After 3 hrs at 37¡C cells were washed with 1% NaCl solution,

serially diluted and plated on LB agar plates supplemented with 20 mM MgSO4. The

colonies were counted after 40 hours incubation at 37¡C.

ACKNOWLEDGMENTS

We would like to thank the editor for detailed and helpful comments on the previous

36

version of the manuscript. We would also like to thank Alyssa Theodore for excellent

technical assistance, E. coli Genetic Stock Center, Radman and Walker Lab for strains,

Eric Stewart, Michael LaFleur and Veronica Godoy for critical reading of the manuscript.

TABLE 1. Bacterial strains.

Strain Relevant genotype Parent strain Reference/Source

MG1655 K-12 Fλ

TD172 ΔrecA::kan JW2669 [73]

TD230 ΔrecB::kan JW2788 [73]

TD160 ΔrecN::kan JW2597 [73]

TD222 recA430 GY3448 [24,75]

E. coli Genetic

Stock Center,

Yale

TD221 lexA3 malE300::Tn10 K996 [23] E. coli

Genetic

Stock Center,

Yale

TD127 lexA300(Def) GW8018 [76] Walker Lab,

MIT, Cambridge

ΔsulA::FRT JW0941 [73]

MV2033 ΔxerD::kan JW2862 [73]

37

MV2037 ΔxerC::kan JW3784 [73]

LLC3 (λ cI+ cro+-gal-) MT1 [31]

Radman Lab,

Necker, Paris

MV1603 λ d(recA::lacZ) cI(Ind-)

AmpR AB1157 [30]

λ d(recA::lacZ) Radman Lab,

cI(Ind-) AmpR Necker, Paris

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55 Schneider R, Travers A, Kutateladze T, Muskhelishvili G (1999) A DNA

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56 Tamayo M, Santiso R, Gosalvez J, Bou G, Fernandez JL (2009) Rapid

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59 Baath E (1998) Growth Rates of Bacterial Communities in Soils at Varying pH: A

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60 Taddei F, Matic I, Radman M (1995) cAMP-dependent SOS induction and

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65 Casjens S (2003) Prophages and bacterial genomics: what have we learned so

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46

Figure 1. Survival of the wild type and the mutants deficient in recombination

and/or SOS induction after ciprofloxacin challenge.

Strains were exposed to ciprofloxacin in exponential growth phase. Viable counts

were determined by plating. Graphs are representatives of at least 5 independent

experiments. Error bars represent standard error.

47

Figure 2. SOS induction and persister level during ciprofloxacin challenge in

exponential growth phase.

Graphs are averages of at least 3 independent experiments and error bars represent

standard error. (A) Induction of the SOS-inducible recA gene expression measured

by assaying the β-galactosidase activity after 15 min of ciprofloxacin challenge. (B)

Persister levels after 6 hours of ciprofloxacin challenge.

48

Figure 3. Fraction of cells undergoing strong SOS induction during ciprofloxacin

challenge.

Cells in exponential growth phase were exposed to ciprofloxacin. A heritable epigenetic

switch based on the reciprocal repression of the phage λ cI and cro genes fused with

the promotorless galactose operon allows detection of clones derived from cells that

have undergone SOS induction as red gal+ colonies on MacConkey galactose plates.

Data points are average of at least 3 independent experiments. Error bars represent

standard error.

49

Figure 4. Ciprofloxacin-induced persistence.

(A) Survival of wild type cells in exponential phase under different ciprofloxacin regimes.

Two cultures were treated with 0.1 µg/ml and 1 µg/ml, respectively, for 6 hours. Third

culture was treated with 0.1 µg/ml for 3 hours after which 1 µg/ml was added (indicated

by an arrow). The data are averages of 3 independent experiments and error bars

indicate standard error.

(B) Wild type cells in exponential phase were treated for 3 hours with increasing

concentrations of ciprofloxacin indicated on x-axis. After the initial treatment, an

additional 1 µg/ml of ciprofloxacin was added to the cultures and incubated for another 3

hours as in (A) (ciprofloxacin MIC is 0.05 µg/ml). As a control, a parallel culture was

exposed to 1 µg/ml for the duration of the experiment. Bars represent the viability at 0, 3

and 6 hr of time course equivalents shown in (A). Open bars; the initial viability count,

50

grey bars; the viability after 3-hour incubation with ciprofloxacin concentration indicated

on the x-axis. Full bars; the final viability count after additional 3-hour incubation with 1

µg/ml ciprofloxacin. Dashed bars; viability of the control culture at 3 and 6 hours. The

data are averages of 3 independent experiments and error bars indicate standard error.

Figure 5. Mitomycin C-induced persistence.

Wild type cells in exponential phase were treated with mitomycin C for either 2 or 4

hours before being exposed to 0.3 µg/ml ciprofloxacin. Open bars; total viable counts,

gray bars; persister fraction. The data are averages of 3 independent experiments and

error bars indicate standard error.

51

Figure 6. Growth phase and persister formation.

Wild type strain and strain unable to induce SOS (lexA3) were cultured with aeration at

37¡C. A sample of each culture taken at the designated time points was treated with

ciprofloxacin for 3 hrs. Cell counts before and after the antibiotic challenge were

determined by plating. Data points are averages of 4 independent experiments. Error

bars represent standard error.

52

CHAPTER 2 Ciprofloxacin Causes Persister Formation by

Inducing the TisB toxin in Escherichia coli

Published in the Feburary 2010 issue of PloS biology

Tobias Dörr1, Marin Vulic

1 and Kim Lewis*

1

1Antimicrobial Discovery Center, Department of Biology, Northeastern University,

Boston, MA 02115

* E-mail: k.lewis@neu.edu

Abstract

Bacteria induce stress responses that protect the cell from lethal factors such as

DNA-damaging agents. Bacterial populations also form persisters, dormant cells that

are highly tolerant to antibiotics and play an important role in recalcitrance of biofilm

infections. Stress response and dormancy appear to represent alternative strategies of

cell survival. The mechanism of persister formation is unknown, but isolated persisters

show increased levels of toxin/antitoxin (TA) transcripts. We have found previously that

one or more components of the SOS response induce persister formation after

exposure to a DNA damaging antibiotic. The SOS response induces several

53

toxin/antitoxin genes in E. coli. Here we show that a knockout of a particular SOS-TA

locus, tisAB/IstR, had a sharply decreased level of persisters tolerant to ciprofloxacin,

an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin

induced persister formation in a tisAB-dependent manner, and cells producing TisB

toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown

to decrease proton motive force (pmf) and ATP levels, consistent with its role in forming

dormant cells. These results suggest that a DNA damage-induced toxin controls

production of multidrug tolerant cells and thus provide a model of persister formation.

Abbreviations: CFU, colony forming units; IPTG, Isopropyl β-D-1-

thiogalactopyranoside; MHB, Mueller-Hinton-Broth; MIC, minimum inhibitory

concentration; pmf, proton motive force

Running title: TisB-induced persister formation in E. coli

Key words: Persister, biofilm, antibiotic tolerance, toxin antitoxin, ciprofloxacin, SOS

response

54

Introduction

Bacterial populations form persisters, dormant cells that are highly tolerant to

antibiotics and play an important role in recalcitrance of biofilm infections [1,2]. Time-

dependent or dose-dependent killing by antibiotics is distinctly biphasic, revealing a

surviving subpopulation of persister cells. Reinoculation of surviving cells produces a

culture with a new subpopulation of persisters, showing that these cells are not mutants,

but rather phenotypic variants of the wild type [3,4]. Reexposure of persisters to a

different bactericidal antibiotic resulted in little or no additional killing, showing that

persisters are multidrug tolerant cells [5]. Gain of function mutants in the Escherichia

coli hipA toxin gene lead to an increase in the frequency of ampicillin- and

fluoroquinolone-tolerant persisters in a growing population from 1 in 10,000 cells or less

(wild type levels) to 1 in 100 cells [6-10], and this hipA7 mutant was shown to form

persisters prior to addition of antibiotic [11]. These persisters were slow- or non-growing

cells. Wild type persisters have been isolated from an exponential culture of E. coli

untreated with antibiotic by sorting out dim cells of a strain expressing a degradable

GFP transcriptionally fused to a ribosomal RNA promoter [12]. This indicated that

persisters are cells that have diminished protein synthesis and are dormant. The

apparent dormancy of persisters accounts for their tolerance to bactericidal antibiotics

whose action requires an active, functional target [13-16].

The mechanism of persister formation is currently unknown. Isolated persisters show

increased expression levels of chromosomal toxin/antitoxin (TA) genes [9,12]. Ectopic

overproduction of RelE, an mRNA endonuclease, [17] inhibits protein synthesis and

creates dormant, multidrug-tolerant cells [9]. The HipA protein is an Ef-Tu kinase

55

[18,19], which also inhibits protein synthesis and produces multidrug tolerant cells upon

overproduction.

However, strains deleted in individual TA loci do not have a phenotype [9,12],

possibly due to their functional redundancy [20-22]. In E. coli, there are at least 15 TA

modules [20,22,23]. Importantly, a screen of an ordered 3,985 open reading frame (out

of a total of 4288) knockout library of E. coli [24] for mutants lacking persisters in

stationary phase produced a largely negative result – not a single strain lacking

persister formation was identified [25]. Similar negative findings were reported with

screens of E. coli transposon insertion (Tn) libraries [26,27] and a Pseudomonas

aeruginosa Tn library [28]. Only mutants with modest reduction in persister levels were

identified, and in the case of E. coli, these were primarily in global regulators [25]. This

strongly suggests that there are multiple, redundant mechanisms of persister formation.

Persisters were originally described by Bigger in 1944 [3], but functional redundancy

has made it very challenging to elucidate the mechanism by which they form.

A useful clue to a possible mechanism of persister formation comes from the

analysis of the SOS response. Interestingly, SOS induces several toxin/antitoxin genes

in E. coli, whose promoters contain a Lex box - symER, hokE, yafN/yafO, and tisAB/IstR

[23,29-35] Another locus, dinJ/yafQ contains a Lex box but is not believed to be under

SOS control [29,30]. Importantly, only the toxin gene is predicted to be upregulated in

the three type 1 TA modules (symER, hokE and tisAB/istR) following SOS induction

while in the type 2 TA modules toxin and antitoxin form an operon and are therefore

both expected to be induced. Fluoroquinolones such as ciprofloxacin induce the SOS

response [36] by blocking the ligase activity of DNA gyrase and topoisomerase,

56

converting them into endonucleases [14,37]. In a separate study we have shown that

the SOS response is also necessary for persister formation in response to the

fluoroquinolone antibiotic ciprofloxacin [38]. In the present study, we examined the

mechanism of this ciprofloxacin-induced persister formation and find that it is governed

by the TisB toxin.

Results

Ciprofloxacin rapidly killed the bulk of E. coli cells, leaving surviving persisters

(Figure 7). Strains deleted in one of the five SOS-TA loci were examined for time-

dependent killing by ciprofloxacin, and one of them, ΔtisAB (GenBank accession

number NC_000913), had a sharply decreased level of persisters (Figure 7A). This

suggests that the majority of persisters, ≥90%, were formed in response to ciprofloxacin

treatment, and their production is dependent on tisAB. Introduction of tisAB in single

copy into the lambda attachment site of the ∆tisAB strain complemented the low

persister phenotype of the knockout strain (Figure 7B). Persister levels observed in

time-dependent killing experiments with ampicillin or streptomycin that do not cause

DNA damage were unchanged in the ΔtisAB strain (not shown). Ampicillin has been

reported to induce the SOS response [39], but apparently the level of induction is

insufficient to influence TisB-dependent persister formation.

IstR-1 is an antisense RNA antitoxin that is expressed constitutively from its own,

LexA-independent promoter and controls the production of the TisB toxin [28]. IstR-2 is

a longer small RNA transcript that is LexA-controlled and contains the entire IstR1 RNA

sequence. IstR-2, however, has been suggested not to be involved in the control of TisB

57

production [40]. tisA is an untranslated open reading frame that contains the antisense

RNA binding site as well as the ribosome binding site for tisB [32]. A schematic of the

tisAB/IstR locus based on [40] is shown in Figure 8.

A strain deleted in istR-1 caused a marked, 10- to 100-fold increase in the level of

persisters (Figure 7A). This is consistent with increased levels of TisB leading to

persister formation. This result is also in apparent contradiction to a published study

showing that ectopic expression of tisB kills cells [41]. It seems likely that the high levels

of expression from the multicopy plasmid used in the above-cited study were

responsible for cell death. Importantly, the minimal inhibitory concentration (MIC) to

ciprofloxacin of tisAB and istR-1 knockouts was the same as in the wild type, showing

that these genes do not affect resistance to this antibiotic, but rather control drug

tolerance by modulating persister production. To test whether IstR-2 was also involved

in tisB regulation in persisters, we produced a knockout of the istR-2 promoter region

and tested it for ciprofloxacin-induced persister formation. Unexpectedly, the ∆PistR-2

strain had reduced persister levels similar to the tisAB knockout (Figure S1). It seems

likely that the istR-2 promoter region contains a binding region of a positive regulator

that is essential for tisB expression.

Using a plasmid-borne promoter-gfp fusion we measured induction of tisAB in

response to ciprofloxacin, and compared this to the expression of other SOS-TA genes

(Figure 9). The tisAB promoter was the most active after 6 hours of exposure to

ciprofloxacin and showed a 1000- fold induction, followed by the symE promoter, which

showed a 100-fold induction. tisAB promoter activity was even higher than that of the

sulA promoter, a standard readout of the SOS response. The dinJ/yafQ promoter was

58

not significantly activated by ciprofloxacin. This is in agreement with a previous report

showing that despite the presence of a putative LexA binding box, the dinJ/yafQ locus

may not be under control of the SOS response [29]. The results of the induction

experiment are consistent with the prominent role of TisB in persister formation in

response to ciprofloxacin.

A common feature of inducible responses is an increase in tolerance upon repeated

exposure to a noxious factor. In a separate study [38] we show that ciprofloxacin

induces persister formation in a typical step-wise induction experiment (exposure to a

low dose of an antibiotic followed by a higher dose). Here, we wanted to test whether

tisB was responsible for this phenotype. Wild type E. coli cells were pre-exposed to low

levels of ciprofloxacin (0.1 µg/ml, 5 x MIC) followed by a higher dose (1 µg/ml) of the

same antibiotic (Figure 10). In a control experiment, the population was exposed to the

high dose from the beginning. Step-wise exposure resulted in a 10- to 100-fold higher

persister level as compared to a population that was exposed to a high dose of the

antibiotic. This pattern is typical of an adaptive response. In contrast to the wild type,

pretreatment with a low dose of antibiotic did not induce a higher level of surviving

persisters in the ∆tisAB mutant. This shows that this adaptive response to ciprofloxacin

depends on tisAB.

Next, we tested the ability of persisters formed in response to tisB expression to

tolerate multiple antibiotics. For this purpose, tisB was cloned into a low copy number

vector pZS*24 with an IPTG inducible promoter and the toxin gene was expressed in

exponentially growing cells. Growth leveled off approximately 1 hour after the addition of

IPTG (data not shown). Cells overproducing TisB were exposed to antibiotics from four

59

unrelated classes, and survival was measured after a 3 hour incubation (Figure 11). As

expected of non-growing cells, the strain overproducing TisB was completely tolerant to

ampicillin, a cell wall synthesis inhibitor that only kills growing cells. Interestingly, cells

overproducing TisB were completely tolerant to ciprofloxacin as well. In contrast to

ampicillin, ciprofloxacin is very effective in killing regular non-growing cells, even those

without ongoing replication [4,9,42]. It appears that TisB produces persisters highly

tolerant to this DNA damaging agent. TisB producing cells also survived exposure to

streptomycin, a protein synthesis inhibitor, 100-fold better than the control strain. This

shows that TisB-dependent persisters exhibit multidrug tolerance. Antibiotics tested in

these experiments act against defined targets. Decreased activity of the target functions

in persisters would lead to drug tolerance. Persisters formed by TisB overproduction

were susceptible to colistin, a polypeptide antibiotic permeabilizing the outer membrane

[43]. This is expected, since an intact outer membrane is essential for cell survival.

Further, TisB overproduction protected a ∆recA mutant against bactericidal antibiotics

from three different classes (Figure 11B).

The SOS response is initiated when RecA senses damaged DNA and activates

cleavage of the global repressor LexA. It was important to establish whether TisB-

dependent formation of persisters was controlled by this well studied SOS response

pathway. The persister level of a ∆recA strain treated with ciprofloxacin was lower as

compared to the wild type, and similar to that of a ∆recA ∆tisB double mutant (Figure

12A).

E. coli can also constitutively express SOS controlled genes if the LexA repressor is

deleted. The level of surviving persisters in E. coli ∆recA lexA300(Def) treated with

60

ciprofloxacin was dramatically increased as compared to the wild type (Figure 12A).

Importantly, the MIC of the E. coli ∆recA lexA300(Def) to ciprofloxacin is 0.002, which is

8-fold lower than in the wild type. RecA is the main recombinase participating in DNA

repair, which explains the increased susceptibility of the mutant to fluoroquinolones that

cause double-strand breaks. This experiment clearly distinguishes between the

decreased resistance of the regular cells, and increased levels of drug-tolerant

persisters in the E. coli ∆recA lexA300(Def) population. Finally, we deleted the tisAB

locus in ∆recA lexA300(Def) and measured survival in response to ciprofloxacin (Figure

12A) and tobramycin (Figure 12B). Persister levels in the ∆tisAB ∆recA lexA300(Def)

triple mutant were drastically reduced as compared to the ∆recA lexA300(Def) strain

and were similar to that of the ∆recA single deletion after exposure to either antibiotic.

Taken together, these experiments show that the SOS response triggers

induction of TisB, causing formation of multidrug tolerant persisters (Figure 13).

Discussion

Previous research clearly indicated redundancy in persister formation

mechanisms, suggesting a unique design of this cell surviving function [2]. Indeed, all

other complex systems of bacteria are made of components usually linked into a single

linear pathway, and a screen of a knockout library readily identifies the genes. By

contrast, a screen of a knockout library did not result in discovery of strains lacking

persisters and the only genes that were identified as contributing to the persister

phenotype were global regulators (hnr, dksA, fis, hns) and genes involved in nucleotide

61

metabolism (apaH, yigB) [25]. The screen was done in stationary phase and the library

did not contain a tisAB knockout strain. TisB-dependent persister formation is observed

under conditions of maximal expression of the SOS response, which is in exponentially

growing cells. Consistent with this, we did not observe a phenotype for the ∆tisAB strain

in stationary phase (not shown), suggesting that under these conditions persisters form

through other mechanisms. The screen [25] did identify the upstream elements of tisB

induction, recA and recB. These knockout strains have increased susceptibility to

fluoroquinolones and were therefore initially not considered as valid candidates for

persister genes.

Another persister component, the glpR regulon was identified in a selection of an

expression library of E. coli for increased drug tolerance [27]. Perhaps this redundancy

of mechanisms evolved in response to antibiotics in the natural environment. If

persisters are specialized survivors, then having multiple mechanisms of formation will

ensure that no single compound will lead to their elimination.

This underscores the challenges in finding approaches to persister eradication.

Redundancy of mechanisms is also challenging for identifying these mechanisms.

Given that persisters are dormant, the search narrows for determinants that can

reversibly block cellular functions. TA loci contain attractive candidates for persister

genes. HipA encoded by the hipBA locus was the first candidate persister gene

identified by a targeted selection for high-persister mutants [6,7]. The hipA7 allele

carries a gain-of-function mutation that causes an increase in persister formation [4,8].

Our recent studies showed that HipA is a protein kinase that phosphorylates EF-Tu,

rendering it non-functional [18,19]. Inhibition of protein synthesis leads to multidrug

62

tolerance and presents a compelling scenario for persister formation. However, deletion

of hipBA has no phenotype ([25]; an earlier report of a phenotype [9] was due to

deleting a flanking region). Expression of other toxins (RelE ; MazF [9,44]) similarly

leads to multidrug tolerance, but deletions do not have a phenotype. Extreme

redundancy of TA genes would explain the lack of a phenotype, and therefore it seemed

useful to search for conditions where a particular toxin would be expressed in a wild

type strain, and then examine a possible link to persister formation.

Several TA genes are expressed under conditions of the SOS response, which is

induced by fluoroquinolone antibiotics. Examination of deletion strains showed that the

level of persisters dropped dramatically in a ∆tisAB mutant and increased equally in a

∆IstR-1 mutant overproducing TisB. During steady-state growth, a fraction of cells

induces the SOS response stochastically, which could have resulted in production of

TisB-dependent persisters [45]. However, the level of persisters surviving treatment with

streptomycin or ampicillin was not affected by the absence of tisB. This suggests that

spontaneous SOS expression is insufficient to produce cells expressing enough TisB to

cause dormancy. This is consistent with our findings that a strain unable to induce the

SOS response exhibits reduced persistence in response to ciprofloxacin, but not

ampicillin or streptomycin [38].

SOS caused by endogenous DNA damage during normal growth has been shown to

induce a “viable but not culturable” state in a subpopulation of cells [45]. It is possible

that this is the consequence of induction of SOS TA modules as well.

Ectopic overexpression of tisB sharply increased the level of persisters. Drug

tolerance following artificial overexpression of a protein, however, may not be a good

63

indicator of a bona fide persister gene. Ectopic overproduction of misfolded toxic

proteins causing stasis produces an artificial state of drug tolerance in E. coli [44]. At the

same time, overexpression experiments are necessary – if induction of a gene does not

lead to increase in drug tolerance, it can be safely eliminated as a candidate. Drop in

persisters in a deletion strain and increase upon overexpression gives reasonable

confidence in functionality of a persister gene. The dependence of TisB-induced

persisters on a particular regulatory pathway, the SOS response, further strengthens

the case for TisB as a specialized persister protein.

The long and unsuccessful search for a mechanism of persister formation has lead

to the provocative hypothesis of dormant cells being formed by random fluctuations in

any protein whose overproduction produces a toxic effect [44]. We previously showed

that persisters are not formed in an early-exponential culture of E. coli, suggesting the

presence of specific persister proteins, rather than random noise in expression of non-

specific genes [4]. However, this debate could only be settled with the identification of a

persister protein. Our finding of an SOS-dependent induction of TisB resulting in

multidrug tolerance suggests that there is in fact a specific mechanism of persister

formation.

The role of TisB in persister formation is unexpected based on what we know about

this type of proteins. TisB is a small, 29 amino acid hydrophobic peptide that binds to

the membrane and disrupts the proton motive force (pmf), which leads to a drop in ATP

levels [41]. Bacteria, plants and animals all produce antimicrobial membrane-acting

peptides [46-48]. Toxins of many TA loci found on plasmids belong to this type as well,

and represent the plasmid maintenance mechanism. If a daughter cell does not inherit a

64

plasmid, the concentration of a labile antitoxin decreases, and the toxin such as the

membrane-acting hok kills the cell [49]. High-level artificial overexpression of tisB also

causes cell death [41]. It is remarkable from this perspective that the membrane-acting

TisB under conditions of natural expression has the exact opposite effect of protecting

the cell from antibiotics. Cells expressing tisB stop growing, and the drop in pmf and

ATP levels will shut down the targets of bactericidal antibiotics. Ciprofloxacin kills cells

primarily by converting its target proteins, DNA topoisomerases, into DNA

endonucleases [14,50]. A drop in ATP will then prevent topoisomerases from damaging

the DNA. β-lactams such as ampicillin kill by activating the autolysins [15,51], and this

requires active peptidoglycan synthesis by the target penicillin-binding proteins.

Peptidoglycan synthesis ceases in non-growing cells. Similarly, the aminoglycoside

streptomycin requires an active ribosome for its killing action. Aminoglycosides kill

primarily by interrupting translation, which creates toxic, misfolded peptides [13,52].

Antibiotics also induce the formation of reactive oxygen species, which contributes to

killing [16], and this requires an active target as well. By creating a dormant state, TisB

will cause a shutdown of antibiotic targets and multidrug tolerance. Fluoroquinolones

such as ciprofloxacin are widely-used broad spectrum antibiotics, and their ability to

induce multidrug tolerant cells is unexpected and a cause of considerable concern.

Induction of persister formation by fluoroquinolones may contribute to the

ineffectiveness of antibiotics in eradicating biofilm infections. Indeed, pre-exposure with

a low dose of ciprofloxacin drastically increases tolerance to subsequent exposure with

a high dose [38].

Induction of persisters by the SOS-induced TisB toxin links together two seemingly

65

opposite strategies of survival – active repair, and entry into a dormant state. It seems

that in the presence of DNA damaging factors, the optimal strategy is to both induce

repair and increase the number of dormant cells, which will survive when everything

else fails. Indeed, a progressive increase in the concentration of fluoroquinolones

rapidly kills regular cells but has little effect on the survival of persisters ([53]; this

study). This means that it is the dormant persisters rather than regular cells with

induced repair that will ultimately survive the DNA damaging antibiotic.

Apart from describing a key element of persister formation, this study also provides a

precedent for a physiological function for a chromosomal TA gene pair. While the role of

TAs in plasmid maintenance is well-established, the function of chromosomal TAs

remains largely unknown. In a recent study, Van Melderen and co-authors produced a

knockout of E. coli lacking 5 toxins, including the well-studied RelE and MazF (mRNA

endonucleases) [21]. The deletion strain had no apparent phenotype and showed

normal growth, susceptibility to antibiotics and stringent response. In Erwinia

chrysanthemi, the chromosomal ccdAB TA module prevented postsegregational killing

of cells that lost an F plasmid, which contains a homologous ccdAB locus [54].

Prevention of postsegregational killing may be a function of some TA genes but would

not explain the presence of >80 TAs in the chromosome of Mycobacterium tuberculosis

[55,56], for example, which is not known to harbor plasmids. Induction of TA genes

under specific conditions such as described in this study may shed some light on their

function.

This study opens an intriguing possibility of a wider link between other stress

responses and persister formation. Pathogens are exposed to many stress factors in

66

the host environment apart from DNA damaging agents, including oxidants, high

temperature, low pH, membrane-acting agents. It is possible that all stress responses

induce the formation of a small but resilient subpopulation of surviving persisters.

Materials and Methods

Media and growth conditions

Experiments were conducted in 0.1 M HEPES-buffered (pH 7.2) Mueller Hinton

Broth (MHB) enriched with 10 mg/L MgSO4 and 20 mg/L CaCl2 according to NCCLS

guidelines for susceptibility testing. Killing experiments were conducted by diluting

overnight cultures 1:100 in 3 ml fresh medium in culture tubes, growing to approximately

2x10^8 CFU/ml and challenging with 0.1 or 1 µg/ml ciprofloxacin. For CFU counts, cells

were plated on LB agar plates containing 20 mM MgSO4 to minimize carryover effects

of ciprofloxacin.

Strain construction

Strains MG1655 ∆tisAB::FRT, ∆IstR-1::FRT and ∆PistR-2::cat, are precise

deletions constructed using the method by Datsenko et al. [57] and cured of their

chloramphenicol resistance cassette with pCP20 where applicable.

P1 transduction was used to move the delta recA::Kan, delta sulA::Kan alleles

(from the MORI KEIO collection[24]) and lexA300(Def) (kindly provided by G. Walker)

into the MG1655 background.

Strain MG1655 pZS*24tisB was constructed by cloning the tisB ORF into the

Kpn1/Cla1 sites of pZS*24 [58] using primers tisBfwKpn1 (5’-

GTAGTAGGTACCATGAACCTGGTGGATATCGCCA-3’) and tisBrevCla1 (5’

67

GTAGTAATCGATACTTCAGGTATTTCAGAACAGCAT-3’).

MG1655 pUA66PtisB-gfp was constructed by cloning the tisAB promoter region

into the XhoI/BamHI sites of vector pUA66gfp using primers PromTisFwXho1 (5’-

GTAGTACTCGAGGCCGGAGCGAGGTTTCGT-3’) and PromTisRevBamH1 (5’-

GTAGTAGGATCCAACACAGTGTGCTCACGCGG-3’). The other promoter-gfp fusions

were taken from a commercial library [59].

For complementation experiments, the tisAB locus was cloned into the CRIM

vector pCAH63 using primers RegiontisBAfwKpn1 (5’-

GTCGTCGGTACCTTGAGTATCGATCACAGTTTGCGT-3’) and RegiontisBArevKpn1

(5’-GTCGTCGGTACCCCTTTGGTGCGACTTGAATCTG-3’) and inserted into the

lambda attachment site of strain MG1655 ∆tisAB::FRT as described by Haldimann et al.

[60].

Promoter activity assay

Cells carrying pUA66-promoter-gfp fusions were grown in MHB to exponential phase as

stated before and exposed to ciprofloxacin. At each time point, aliquots were removed,

washed 2 x in 1% NaCl and transferred to a 96 well plate. GFP fluorescence was

measured with Ex/Em 485/515 on a Gemini XS spectrophotometer (Molecular Devices).

Induction was normalized to background (pUA66gfp), CFU/ml and initial fluorescence.

68

TisB overexpression and persistence

MG1655 carrying either pZS*24 or pZS*tisB was grown to exponential phase in 12 ml

MHB in 125 ml baffled flasks containing 20 µg/ml kanamycin. TisB expression was

induced for 2 hours in mid-exponential phase by addition of 500 µM IPTG. The culture

was then split and exposed to either ciprofloxacin (1 µg/ml), ampicillin (50 µg/ml),

streptomycin (25 µg/ml) or colistin methane sulfonate (10 µg/ml) for 3 hours.

69

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76

Figure 7. Survival of the tisAB/istR mutants after ciprofloxacin exposure and

complementation of the phenotype

(A) Knockout strains of the toxin locus tisAB and its antitoxin istR-1 were exposed to 1

µg/ml ciprofloxacin in exponential growth phase and survival determined by spot plating

for colony forming units. The graph is a

representative of at least 5 independent experiments with similar results, error bars

indicate the standard error. (B) MG1655 ∆tisAB carrying the tisAB region as a single

copy insertion in the lambda attachment site was treated as described in (A).

77

Figure 8. Schematic of the tisAB/IstR locus.

Only the LexA-controlled toxin tisB is translated in vivo, tisA contains the binding site for

the constitutively expressed antitoxin RNA IstR-1 [36]. The IstR-2 RNA is under LexA

control and contains the entire IstR-1 RNA. Its role in tisAB regulation is currently

unclear.

78

Figure 9. Induction of LexA-controlled promoters by ciprofloxacin.

Cells carrying plasmid borne promoter-gfp fusions were exposed to 0.1 µg/ml

ciprofloxacin in exponential phase. Fold induction is GFP fluorescence after 3 hours

(open bars) and 6 hours (green bars) of exposure normalized to initial fluorescence.

This graph is a representative of 3 independent experiments with similar results, error

bars indicate the standard error.

79

Figure 10. Adaptive ciprofloxacin tolerance in E. coli

Wild type MG1655 and its ∆tisAB derivative were grown to exponential phase and

exposed to 0.1 µg/ml ciprofloxacin for 3 hours, after which 1 µg/ml was added

(ciprofloxacin MIC is 0.016 µg/ml). As a control, a parallel culture was immediately

exposed to 1 µg/ml. Viable cell number was determined by serial dilution and plating for

colony forming units (CFU/ml). The data points are averages of 3 independent

experiments, error bars indicate the standard error.

80

Figure 11. TisB overproduction and antibiotic tolerance

tisB was overexpressed in (A) MG1655 and (B) MG1655 ∆recA in exponential phase

from a low copy number vector and exposed to ciprofloxacin (1 µg/ml), ampicillin (50

µg/ml), streptomycin (25 µg/ml) or colistin (10 µg/ml). Survival after 3 hours was

compared to a control strain carrying vector without tisB. The graph shows averages of

three independent experiments, error bars are standard error.

81

Figure 12. TisB-dependent persister formation in SOS response mutants

E. coli MG1655 and its derivatives ∆recA, ∆recA ∆tisAB, ∆recA lexA300(Def) and ∆recA

lexA300(Def)∆tisAB were grown to exponential phase and exposed to (A) ciprofloxacin

at 1 µg/ml or (B) tobramycin at 20 µg/ml. Data are averages of at least 3 independent

experiments, error bars indicate the standard error.

82

Figure 13. Model of ciprofloxacin-induced persister formation.

Ciprofloxacin induces the SOS response, which upregulates DNA repair functions. In a

subpopulation of cells the SOS response also induces the TisB toxin to a high level,

which causes a decrease in proton motive force and ATP level, leading to multidrug

tolerance.

83

Supporting Information

Figure S1 Persister formation in a strain with an istR-2 promoter deletion. Cells were

grown to exponential phase and exposed to 0.1 µg/ml ciprofloxacin for 3 hours to induce

TisB, followed by a higher dose (1 µg/ml) for another 3 hours. Cell survival was

assessed by spot-plating for colony forming units.

84

CHAPTER 3 TISB MECHANISM OF ACTION

Tobias Dörr, Ron Ortenberg and Kim Lewis

Manuscript in preparation

Introduction

TisB is a largely hydrophobic, alpha helical peptide with 29 amino acids

(MSLVDIAILILKLIVAALQLLDAVLKYLK). It is well conserved throughout

Enterobacteriaceae and is induced as part of the LexA – controlled SOS DNA damage

response (Fernandez De Henestrosa et al., 2000, Vogel et al., 2004, Unoson &

Wagner, 2008). We have previously shown that tisB is the mediator of SOS-induced

adaptive tolerance to ciprofloxacin in Escherichia coli and that ectopic overexpression

from a low copy number vector leds to cessation of growth and subsequently to

multidrug tolerance (Dörr et al., 2010).

TisB was associated with the inner membrane and led to a drop in intracellular

ATP concentrations and subsequently cell death upon overproduction from a high copy

number vector (Unoson & Wagner, 2008, Fozo et al., 2008a). Transcriptome analysis

after high-level overexpression revealed an cellular stress response that is expected for

a membrane acting agent, importantly an upregulation of the envelope stress response

(cpx regulon) and also the superoxide stress response (soxS) (Fozo et al., 2008a).

Thus, it seemed possible that TisB disrupts the inner membrane in a detergent-

like fashion. Since low-level expression, however, makes cells dormant rather than

killing them, this seemed unlikely. Rather, association with the inner membrane and

85

drop in ATP concentrations inducing growth arrest suggests an interference of TisB with

the proton motive force or the protein components essential for its establishment.

Results

TisB from pathogenic E. coli 0157:H7 confers multidrug tolerance

The laboratory strain MG1655 has a Ser->Asn change in tisB in the second

amino acid residue. To test whether this affected toxicity or multidrug tolerance, we

amplified tisB from the chromosome of 0157:H7 Sakai and cloned it into a low copy

number vector. Toxicity and persistence after overexpression of tisB 0157 were the same

as tisB from the wild type strain MG1655 (Fig. 14).

The C-terminus is important for TisB – mediated persister formation

TisB has been predicted to insert into the inner membrane with its C-terminus

while the two N-terminal amino acid residues extend into the cytoplasm (Unoson &

Wagner, 2008). While constructing N-terminal and C-terminal translational fusions with

mCherry, we noticed that the TisBmCherry strain had a low persister phenotype while

the mCherryTisB strain had wild type persister levels (Fig. 15). This strongly suggested

that the C-terminus is important for toxicity and lends support to the prediction that the

C-terminus interacts with the inner membrane.

TisB toxicity is reduced when cells are capable of substrate-level

phosphorylation

86

When TisB is ectopically overproduced in medium containing glucose, cell

proliferation stops after around 1 hour and cells remain in a non-growing state for at

least 6 h, after which growth resumes (Fig. 16). This resumption could reflect a)

decrease in the concentration of the inducer (IPTG), b) the rise and overgrowth of TisB

resistant mutants or c) reprogramming of metabolism towards the ability to grow in the

presence of TisB/expression of a resuscitation factor.

Varying the initial IPTG concentration did not affect resuscitation kinetics (not

shown) and we therefore conclude that the inducer is stable enough to cause tisB

expression throughout the experiment. Rediluting the overgrown pZS*34tisB (+IPTG)

cultures into fresh medium with IPTG resulted in the same kinetics of growth arrest as

the parental cultures (not shown), demonstrating that these were not resistant mutants.

Thus, we concluded that resuscitation from TisB overproduction reflects the expression

of a resuscitation factor or reprogramming metabolism.

To test this further, we overexpressed tisB anaerobically in the presence of

different carbon sources. Since TisB is likely to affect oxidative phosphorylation through

its interaction with the inner membrane, allowing cells to use fermentation for ATP

generation could make it possible to bypass TisB toxicity. TisB was therefore

oveproduced in a minimal medium containing succinate (which E. coli cannot ferment),

or in a medium containing the fermentable sugar glucose. Nitrate was added as an

electron acceptor to one culture containing glucose and to the other containing

succinate. Thus, cells could either use only fermentation (glucose), fermentation +

electron transport chain (glucose/nitrate) or only the electron transport chain (succinate)

for ATP generation. Toxicity was reduced in the medium containing only glucose and

87

even more reduced in the medium containing glucose and nitrate. Toxicity was most

severe in the medium containing only succinate (Fig. 17). These results suggested that

oxidative phosphorylation is affected in cells overexpressing tisB and that cells can

partially bypass this toxicity by generating ATP through substrate level phosphorylation.

It also suggested that ongoing oxidative phosphorylation was not essential for TisB

toxicity since the peptide was still toxic in the medium containing only glucose.

We therefore wanted to confirm whether a functional electron transport chain was

necessary for TisB toxicity. Ectopic overexpression of tisB in a strain deleted in atpA

(coding for ATP synthase) or in the electron transport chain - less ∆hemA strain still

caused toxicity (Fig. 16), confirming that ongoing oxidative phosphorylation was not

necessary for TisB action. Therefore it seemed possible that TisB acts by disturbing the

proton motive force, likely by being an uncoupler, rather than by specific inhibition of the

electron transport chain.

To test whether uncoupling would be able to cause multidrug tolerance similar to

that associated with TisB overproduction, we treated a growing culture of E. coli with

increasing concentrations of the protonophore Carbonyl cyanide m-

chlorophenylhydrazone (CCCP) and subsequently with ciprofloxacin. Cccp abolishes

the proton motive force and is used as a standard treatment for membrane

depolarization. Only the two highest doses used (100 and 500 µM) led to a total growth

arrest (Fig 18A, red line) and only the highest concentration protected cells appreciably

against ciprofloxacin (Fig. 18A, blue line). Thus, uncoupling can indeed induce

ciprofloxacin – tolerance. We then used the highest concentration of cccp to treat cells

followed by treatment with different classes of antibiotics. At a concentration of 500 µM,

88

cccp protected E. coli against ampicillin, streptomycin and ciprofloxacin, in all cases to a

higher degree than overexpression of tisB (Fig 18B). Thus, uncoupling of oxidative

phosphorylation can induce a persister-like state in E. coli.

TisB overproduction decreases proton motive force

Since TisB was associated with the inner membrane and decreased intracellular

ATP concentrations (Unoson & Wagner, 2008), we wanted to learn whether TisB

abolished the proton motive force. We used the cyanine dye DiOC(2)3, which

accumulates in the membrane in the presence of a proton motive force, shifting its

emission spectrum from green towards red. Thus, all cells fluoresce green while only

those with a proton motive force fluoresce red. Dividing red by green fluorescence

values gives a good approximation of the presence of a proton motive force

independent of cell size.

The results are shown in Fig. 19. After 30 minutes of induction the proton motive

force decreased and within 90 minutes reached a similar level to that of the control

population depolarized with cccp. Thus, TisB production leads to membrane

depolarization.

Cells do not acquire genetic resistance against tisB overexpression

In order to find possible interaction partners or resuscitation factors of TisB we

attempted to isolate mutants resistant to tisB overexpression. Briefly, E. coli MG1655

was mutagenized with ethyle methane sulfonate (EMS), which led to 1000-fold increase

in mutation frequency to rifampicin (not shown). The mutagenized culture was

89

transformed with pZS*34tisB and plated on MOPS minimal medium containing 0.2 %

succinate as the sole carbon source as well as chloramphenicol and IPTG. Colony

forming units/ml were assessed before and after plating and revealed a mutation

frequency for tisB resistance of ∼2 x 10-5. The transformation was performed 5 times

with cultures from 4 different rounds of mutagenesis. Plasmids were extracted from

mutants and retransformed into wild type to re-assess toxicity. Around 90 % of mutant

plasmids had lost tisB toxicity and were thus discarded. The remaining mutants were

subjected to whole genome sequencing and single nucleotide polymorphism (SNP)

analysis. The first three strains from two rounds of mutagenesis each had 30-40 SNPs

compared to the reference strain MG1655. Only 4 of these SNPs overlapped between

the three strains, lacI (lac operon repressor), ylbE (NO – inducible gene of unknown

function), mdfA (major facilitator family drug efflux pump) and pgpB (coding for

phosphatidylglycerophosphate phosphatase). The genes ylbE, mdfA and pgpB had

exactly the same mutations in all the three strains, indicating that these were

background mutations in the parental strain. All three strains, however, had different

mutations in lacI, which likely decreased expression of tisB from the IPTG-inducible

system used to isolate mutants.

Thus, the whole process was repeated in a strain deleted in lacI. Ten mutants

each from two different rounds of mutagenesis were picked and tested for plasmid

mutants. All mutant plasmids had lost tisB toxicity.

Further, we attempted to isolate mutants resistant to tisB overexpression after

transposon mutagenesis. This yielded a negative result as well; 32 mutants from two

90

rounds of transposon mutagenesis (after transforming approximately 30.000 mutants

each time) that scored as tisB resistant had mutated plasmids.

TisB forms a stable pore in vitro

Since the results described above indicated that TisB may act as an uncoupler,

we measured the effect of synthesized TisB peptide on membrane conductance in an in

vitro artificial black membrane setup. In short, current was applied to two chambers

separated by a lipid bilayer and conductance measured as electric current passing from

one chamber to the other. The lipid bilayer acts as an isolator and thus, before the

experiment, conductance across the bilayer is zero. When synthesized TisB was

present in either chamber, conductance increased in a step-wise fashion after a short

delay of 1 s, which is indicative of pore formation in the membrane (Fig. 20 A).

Moreover, reversion of voltage did not affect current, which indicates the pore is stable

once it has formed (Fig. 20 B). Current/voltage plots further showed that the TisB pore

was selective for chloride anions (Fig. 20 C). It has to be noted, however, that other

anions were not tested.

Discussion

TisB belongs to a group of small, toxic, membrane-associated peptides (STMPs)

that are part of type1 toxin/antitoxin systems and are found in enterobacterial plasmids

and chromosomes (Gerdes et al., 1986a, Fozo et al., 2008b, Pedersen & Gerdes, 1999,

Fozo et al., 2010). Early results with plasmid-encoded hok toxins implicated these

peptides in plasmid maintenance and programmed cell death (Gerdes et al., 1986b)

while the physiological role of chromosomal STMPs remained unknown. We have

91

previously shown that expression of tisB in vivo causes multidrug tolerance after

exposure to a DNA damaging antibiotic (Dörr et al., 2010) and in this study investigated

the mode of action of this peptide.

Since TisB had been shown to associate with the inner membrane and to cause

a drop in intracellular ATP concentrations, it seemed likely that the peptide interfered

with electron-transport chain - mediated ATP generation. E. coli maintains a proton

motive force (PMF) mainly by proton extrusion through an electron transport chain

during oxidative phosphorylation and generates ATP by using the energy from PMF -

driven proton-influx through an ATP synthase (Maloney et al., 1974, Haddock & Jones,

1977). Interference with this process would halt ATP production, at least temporarily,

and thus quickly lead to the observed depletion of intracellular ATP. Indeed, when

ectopically overproduced, TisB was most toxic in minimal medium containing succinate,

which absolutely requires oxidative phosphorylation to generate energy. When supplied

with a fermentable sugar source such as glucose, however, toxicity was reduced. Thus,

TisB interfered with electron transport chain - mediated ATP generation.

TisB was predicted to insert into the inner membrane with its C-terminus, while

two N-terminal residues extend into the cytoplasm. Two observations we made were in

agreement with these predictions: adding an N-terminal fluorescent protein tag or an

amino acid substitution in the second residue (which exists in the laboratory strain of E.

coli, MG1655) did not affect toxicity while adding a C-terminal fluorescent protein

abolished TisB-dependent persister formation.

These results led us to hypothesize that TisB may function as an uncoupler. A

chemical uncoupler, cccp, could induce multidrug tolerance similar to what was

92

observed with TisB overproduction, showing that dissipation of the proton motive force

could lead to persister formation. Ectopic overexpression of tisB led to a drop in proton

motive force similar to that induced by cccp. Further, TisB was toxic in a ∆hemA strain

lacking cytochromes as well as a strain deleted for ATP synthase, suggesting that TisB

toxicity was not mediated by a specific inhibitory interaction of TisB with parts of the

electron transport chain. Moreover, TisB toxicity was almost completely alleviated under

anaerobic conditions in a medium containing nitrate as an electron acceptor and

glucose as a carbon source. Providing the ability to use the electron transport chain

while also relying on substrate level phosphorylation for ATP generation would make it

possible to maintain a (weak) proton motive force even in the presence of a mild

uncoupler.

Additionally, we failed to isolate mutants resistant to TisB overproduction.

Resistant mutants were expected to include an upregulation of a resuscitation factor

(e.g. a protease) or a mutation in a target, precluding TisB binding. The only mutants we

could isolate were in lacI, which likely produced lac repressor alleles that were unable to

bind IPTG and therefore repressed tisB expression from the vector. Even though we

cannot exclude the possibility that resistant mutants were not scored in the specific

conditions we used for mutant selection, a negative result strongly suggests that there is

no specific mutable target for TisB action. Upregulation of a resuscitation factor through

mutation would only be expected to occur if this factor contained a repressor binding

site or other regulatory sites which could be mutated to increase expression, which for

example would be the case for the LexA - regulated Lon protease.

93

Finally, we show that TisB peptide forms a stable pore in an in vitro black

membrane system. When voltage was applied to a two-chamber system, which was

separated by an artificial membrane and contained synthesized TisB peptide, current

could be measured approximately 1 second after application of voltage. This delay

indicates that TisB did not form a pore simply by accumulation in the membrane but

rather needed voltage as a signal. This could reflect the mechanism of pore formation in

vivo, where transmembrane voltage in the form of a proton motive force is around -200

mV during aerobic, exponential growth (Kashket, 1985).

The TisB pore was further shown to be selective for anions. Thus, the pore does

not work as a protonophore but could rather make the membrane permeable for OH-

ions, which would dissipate the proton motive force.

Taken together, we propose the following model for TisB mode of action.

Following DNA damage, TisB molecules accumulate in the inner membrane. After

crossing a certain concentration threshold, several TisB molecules establish a pore in

the inner membrane, a process for which the proton motive force is essential. The pore

decreases the proton motive force probably by increasing membrane permeability for

hydroxyl anions.

Materials and Methods

Bacterial strains and plasmids

MG1655∆hemA::Kan is a precise deletion of the hemA open reading frame.

∆atpA::Kan was constructed using P1 phage transduction from the corresponding strain

in the KEIO collection (Baba et al., 2006). pZS*34tisB0157 was constructed by PCR

94

amplifying the tisB open reading frame from the chromosome of E. coli 0157:H7 Sakai

and cloning it into the Kpn1/Cla1 sites of chl resistant, IPTG inducible, low copy number

vector pZS*34 (Lutz & Bujard, 1997).

Growth conditions

Cells were grown in 0.1 M HEPES – buffered Mueller Hinton Broth, or, where

mentioned, in MOPS minimal medium supplemented with 100 µg/ml uracil, 13.4 µM

H2PO4 and 0.2 % glucose, maltose or succinate.

The ∆hemA and ∆atpA strains were always grown in medium containing 0.2 % glucose.

TisB overproduction resistance screen

Cells were grown to mid-exponential phase (3 x 108 CFU/ml), washed twice and

resuspended in 0.1 M HEPES buffer (pH 7.2), after which 15 µL/ml culture of ethane

methyle sulfonate was added for 35 minutes. Cells were then washed twice,

resuspended in 1ml of fresh growth medium, grown overnight and diluted onto agar

plates to reach approximately 10,000 colonies/plate. These colonies were scraped off

the plate, diluted into fresh growth medium, grown for 1h and transformed by

electroporation with pZS*34tisB. Cells were then washed three times and plated on

MOPS minimal agar supplemented with 0.2 % succinate, 15 µg/ml chloramphenicol and

1 mM IPTG. Candidates for resistant mutants were picked, outgrown and transformed

with kanamycin resistant pZS*24tisB. Mutants that did not exhibit toxicity after TisB

overproduction from this vector were tested further by extracting their pZS*34tisB,

transforming it into wild type and testing for remaining toxicity.

Proton motive force measurements

95

MG1655 pZS*34tisB was grown to mid-exponential phase and 1mM IPTG

added. Membrane potential was assessed using the protocol of the Invitrogen Baclight

membrane potential kit. In short, cells expressing tisB were diluted 100-fold into pre-

warmed phosphate buffered saline (PBS), incubated with DiOC(2)3 for 30 minutes and

analyzed in a BD FACSAria. As depolarized control, 5 µM cccp was added to one

sample for 10 min. Membrane potential was estimated by dividing population mean red

fluorescence values by population mean green fluorescence values.

Black membrane experiments

All artificial lipid bilayer experiments were done in collaboration with Sergey M.

Bezrukov at the Laboratory of Physical and Structural Biology/NIH/Bethesda, Maryland

essentially as described by Kullman and Bezrukov (Kullman et al., 2002). In short,

synthesized TisB (provided by Wayne Anderson, Northwestern University, Chicago,

Illinois) was added to a two-chamber system filled with KCl and separated by an

artificial lipid bilayer. Voltage was applied and the current through the two chambers

measured.

References

Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K. A. Datsenko, M.

Tomita, B. L. Wanner & H. Mori, (2006) Construction of Escherichia coli K-12 in-

frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:

2006.0008.

Dörr, T., M. Vulic & K. Lewis, (2010) Ciprofloxacin causes persister formation by

inducing the TisB toxin in Escherichia coli. Plos Biol 8: e1000317.

96

Fernandez De Henestrosa, A. R., T. Ogi, S. Aoyagi, D. Chafin, J. J. Hayes, H. Ohmori &

R. Woodgate, (2000) Identification of additional genes belonging to the LexA

regulon in Escherichia coli. Mol Microbiol 35: 1560-1572.

Fozo, E., M. Kawano, F. Fontaine, Y. Kaya, K. Mendieta, K. Jones, A. Ocampo, K.

Rudd & G. Storz, (2008a) Repression of small toxic protein synthesis by the Sib

and OhsC small RNAs. Mol Microbiol 70: 1076-1093.

Fozo, E. M., M. R. Hemm & G. Storz, (2008b) Small toxic proteins and the antisense

RNAs that repress them. Microbiol Mol Biol Rev 72: 579-589, Table of Contents.

Fozo, E. M., K. S. Makarova, S. A. Shabalina, N. Yutin, E. V. Koonin & G. Storz, (2010)

Abundance of type I toxin-antitoxin systems in bacteria: searches for new

candidates and discovery of novel families. Nucleic Acids Res 38: 3743-3759.

Gerdes, K., F. W. Bech, S. T. Jorgensen, A. Lobner-Olesen, P. B. Rasmussen, T.

Atlung, L. Boe, O. Karlstrom, S. Molin & K. von Meyenburg, (1986a) Mechanism

of postsegregational killing by the hok gene product of the parB system of

plasmid R1 and its homology with the relF gene product of the E. coli relB

operon. Embo J 5: 2023-2029.

Gerdes, K., P. B. Rasmussen & S. Molin, (1986b) Unique type of plasmid maintenance

function: postsegregational killing of plasmid-free cells. Proceedings of the

National Academy of Sciences of the United States of America 83: 3116-3120.

Haddock, B. A. & C. W. Jones, (1977) Bacterial respiration. Bacteriol Rev 41: 47-99.

Kashket, E. R., (1985) The proton motive force in bacteria: a critical assessment of

methods. Annu Rev Microbiol 39: 219-242.

97

Kullman, L., M. Winterhalter & S. M. Bezrukov, (2002) Transport of maltodextrins

through maltoporin: a single-channel study. Biophys J 82: 803-812.

Lutz, R. & H. Bujard, (1997) Independent and tight regulation of transcriptional units in

Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements.

Nucleic Acids Res 25: 1203-1210.

Maloney, P. C., E. R. Kashket & T. H. Wilson, (1974) A protonmotive force drives ATP

synthesis in bacteria. Proceedings of the National Academy of Sciences of the

United States of America 71: 3896-3900.

Pedersen, K. & K. Gerdes, (1999) Multiple hok genes on the chromosome of

Escherichia coli. Mol Microbiol 32: 1090-1102.

Unoson, C. & E. Wagner, (2008) A small SOS-induced toxin is targeted against the

inner membrane in Escherichia coli. Mol Microbiol 70: 258-270.

Vogel, J., A. L, W. EG & A. S, (2004) The small RNA Istr inhibits synthesis of an SOS-

induced toxic peptide. Current Biology 14: 2271-2276.

98

Figure 14. Survival in antibiotics after overexpression of tisB from O157:H7 Sakai

E. coli MG1655 pZS*34tisB0157 was grown to mid-exponential phase and induced with

IPTG for 2 hours. Antibiotics were added to 0.1 µg/ml(ciprofloxacin), 50 µg/ml

(ampicillin) and 50 µg/ml (streptomycin) for three hours. Log percent survival is colony

forming units normalized to cell density before addition of antibiotics. The error bars

represent standard error of 3 independent experiments.

99

Figure 15. Survival of strains carrying translational TisB – mcherry fusions in

ciprofloxacin.

Cells were grown to mid-exponential phase and exposed to 0.1 µg/ml cipro for 3 hours,

followed by 3 hours of 1 µg/ml cipro. N1, N2 = N-terminal fusion strains, C1, C2 = C-

terminal fusion strains.

100

Figure 16. Overexpression of tisB in respiratory chain mutants. Cells were grown to mid-exponential growth phase in 24 well plates in Mueller Hinton

Broth with 0.2 % glucose and induced with 1 mM IPTG at time zero. OD600 was

followed at 25 minute intervals using an automated plate reader. Shown here is a

representative of three different experiments with similar results.

101

Figure 17. TisB overproduction under anaerobic conditions in different carbon sources. Cells were grown in an anaerobic chamber in MOPS minimal medium plus either

glucose, glucose + nitrate or succinate + nitrate. The y-axis is log increase in CFU/ml

(normalized to CFU/ml before addition of IPTG), error bars are standard error of three

independent experiments.

102

Figure 18. Effects of cccp on multidrug tolerance MG1655 was grown to mid-exponential phase and exposed to cccp at different

concentrations for 10 minutes before addition of antibiotic. A, blue line: Log percent

survival after three hours of exposure to 0.1µg/ml ciprofloxacin, red line: fold growth

inhibition relative to control (no cccp). B) fold protection (= survival of treated /untreated)

of cccp at 500 µM compared to overexpression of tisB. Strep = streptomycin (25 µg/ml),

amp = ampicillin (50 µg/ml), cipro = ciprofloxacin

103

Figure 19. Proton motive force after TisB overproduction Cells carrying pZS*34tisB were grown to mid-exponential phase and induced with IPTG.

At the indicated time points, an aliquot was removed in diluted 1:100 into prewarmed

PBS + DioC(2)3, stained for 30 minutes and analyzed by FACS (A-C are representative

scatter plots green versus red fluorescence). Proton motive force was estimated by

normalizing population mean red fluorescence to population mean green fluorescence

(D). Controls were the chemical uncoupler cccp, a protonophore, and the electron

transport chain – less ∆hemA strain, which has a naturally low proton motive force.

104

Figure 20. TisB-dependent conductance of a black membrane lipid bilayer. Synthesized TisB was added to a two-chamber system separated by an artificial lipid

bilayer (black membrane) and conductance recorded upon application of voltage.

The graphs show time dependent conductance after addition of TisB (A), voltage-

dependent current through the TisB pore upon voltage reversion (B), Voltage-

dependent current through the TisB pore with unequal concentrations of KCl in both

chambers (C).

105

DISCUSSION

The persister phenomenon is still puzzling researchers more than 60 years after

its discovery. Though more and more is known about the potential clinical importance of

persisters, the mechanism(s) of formation have remained largely unknown.

Based on several studies, persisters were suggested to be an entity of

stochastically-formed, non-growing, multidrug tolerant cells, formed by several possible

mechanisms resulting in the same phenotype (Balaban et al., 2004, Shah et al., 2006

Lewis, 2007). Several pieces of evidence, however, suggested otherwise. Cells

expressing an unstable GFP from a growth-rate-dependent promoter produced a dim

subpopulation enriched in persisters (Shah et al., 2006), which still exhibited a high

variance in fluorescence. This could be an artifact of detection or heterogeneity in

degradation of GFP after growth arrest, but could also indicate that this subpopulation

did not consist entirely of non-growing cells but rather a mixture of non-growing and

slowly growing cells. Further, Wiuff et al. showed that persisters were multidrug tolerant

by exposing a population to a bactericidal antibiotic, then washing and re-exposing the

bona fide persisters to a different antibiotic and concluding that further survival was not

much affected (Wiuff et al., 2005). In actuality, however, the persisters died by up to one

order of magnitude when exposed to the second dose of antibiotic. Since persisters in

this study were washed and resuspended in a fresh medium, these results are difficult

to interpret and could reflect resuscitation from dormancy. Another explanation is that

90 % of persisters are specific to the antibiotic they were exposed to.

106

Indeed, here we have shown that exposure of a population of growing E. coli

cells to a low dose of ciprofloxacin increases their tolerance level to a subsequent

exposure to a high dose of the same antibiotic, indicating that persistence can be an

inducible response to an antibiotic. This increase in tolerance was dependent on the

SOS DNA damage response. Removal of the cell’s ability to induce the SOS response

did not entirely eradicate survivors to ciprofloxacin. Therefore, a subfraction of persister

cells pre-exists in every population as a kind of “life-insurance” for the whole population

after a catastrophic event. This persister fraction, however, can be increased as a

response to DNA damage and likely other stresses as well.

These results add a new, deterministic component to our understanding of

persisters and induced persistence may play an important role in human infectious

disease. Fluoroquinolones are widely used, broad-spectrum antibiotics and induction of

a dormant state may aide pathogens in surviving high doses of antibiotics in the

absence of resistance. Further, pathogens can experience SOS induction after

exposure to the host immune system. Urinary tract infection (UTI) causing strains of E.

coli, for example, induce SOS and filament during infection (Justice et al., 2006). This

may induce persisters and consequently leave a survivor reservoir after treatment with

any antibiotic, which may contribute to the notorious persistence of these infections.

First clues towards the mechanism of persister formation came from

transcriptome analysis of persister cells, which showed that chromosomal toxin antitoxin

modules were upregulated, more specifically several mRNA interferases.

Overexpression of chromosomal toxins (and other toxic proteins) can emulate a

persister-state and cause multidrug tolerance ( Keren et al., 2004, Correia et al., 2006,

107

Vazquez-Laslop et al., 2006, Shah et al., 2006). TA modules are abundant on bacterial

chromosomes, which offered an explanation for the lack of strong phenotypes for single

knockouts. Here, we found that a chromosomal toxin TisB is the effector of induced

persistence. Overexpression of tisB led to growth arrest and multidrug tolerance and,

more importantly, a knockout led to a decrease in persistence. TisB belongs to a group

of small, hydrophobic membrane peptides with RNA antitoxins. Other members of this

group, such as the hok and ibs toxins had been shown to kill and lyse cells either when

produced as part of a plasmid maintenance system (hok) (Faridani et al., 2006) or upon

overexpression (ibs) (Fozo et al., 2008). It was therefore surprising that tisB expression

actually protected cells. How does a small detergent-like peptide such as TisB induce

growth arrest? TisB overproduction was transiently toxic when cells were provided with

a fermentable sugar such as glucose and permanently toxic when supplied with

succinate, which requires oxidative phosphorylation for energy generation. TisB toxicity

was not alleviated in a strain, which lacks an electron transport chain or ATP synthase.

These results suggested that TisB interfered with the proton motive force. Using an

artificial black membrane system we showed that upon a voltage signal, TisB forms a

stable, anion-selective pore. Overexpression dissipated the proton motive force to a

similar level to that of cccp, a protonophore, which we found was able to induce a

multidrug tolerant state as well. These results could provide the following explanation for

the observed growth-arrest and multidrug tolerance: TisB forms a pore in the inner

membrane and abolishes the proton motive force. While this by itself excludes

antibiotics such as aminoglycosides from the cell (Bryan & Van Den Elzen, 1977), it

would also lead to a depletion of intracellular ATP and a subsequent slowdown or

108

shutdown of metabolism. Gyrase, one of the targets for ciprofloxacin, for example, has a

Km for ATP of approximately 300 µM (Higgins et al., 1978). TisB overproduction led to a

10-fold drop in ATP concentrations after expression from a high copy number vector for

15 minutes (Unoson & Wagner, 2008). Given that intracellular [ATP] is in the low mM

range (Albe et al., 1990) a tenfold drop would decrease gyrase activity to half-maximal

levels, which would significantly decrease the amount of damage ciprofloxacin binding

can cause.

Why does TisB protect only a small subpopulation of cells and not the whole

population? This could be reflective of the stochasticity of either TisB expression or that

of its antitoxin, IstR-1. One possibility is that after exposure to ciprofloxacin, persisters

are the cells that express a certain level of TisB within a normal distribution. Another

possibility is that stochasticity of antitoxin expression leads to TisB-mediated survival,

with persisters being those cells that express IstR-1 to the lowest level. Indeed, an

antitoxin knockout increased persister levels 10- to 100-fold (Dörr et al., 2010),

suggesting that a high degree of persister formation is actually blocked by high level

expression of antitoxin. Still even the ∆istR-1 strain did not allow the whole population to

survive in a TisB-dependent way. Further, a high dose of ciprofloxacin killed the bulk

completely within the first 30 minutes of exposure, leaving surviving persister cells. The

tisB promoter has the strongest lexA binding of all SOS inducible promoters (Fernandez

De Henestrosa et al., 2000, Vogel et al., 2004, Unoson & Wagner, 2008) and it seems

unlikely that cells are actually able to induce TisB before the bulk dies. The explanation

of persister formation in these conditions of high ciprofloxacin exposure purely by

stochasticity of istR or TisB expression levels is thus doubtful.

109

A third possible explanation is therefore that persisters are pre-conditioned to

induce TisB and survive. Shah et al. showed that populations of E. coli harbor a

subpopulation of non- or slowly growing cells, which are enriched in persisters to

ciprofloxacin. A plausible model of persister formation in these conditions is that these

cells survive the initial exposure to ciprofloxacin. Due to their low metabolic activity they

are in fact damaged by the antibiotic, but not to a lethal degree. For a while this low

level DNA damage can be compensated by repair functions but extended exposure to

the antibiotic will kill off even these cells, unless they are able to induce TisB, which

shuts down metabolism and turns these “pre-persisters” into multidrug tolerant persister

cells (Fig. 4-1). Apart from rapid death after exposure to high ciprofloxacin and

emergence of persisters in a time frame that is likely too short to allow for induction of

TisB, several other experimental observations support this model: survivors to

ciprofloxacin have a low level of SOS induction but rely on repair functions such as recA

(Dörr et al., 2009). If stochasticity of persister formation purely relied on TisB expression

levels, the upstream event of SOS induction should be highly uniform in the bulk.

Further, the TisB-dependent persister phenotype is visible only after extended exposure

to ciprofloxacin (3 h), suggesting that for the first three hours cells have different means

of coping with antibiotic stress.

In summary, we have shown that the flouroquinolone antibiotic ciprofloxacin can

induce the formation of multidrug tolerant persister cells by inducing the small peptide

toxin TisB. TisB forms a pore in the inner membrane and dissipates the proton motive

force, leading to a drop in intracellular ATP concentrations and subsequent shutdown of

growth and possibly metabolism. TisB dependent persister formation is likely regulated

110

by expression levels of its antitoxin RNA IstR-1 and by stochasticity in SOS induction

intensities.

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Figure 21. Model of stochasticity in TisB-mediated persister formation Upon exposure to ciprofloxacin rapidly growing cells (green) die off quickly (A). Existing

persisters (red) and “pre-persisters” (light green) survive, the latter induce the SOS

response (B). Only those pre-persisters with low initial levels of IstR-1 can produce TisB

(C), which shuts down metabolism and induces dormancy.