RESEARCH ARTICLE

Cho et al., Microbiology 2017;163:308–321

DOI 10.1099/mic.0.000436

EDITOR’S CHOICE
MazEF toxin-antitoxin proteins alter Escherichia coli cell
morphology and infrastructure during persister formation and
regrowth
Junho Cho,1 Anita Nicole Carr,1 Lisa Whitworth,2 Brent Johnson2 and Kevin Scott Wilson1,*

Abstract
When exposed to antibiotics, many bacteria respond by activating intracellular ‘toxin’ proteins, which arrest cell growth and
induce formation of persister cells that survive antibiotics. After antibiotics are removed, persisters can regrow by
synthesizing ‘antitoxin’ proteins that sequester toxin proteins. In Escherichia coli, MazE antitoxin sequesters the activity of
MazF toxin, which extensively cleaves cellular RNAs. Although the functions of MazEF proteins are well characterized, there
is surprisingly little known about their effects on cell structure. Here, using a combination of microscopy techniques, we
visualized the effects of MazEF and three bactericidal antibiotics on E. coli cell morphology and infrastructure. When
ectopically expressed in E. coli, MazF temporarily stalled cell growth and induced persister formation, but only mildly
elevated DNA mutagenesis. Viewed by electron microscopy, MazF-expressing persister cells were arrested in cell growth
and division. Their chromosomal DNAs were compacted into thread-like structures. Their ribosomes were excluded from
their nucleoids. After exposure to ciprofloxacin, persister regrowth was activated by MazE. Cell division remained inhibited
while cells became extraordinarily elongated, then divided multiple times during stationary growth phase. This extreme
filamentation during persister regrowth was unique to ciprofloxacin-treated persisters, likely caused by inhibition of cell
division during regrowth, and was not observed with kanamycin-treated persisters.

INTRODUCTION E. coli toxin-antitoxin proteins are among the best charac-

Many bacteria harbour ‘toxin’ and ‘antitoxin’ genes that regu-
late many aspects of cell growth and survival [1]. The
expressed toxin proteins arrest cell growth. In growing cells,
toxin proteins are sequestered by cognate antitoxin proteins.
When bacteria encounter external stresses such as bactericidal
antibiotics, they degrade their antitoxins, which unleash their
toxins. Toxin-expressing cells revert to a persister phenotype,
whereby they can tolerate multiple types of antibiotics by not
growing. When removed from antibiotics, persisters can
regrow and potentially evolve into genetically antibiotic-resis-
tant cells. Persisters are associated with major chronic infec-
tious diseases in humans. Persisters can form stochastically or
can be induced by antibiotics [1]. Persister formation and
regrowth appears to be regulated redundantly by multiple
pairs of toxin-antitoxin genes. Pathogenic strains of Mycobac-
terium tuberculosis have over 80 gene pairs [2], while labora-
tory strains of Escherichia coli have at least ten [3].
terized currently. MazF toxin and MazE antitoxin were
among the early discovered proteins [4]. MazF is a ribonu-
clease that extensively degrades mRNAs [5], and it also
cleaves rRNAs in ribosomes [2, 6]. MazE inhibits MazF by
binding to its active site [7]. Unleashed MazF inhibits
nucleic acid and protein synthesis [8], halting cell elongation
and division. Fig. 1 illustrates our current understanding of
stress response pathways in cells exposed to two antibiotics
that inhibit cell elongation and division. b-Lactam antibiot-
ics kill growing cells and induce synthesis of ppGpp (guano-
sine 3¢, 5¢-bisdiphosphate) in surviving cells [9]. This leads
to degradation of MazE, activation of MazF, and formation
of persister cells. Early studies concluded that bacteria
exposed to bactericidal antibiotics generate mutagenic reac-
tive oxygen species (ROS), which can damage DNA, creat-
ing mutant cells or causing cell death [10, 11]. In surviving
persister cells, ROS mutagenesis could lead to genetically
resistant cells that can grow in the presence of antibiotics

Received 30 May 2016; Accepted 20 January 2017

Author affiliations: 1Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA; 2Microscopy
Laboratory, Oklahoma State University, Stillwater, OK 74078, USA.
*Correspondence: Kevin Scott Wilson, kevin.s.wilson@okstate.edu
Keywords: antibiotics; electron microscopy; nucleoid; ribosomes; SOS and stringent response.
Abbreviation: ROS, reactive oxygen species.
Four supplementary figures, a supplementary video and a supplementary table are available with the online Supplementary Material.

000436 ã 2017 The Authors

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 Cho et al., Microbiology 2017;163:308–321

b-Lactam
Cell (ampicillin)
death Cell death
SOS
Cell wall ppGpp Antitoxin Quinolone response
synthesis (MazE) (ciprofloxacin)
DNA
breaks

Cell RNA/protein Toxin Cell

DNA synthesis
elongation synthesis (MazF) division
Persister
cell

ROS

DNA
damage

Mutant cells Cell death

Fig. 1. Antibiotic-induced persister formation, regulated by MazEF proteins in E. coli. This network highlights the regulation of cell
elongation and division by MazEF proteins. It also shows the overall impacts of the bactericidal antibiotics ampicillin and ciprofloxacin.
Each arrow entails multiple steps (not shown, for simplicity); dashed arrows indicate pathways that are less well defined. Cell elonga-
tion: ampicillin (and other b-lactams) inhibit cell-wall synthesis, leading to cell death by lysis. E. coli cells respond by making ppGpp
[9], leading to MazE degradation, which liberates MazF and induces persistence [4]. ppGpp also inhibits RNA and protein synthesis via
the stringent response pathway [42]. Persister cell: MazF-expressing cells generate ROS [21], which damage DNA and may lead to anti-
biotic-resistant mutant cells or death in severe cases [11]. MazF degrades most mRNAs and cleaves rRNA, thereby inhibiting RNA,
protein and DNA synthesis [8]. Cell division: ciprofloxacin (and other quinolones) interfere with supercoiling during DNA synthesis, lead-
ing to DNA breaks and cell death. E. coli cells activate the SOS response, which inhibits cell division until DNA damage is repaired [40].

[12]. However, other studies have called these conclusions substantial uncertainties about the roles of toxin-antitoxin
into question [13, 14]. proteins in bacterial physiology, even for the better charac-
terized MazEF proteins. Surprisingly little is known about
Multiple toxin-antitoxin gene pairs are necessary for effi-
how toxin-antitoxin proteins affect intracellular structures.
cient formation of E. coli persisters [3]. This redundancy
To fill this knowledge gap, in the current study we visualized
complicates our understanding of the specific cellular roles
the effects of ectopic expression of MazEF proteins in E. coli
of each pair. Ectopic expression of single toxin genes leads
cells using a combination of light and electron microscopy.
to the formation of antibiotic-tolerant cells with phenotypes
We analysed the effects of different bactericidal antibiotics
similar to naturally formed persisters [1]. E. coli cells ectopi-
during the formation and regrowth of persisters (Fig. 1).
cally expressing MazF from low-copy plasmids have served
Our results reveal that MazEF proteins cause substantial
as tractable models of natural persister cells, which are sto-
alterations in cell shape as well as reorganization of intracel-
chastically formed, rare and transient [8, 15]. These MazF-
lular nucleoid and ribosome structures. We found that
expressing cells remain alive, although not growing and
MazF greatly enhances the survival of persisters, but only
dividing, and can be revived by ectopically expressing MazE
mildly elevates cellular mutagenesis.
[8, 16]. MazF-expressing cells remain metabolically active,
undergoing futile cycles of RNA synthesis and degradation
[17]. However, other studies suggest that MazF-expressing METHODS
cells can become irreversibly dormant [18]. MazF has been Bacteria and plasmids
proposed to elevate cellular mutagenesis through ROS pro-
E. coli strain SC28 and plasmids pSC3326 and pSC228 were
duction [19, 20] and the SOS response [21]. MazF may
obtained from Kenn Gerdes [8]. E. coli strain MC1000 was
induce programmed cell death, dependent on quorum sens-
purchased from the Coli Genetic Stock Center (http://cgsc.
ing [22], for the benefit of a bacterial population exposed to
biology.yale.edu/).
antibiotics. MazEF proteins are distributed broadly in bac-
teria, especially pathogens, with nine mazEF gene pairs Microbiology
identified in M. tuberculosis [2].
Bacterial growth and lysis was monitored by the optical den-
The studies cited above establish the importance of MazEF sity of liquid cultures. E. coli cells (from 1 ml of saturated
proteins for regulating growth of bacteria and their toler- overnight culture in Luria broth (LB) +30 µg ml 1 chloram-
ance of antibiotics. These studies also highlight the phenicol or 50 µg ml 1 ampicillin) were inoculated (t=0 min)

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 Cho et al., Microbiology 2017;163:308–321
into 500 ml LB in a 2 l flask. Cell growth at 37  C was moni-
tored by measuring the optical density (absorbance of 600 nm
light, OD600) of culture samples (1 ml) every 30 min. Doubling
times were calculated from five time points during maximal
logarithmic growth by plotting log2(OD600) versus time and
determining the slope of the fitted line, representing the recip-
rocal of the doubling time. MazF was expressed from
pSC3326 by adding arabinose (0.4 %) when the OD600
reached 0.25 (t=255 min). MazE was expressed from pSC228
by adding IPTG (2 mM) when the OD600 reached 0.6
(t=270 min). To lyse or kill non-persister cells, ampicillin
(100 µg ml 1), ciprofloxacin (1 µg ml 1) or kanamycin
(100 µg ml 1) were added to the cultures.
For regrowth, persister cells (SC28/pSC3326/pSC228) were
harvested from 250 ml culture, washed with cold LB, resus-
pended in fresh LB+IPTG (2 mM) to express MazE, and
regrown at 37  C. Viable cell numbers were measured as
colony-forming units (c.f.u.) per ml of cell sample. Cell
samples (1 ml) were 10-fold diluted (to 10 6 dilution) in
ice-cold liquid LB. Aliquots (100 µl) of each dilution were
spread onto solid LB-agar+chloramphenicol (30 µg ml 1)
and incubated overnight at 37  C.
Genetics
Mutations were identified and analysed by sequencing the
plasmid-encoded mazF gene. Single culture samples were
spread onto LB agar. The mazF gene was amplified by PCR
from several colonies. Colonies were transferred into separate
tubes containing 200 µl water and heat-denatured (90  C,
5 min). The lysate was mixed thoroughly and cooled to room
temperature. The mazF gene was amplified by PCR using Q5
High-Fidelity DNA polymerase (M0491; New England
Biolabs) and DNA primers flanking the mazF gene in the
plasmid at 196 nucleotides upstream of the start codon and
238 nucleotides downstream of the stop codon. Upstream
primer: 5¢-TTATTTGATGCCTGGCAGTTCC-3¢; down-
stream primer: 5¢-CATTGATTATTTGCACGGCGTC-3¢.
PCR products were separated by agarose gel electrophoresis
and stained with ethidium bromide. Gel bands containing
PCR products were purified by the QIAquick gel extraction
and PCR purification kits (Qiagen). Both DNA strands of the
PCR products were sequenced using BigDye-terminated reac-
tions and the original DNA primers. The sequences were
searched against E. coli MG1655 complete genome (GenBank
accession number CP009685.1) through BLAST (www.ncbi.
nlm.nih.gov) and matched the five copies of IS1 transposable
element in the genome.
Mutation frequencies were measured by the fluctuation test
[23]. Two parallel cultures of E. coli SC28/pSC3326 were
grown in 250 ml LB in a 1 l flask at 37  C. To one culture,
MazF protein was induced by adding arabinose when the
OD600 reached 0.250. Both cultures were further incubated
to saturation (total time=12 h). Cells from each culture
were diluted in LB by 106. Aliquots (1 ml) of diluted cells
were distributed to seven test tubes and were incubated
with or without arabinose for 12 additional hours. Aliquots
(200 µl) of cells were spread onto solid LB (without or with
310
100 µg ml 1 rifampicin) and incubated at 37  C overnight.
From the c.f.u. per ml, mutation rates were calculated using
the MSS-Maximum Likelihood Estimator Method [23].
Light microscopy
Fluorescence light microscopy was used to assess the viability,
growth and length of individual cells. Cell samples were
immobilized in LB+1 % agarose, sandwiched between glass
slide and coverslip, and visualized through an epi-fluorescence
microscope (XYL-146Y; Amscope. Light source: mercury
lamp; 100 objective lens: 100 and 20 camera magnifica-
tion) equipped with a phase contrast lens (PCT200-INF;
Amscope) and camera (MU1000-CK; Amscope).
sCell viability was assessed using the Live/Dead BacLight Via-
bility Kit for microscopy (L7007; Invitrogen) with two nucleic
acid stains: green dye (SYTO 9) and red dye (propidium
iodide), which stain live and dead cells, respectively [24]. All
cells were visualized by phase-contrast microscopy. Live+dead
cells were visualized by exciting both dyes with light of 420–
490 nm, and detecting the emission of green+red light using a
cut-off filter >520 nm (dichroism: 505 nm). Dead cells were
visualized by exciting propidium iodide with light of 500–
550 nm and detecting the emission of red light using a cut-off
filter >590 nm (dichroism of 575 nm).
Culture samples (1 ml) were removed at various time points.
Cells were pelleted by centrifugation (10 500 g, 1 min), re-
suspended in 1 ml of 150 mM NaCl, re-centrifuged
(10 500 g, 1 min) and re-suspended in 0.5 ml of 150 mM
NaCl. As a control, a portion of these cells was treated with
70 % 2-propanol (30 min, room temperature, with mixing)
and washed with 150 mM NaCl; this control served to cali-
brate 100 % dead (killed) cells. The re-suspended cells were
mixed with 1.5 µl of the green/red-dye mixture, and incu-
bated in the dark at room temperature for 15 min on a
rotary mixer. The samples were centrifuged (10 500 g,
1 min), 450 µl of supernatant was removed, and the cell pel-
lets were re-suspended in the remaining supernatant.
Cell lengths were measured using the Amscope software.
Lengths were measured (to the nearest 0.1 µm) of between
150 and 500 cells from biological replicates (independently
grown cell samples). From distributions of cell lengths,
we determined the average length and standard deviation
(Fig. S1, available in the online Supplementary Material).
Cell elongation and division was monitored by time-lapse
microsopy. Cells were immobilized in LB+0.2 % agarose,
with or without ciprofloxacin or IPTG, and mounted on the
microscope at 37  C. Video was recorded by capturing
images of the cells every 60 s over a period of up to approxi-
mately 150 min.
Electron microscopy
Transmission electron microscopy was used to analyse cel-
lular morphology and internal structures. In preparation for
electron microscopy, cell samples from liquid culture were
pelleted by centrifugation (735 g, 5 min, 4  C) and re-
suspended in 1 ml phosphate-buffered saline solution. Cells
 Cho et al., Microbiology 2017;163:308–321
were pelleted again by centrifugation and 900 µl of superna-
tant was discarded. The cell pellets were re-suspended in
1 ml fixative buffer (2 % glutaraldehyde, 100 mM sodium
cacodylate, 4.5 mM CaCl2) and incubated at 4  C for 2 h.
The fixed cells were centrifuged and re-suspended in wash
buffer (60 mM sodium cacodylate, 6 % sucrose) for 15 min,
repeated three times and stored at 4  C overnight. On the
following day, the samples were centrifuged (735 g, 5 min,
4  C) and 900 µl of supernatant was discarded. The pellets
were fixed with 1 % OsO4 (1 h, 4  C), washed three times
with wash buffer and dehydrated through a series of washes
with increasing concentrations of ethanol (1 each with 50,
70, 90 and 95 %; and 3 with 100 %).
Fixed, dehydrated cells were prepared for electron micros-
copy by infiltration, embedding, polymerization, thin-
sectioning and staining [25]. Samples were washed (15 min,
three times) with propylene oxide and infiltrated with a 1 : 1
mix of propylene oxide and poly/bed 812 resin. On the fol-
lowing day, the mixture was embedded in poly/bed 812 and
polymerized (60  C, 48 h). Samples were thin-sectioned, and
microscopy grids were stained with 2 % uranyl acetate and
Reynold’s lead citrate. Sectioned samples were visualized by
transmission electron microscopy (JEM-2100; JEOL. Direct
magnification: 8000 to 25 000; HV=200 kV).
RESULTS
MazF temporarily stalls E. coli growth, forming
persisters that survive ampicillin
To study how MazF affects cell physiology, a previously
characterized E. coli strain SE28 harbouring inducible mazE
and mazF genes on separate plasmids, and lacking the cor-
responding genes on its chromosome [8], was chosen. Cell
growth of this strain was monitored in liquid culture
(Fig. 2a). When MazF expression was induced, cell growth
quickly stalled and then resumed after ~200 min, yielding a
biphasic growth curve. After growth resumed, the cell dou-
bling rate was three-fold slower than the control culture not
expressing MazF. Ampicillin (a b-lactam antibiotic causing
cell lysis; Fig. 1) was added at two time points. When ampi-
cillin was added during the stalled growth phase, there was
no detectable cell lysis, suggesting that virtually all cells in
the culture were persisters. When ampicillin was added after
growth resumed, a fraction of the cells lysed. This result sug-
gested a mixed population of antibiotic-sensitive cells and
persisters in the culture after growth resumed, consistent
with the slower average cell doubling rate at this time point.
The E. coli SE28 strain was derived from E. coli MC1000 [8],
which was subsequently found to be defective in genes
required for ppGpp synthesis and the stringent response
(relA1 spoT1; http://cgsc.biology.yale.edu/Strain.php?ID=
8823; K. Gerdes, personal communication). The deficiency
of E. coli SE28 in both mazEF and the stringent response
may lower its overall fitness and stress tolerance in compari-
son to E. coli MC1000 [26]. We found that both strains had
identical growth curves when the plasmid-encoded mazF
311
was suppressed (Fig. S1a). This is consistent with earlier
studies showing that the mazEF genes on the chromosome
do not affect cell viability or growth, independent of the
stringent response [27].
MazF mildly elevates cellular mutagenesis
Previous studies have shown that stalled growth and antibi-
otic tolerance of MazF-expressing cells arise from extensive
RNA degradation that inhibits translation [8, 15]. In seeking
an explanation for the resumed growth of MazF-expressing
cells, we hypothesized that the resumed growth may be
caused by DNA mutagenesis (Fig. 1), since MazF stimulates
ROS production [19, 20]. Mutations in the plasmid-encoded
mazF gene seemed to be a good possibility. Moreover, the
deficiency of the stringent response might exacerbate muta-
genesis induced by the ectopic mazF expression. To test this
hypothesis, the mazF gene was analysed from single colonies
of cell samples from the biphasic growth and regrowth
(Figs 2b and S1b–d). The DNA products included not only
the expected mazF length, but also many longer, and a few
shorter, than predicted. The longer products resulted from
insertions of IS1 transposable element into mazF (Table S1).
IS1 insertions were found at highest frequency at the end of
the stalled phase. After growth resumed, IS1 insertion muta-
tions were replaced by a variety of frameshift and substitu-
tion mutations.
The inactivating mutations in mazF could be driven by
one of two mechanisms: 1) MazF expression could elevate
the general rate of DNA mutagenesis in cells, consistent
with its role in inducing ROS [19]; or 2) even if MazF
expression has no effect on DNA mutagenesis, inactivating
mutants in mazF would be strongly selected in the popula-
tion since they would allow cell regrowth. To distinguish
these mechanisms, we determined how MazF expression
affects cellular mutagenesis by applying a classic fluctuation
test [28]. The frequency of spontaneous chromosomal
mutations conferring resistance to another antibiotic, rifam-
picin, which generally occur in the rpoB gene of RNA poly-
merase, [29] was measured. MazF expression increased the
rate of mutagenesis by about two-fold (Table 1). These
results suggest that MazF contributed a small increment to
the overall cellular mutation frequency. These results are
inconsistent with ROS-induced programmed cell death
[19], but consistent with other studies [8, 27].
MazF alters persister cell infrastructure
To shed light on the cellular effects of MazF, we analysed
cell dimensions by light microscopy (Figs 2c and S2). The
mean length of the control cells (not expressing MazF)
under rapid growth conditions was 1.9 µm, which is typical
for E. coli K12 [30]. As the control cells progressed through
the exponential growth phase into stationary phase, they
became progressively shorter (Fig. 2c; samples afibfiffii).
By contrast, persister cells did not follow this developmental
progression. Rather, persisters remained the same length
after MazF induction and ampicillin treatment (samples
cfihfil). This suggests that MazF arrests cell growth and
 Cho et al., Microbiology 2017;163:308–321

(a) (b)
1.8 Sample

Maker
–MazF f i
1.6 +MazF c d
1.4 +MazF +Amp

Gene length (kb)

j
1.5 mazF :: lS1
+MazF +Amp g
1.2
Amp
Optical density

b 0.7 mazF
1.0 0.5
0.8
k
0.6 +MazF d
0.4 (c)
a c e h l
0.2
Amp 3
0

Time (min) 0 100 200 300 400 500 600 700

Cell length (µm)

2

(d)
All cells (sample g) Live (green) cells Dead (red) cells

0
Sample a b c f g h i j k l
MazF + + + + + +
Amp + + +

Fig. 2. Characteristics of MazF-expressing persister cells surviving ampicillin treatment. (a) Effect of MazF and ampicillin on E. coli
cell growth. The growth of four separate cultures of E. coli SE28/pSC3326 [8] was monitored by optical density over time (see Meth-
ods). Error bars represent ±standard deviations of three biologically independent cultures for each growth curve: control cells not
expressing MazF (blue curve), MazF-expressing cells treated with ampicillin (Amp; purple and green curves) or not treated with Amp
(orange curve). Cell doubling times (see Methods): control cells (37 min); MazF-expressing cells doubled every 36 min during the first
exponential phase, and 97 min during the second exponential phase. Eleven cell samples (a–l) were removed at various times in the
grown curves (see main text). (b) Mutagenesis of the plasmid-encoded mazF gene. Single culture samples were spread onto solid LB.
Individual colonies were analysed by PCR and DNA sequencing (Table S1). The agarose gel image shows examples of mazF genes con-
taining IS1 insertions from colonies arising from culture samples c and d. PCR products: mazF gene (336 bp) + flanking plasmid
sequences (434 bp); mazF:: IS1 also includes IS1 (768 bp) inserted in the coding sequence of mazF. Table S1 summarizes the insertion
sites of IS1 in mazF and other identified missense and frameshift mutations in mazF. (c) Cell lengths. Cell samples (a–l) from growing
cultures were visualized by light microscopy. The average values (±standard deviations) of cell lengths are plotted from data of cell
population distributions (Fig. S2). (d) Cell viability was assessed by the Live/Dead assay. Shown here is cell sample g; other samples
are shown in Fig. S3. Bar, 10 µm.

division in the SE28 strain (Fig. 1). We also examined cell In all samples, greater than 95 % of cells were alive before
viability using the Live/Dead assay (Figs 2d and S3a) [24]. and after MazF expression.
To examine effects of MazF on intracellular organization,
transmission electron microscopy was used and revealed
Table 1. Effect of MazF expression on cellular mutagenesis rate as four distinct cell phenotypes (Figs 3 and S4).
measured by fluctuation test
(1) Control cells during exponential growth phase (samples
E. coli SE28/ Mutation rate* Confidence interval (95 %) a, b). Numerous ribosomes can be seen as electron-
pSC3326
Upper bound Lower bound
dense dots scattered inside the cytoplasm. Ribosomes
were intermingled with the more diffuse nucleoid.
8
Control 1.7010 2.28 1.18 Most cells were seen undergoing cell division and the
MazF-expressing 3.29108 4.11 2.54 nucleoids were located away from cell division.
(2) Control cells during stationary phase (samples f, i).
*Calculated from the fluctuation test [23], see Methods.
The cytoplasm was shrunken and the periplasmic

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 Cho et al., Microbiology 2017;163:308–321

–MazF f i 4
+MazF (i)
1.5
+MazF +Amp j

Optical density
+MazF +Amp Amp

1.0 g
b
k
Amp d
0.5 +MazF

a c e h l
0

Time (min) 0 100 200 300 400 500 600 700

(a) (f) (j)

(b) (g) (k)

1
5
6

(c ) (h) (l)

Fig. 3. Infrastructure of persisters surviving ampicillin treatment. For reference, growth data are reproduced from Fig. 2(a). Cell sam-
ples (a–l) were imaged by electron microscopy (see Methods). Bar, 0.2 µm. Cell features: 1, diffuse nucleoid [43] of rapidly growing cell
not expressing MazF; 2, membrane indentation in a MazF-expressing cell beginning, but not completing, cell division; 3, widened peri-
plasmic space in cell at stationary phase, not expressing MazF; 4, ‘cytoplasmic granule’ [37] in cell at stationary phase; 5, ribosomes in
cytoplasm, outside of condensed nucleoid, in MazF-expressing persister cell; 6, condensed chromosomal DNA threads [25]; 7, glass-
like translucent cavity [38].

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 Cho et al., Microbiology 2017;163:308–321
space was widened. Small granules were frequently
found in the periplasmic spaces. The granules were
connected to the cytoplasm by a loop, or present as
isolated bodies.
(3) MazF-expressing evolving cells (samples g, j). Nucleoids
were much more defined. Ribosomes were largely
located outside of the nucleoid. The nucleoids were
not localized towards dividing cells.
(4) Persister cells expressing MazF and surviving ampicillin
(samples c, h, l, k). Persister cells shared many features
in common with MazF-expressing evolving cells.
Nucleoids in persister cells were further collapsed and
appeared translucent with glass-like cavities.
Chromosomal DNAs were condensed into irregular
networks of slender, electron-dense strings.
The distinctive infrastructure of persister cells suggests that
translation and transcription are largely shut down, result-
ing from the ribonuclease activities of MazF and ampicillin
stress response.
Regrowth of persisters
After surviving antibiotic exposure, how do persisters
recover and resume growth? When MazF is expressed,
mRNAs and rRNAs in ribosomes are largely cleaved. How
do surviving MazF-expressing cells synthesize new RNAs
and proteins and begin to grow again? An early step in their
recovery is plausibly the nullification of MazF through syn-
thesis of more MazE.
To address the question of persister regrowth, we employed
E. coli SE28 containing two plasmids independently express-
ing MazE and MazF [8]. Since the MazE plasmid confers
genetic resistance to ampicillin, we chose to examine
ciprofloxacin treatment. While both antibiotics are bacteri-
cidal, ciprofloxacin inhibits DNA gyrase and induces cyto-
plasmic vacuoles, rather than cell lysis [31]. First, growth
curves of cells treated with ciprofloxacin and expressing
MazF were compared (Fig. 4a). Unlike ampicillin, ciproflox-
acin had a delayed inhibition on cell growth, as reflected by
the increased optical density of cultures over time. MazF
expression generated a biphasic growth curve, similar to our
previous experiment. MazF and ciprofloxacin terminally
stalled growth.
Regrowth of persisters was then examined (Fig. 4b). The
surviving persisters were inoculated into fresh growth media
and MazE was expressed. After a 2 h delay, persisters began
to regrow and finally reached an optical density similar to
the control cells. The regrown cells were not genetically
resistant to ciprofloxacin (Fig. S1e), indicating that they
arose from true persisters. Without MazE expression, per-
sisters failed to regrow (Fig. 4b) and appeared largely dead
by the Live/Dead assay (data not shown).
Regrowing persisters become very long
Cell viability and multiplication during the formation and
regrowth of persisters was considered. Whereas the Live/
Dead assay provides information about cell viability in
314
terms of membrane integrity [24], it does not assess viability
in terms of cell multiplication. The optical density reflects
cell density in liquid culture, but does not distinguish cell
numbers from cell size. To address these points, the OD600
of MazF-expressing cells growing in liquid culture
was compared to the number of colony-forming units per
millilitre (c.f.u.) spread onto solid growth media containing
the inducer for MazE (Fig. 4c). The c.f.u. exponentially
increased during the first growth phase, then decreased in
the stalled phase and exponentially increased during the
resumed growth to stationary phase. The c.f.u. of samples
taken from the regrowth of persisters was then measured.
For the control cells, during exponential growth from
OD600 of 0.6 to 1.6, the c.f.u. increased more than 30-fold
(Fig. 4d; samples a, b). During persister regrowth, the OD600
increased from 0.6 to 1.5 while the c.f.u. increased only two-
fold (samples d, e). After reaching stationary phase, the
regrown persisters were incubated for 12 more hours (sam-
ple e+12). Surprisingly, the c.f.u. increased more than ten-
fold, while the optical density increased slightly. These
discrepancies suggested that the optical density of the cul-
ture did not simply reflect viable cells.
These discrepancies were resolved by visualizing regrowing
persisters using fluorescence light microscopy (Fig. 5a).
Almost all persister cells were scored as alive according to
the Live/Dead assay (Fig. 5b). However, only half of them
formed colonies (Fig. 4d; ratio of samples a/d). During
regrowth, some persisters became exceedingly long
(Fig. 5c). Their mean length was 21 µm, with a very broad
distribution of length (Fig. S2). Some cells were >100 µm
long, while others were <10 µm. They reached maximum
length at early stationary phase. However, after 12 more
hours in later stationary phase, all of the regrown cells
reverted to near normal length (~3 µm).
Thus, the very long cells accounted for the increase in the
optical density during logarithmic regrowth of persisters.
While practically all persisters remained viable, only about
half could be cultured. Of the revived persisters, many
became activated in cell elongation but remained inhibited
in cell division. Later in stationary phase, the regrown cells
were predominantly short, which accounted for the ten-fold
increase in c.f.u. while the OD600 remained nearly constant.
Ciprofloxacin reduced the c.f.u. of the control cells by 105-
fold. Ciprofloxacin-treated cells were also elongated
(Fig. 5b), as previously observed [31]. However, unlike per-
sisters, ciprofloxacin-treated cells did not divide. Instead,
vacuoles formed in the cytoplasm (Video S1) [31]. The cells
stained green (Fig. S3b), inconsistent with the much-
reduced c.f.u. (Fig. 4d), suggesting that the cells may remain
viable but not culturable.
Very long cells divide into multiple cells
Since many of the long cells appeared to be alive (Fig. 5a, c),
we hypothesized that they underwent cell division during
later stationary phase. This hypothesis was tested by follow-
ing persister regrowth in real time using time-lapse
 Cho et al., Microbiology 2017;163:308–321

(a) (b)
1.8 b
Control
1.6 +CFX
+MazF f
1.4 +MazF +CFX
e
1.2
Optical density

1.0

0.8 c d
d
0.6
a
a +Maz E
0.4
CFX
0.2 +MazF Control
+MazE
0 OD600 (e+12 h)=
–MazE 1.600±0.019

Time (min) 0 100 200 300 400 500 0 100 200 300 400

(c) (d)
1.6

1.4 c.f.u. (f) =

12 1.62±0.24×103
Viable count (c.f.u. ml–1 linear scale × 108)

1.2 109

Viable count (c.f.u. ml–1 log scale)

10
1.0
Optical density

0.8 8
c

0.6 6
108
0.4 a
4
0.2

2
0

0
107
0

120
150
180

240

300

390

510

a b c d e e+12
Time (min)

Fig. 4. Formation and regrowth of persisters surviving ciprofloxacin treatment. (a) Effect of MazF and ciprofloxacin on E. coli cell
growth. E. coli SE28 with pSC3326 containing mazF and pSC228 containing mazE [8] was grown in four separate cultures (see Meth-
ods). Error bars represent ±standard deviations of three biologically independent cultures for each growth curve: control cells not
expressing MazF (blue curve), control cells treated with ciprofloxacin (CFX; orange curve), MazF-expressing cells treated or not treated
with CFX (purple and green curve, respectively). Cell samples (a–f) were removed at various times in the grown curves (see main text).
(b) Regrowth of control cells (sample a) and persisters (sample d). Cells were washed, MazE was induced, and regrown in fresh LB.
Two additional cell samples were collected: regrown persister cells at early stationary phase (sample e) and late stationary phase
(sample: e+12). The OD600 of sample e+12 is boxed below the curve. (c) Comparison of the OD600 and viable cell counts of MazF-
expressing cells. The green curve is reproduced from panel (a), for comparison. Red bars: cell samples were removed at various times
and spread onto solid growth media containing inducer for MazE (see Methods). Error bars indicate standard deviations of three bio-
logical replicates. (d) Number of viable cells during regrowth of persisters. Boxed data: c.f.u. of the ciprofloxacin-treated control cells
(sample f). Error bars indicate standard deviations of three biological replicates.

315
 Cho et al., Microbiology 2017;163:308–321

(a) All cells Live (green)+dead (red) cells Dead cells only
d
e
e+12 h

(b) (c)
100
40

90
30
Cell length (µm)
Cell viability

80
20

70 10

60 0
CFX + + CFX + +
MazF + + MazF + +
MazE + + MazE + +
Sample a b f c d e e+12 Sample a b f c d e e+12

Fig. 5. Regrowing persisters become very long. (a) Cells were imaged by fluorescence light microscopy. The viability of individual cells
was assessed by the Live/Dead assay (see Methods). Shown here are samples d, e and e+12 (see Fig. 4a, b); other samples are shown
in Fig. S3(b). Bar, 10 µm. (b) Cell viability by the Live/Dead assay. Plotted are the average viabilities (±standard deviations) from the
percentage of live (green) cells. (c) Cell lengths. Mean values (±standard deviations) are plotted from data of cell population distribu-
tions (Fig. S2).

316
 Cho et al., Microbiology 2017;163:308–321
microscopy. Over a period of 90 min, the long cells could be
observed to grow even longer (Fig. 6a; Video S1). Each cell
divided multiple times, preferentially at their tips and occa-
sionally in their centre. By contrast, non-persisters express-
ing MazF and MazE grew and divided like the control cells.
Ciprofloxacin-treated cells failed to grow or divide. Thus,
persisters followed a unique pathway of regrowth.
Looking at the elongated cells by transmission electron
microscopy, we observed two distinct cell internal anato-
mies. In one cell type, the nucleoid was compacted near the
centre, surrounded by a large cytoplasm, and the periplasm
was expanded at the cell tips (Fig. 6b). In the other cell type,
the nucleoid was dispersed and intermingled with ribosomes
throughout the elongated cell (Fig. 6c). It is unclear which
cell type was most likely to divide into multiple cells. From
the collective results (Figs 4–6), we conclude that persister
cells, induced by MazF and surviving ciprofloxacin treat-
ment, regrow by cell elongation followed by multiple cell
divisions.
Persister regrowth depends on prior antibiotic
exposure
We wondered whether the extreme filamentation of recov-
ering persisters was a specific effect of MazF-expressing cells
after treatment with ciprofloxacin, or whether it is a general
feature of recovering persisters. Ciprofloxacin is known to
induce filamentation by inhibiting DNA synthesis (via
DNA gyrase), which induces the SOS response and blocks
cell division (Fig. 1) [32].
Therefore, the recovery of persisters treated with a different
bactericidal antibiotic was assessed. Kanamycin, an amino-
glycoside that causes ribosomes to mistranslate mRNAs,
was the antibiotic examined. In the absence of MazF, kana-
mycin caused a slow decrease in the OD600 of an exponen-
tially growing culture, whereas MazF expression protected
the culture from apparent lysis (Fig. 7a). The OD600 of the
regrowing persisters decreased further before reaching a
normal saturating stationary level (Fig. 7b). The kanamy-
cin-treated persisters before and after regrowth were exam-
ined by light microscopy and Live/Dead assays (Fig. 7c–e).
Before regrowth, the cell length distributions were very sim-
ilar to the persisters treated with ciprofloxacin. After
regrowth to early stationary phase, the recovered cells
appeared normal, in stark contrast to the regrown ciproflox-
acin-treated persisters. MazF protected persisters from the
bactericidal effects of kanamycin, and the regrown cells
were largely viable.
From these results, we conclude that the extreme filamenta-
tion of recovering persisters is specific to their prior expo-
sure to ciprofloxacin. This is likely a consequence of a
continued inhibition of cell division during the regrowth
phase. Cell division only becomes reactivated upon entry
into stationary phase. By contrast, cell elongation and divi-
sion remain balanced in the kanamycin-treated persisters.
317
DISCUSSION
During rapid growth of bacteria in the absence of antibiot-
ics, persisters are generally rare and form stochastically [1].
When exposed to bactericidal antibiotics, persisters also
form through stress responses that stimulate ppGpp synthe-
sis, leading to antitoxin degradation and toxin activation
[1]. In E. coli, MazF is one of ten type II toxins that collec-
tively lead to persister formation [3].
Many questions about persisters remain unanswered [33].
Bacteria vary considerably in number and type of toxin-
antitoxin proteins. Even in E. coli, persisters encompass
broad and heterogeneous physiological states dependent
on growth conditions and antibiotics [33]. Persisters can
form through multiple toxin pathways. Persisters can still
form without the stringent response pathway and ppGpp
synthesis [34].
Fig. 1 summarizes the known pathways involved in E. coli
persister formation. When exposed to ampicillin, bacteria
respond by synthesizing ppGpp, leading to MazE degrada-
tion and MazF activation. MazF inhibits RNA and protein
synthesis, effectively blocking cell elongation, and protecting
persister cells from death by lysis. Ciprofloxacin generates
DNA breaks which, if not repaired through the SOS
response pathway, quickly kill dividing bacteria that are
actively synthesizing DNA. By inhibiting DNA synthesis,
MazF blocks cell division and protects persisters from cipro-
floxacin. Bactericidal antibiotics were reported to stimulate
ROS production that could lead to mutant cells [11], but
these conclusions were challenged by subsequent studies
[13, 14].
We have shown that MazF temporarily halted E. coli
growth, then resumed growth because of a variety of
selected mazF mutations in the expressing plasmid. MazF
expression elevated DNA mutagenesis by only two-fold.
Our findings disfavour the hypothesis that MazF may
stimulate stress-induced mutagenesis that could lead to
antibiotic-resistant cells [19]. We found that MazF-
expressing persisters remained largely viable, in agreement
with some studies [8, 16, 17] but at variance to others [5,
18, 20, 21]. However, only a fraction of persisters could
resume growth after expressing MazE to neutralize MazF.
The remaining cells appeared to exist in a viable but non-
culturable state.
MazF alters the morphology and infrastructure of
persister cells
Through electron microscopy, we observed dramatic effects
of MazF on E. coli cell structure. In rapidly growing control
cells, nucleoids were diffuse. Ribosomes were distributed
throughout the cytoplasm and intermingled within the
nucleoid, suggesting transcriptional-translational coupling
[35, 36]. At stationary phase, the nucleoids of control cells
became condensed and separated from ribosomes in the
cytoplasm, consistent with previous studies [37].
 Cho et al., Microbiology 2017;163:308–321

(a)
0 min 30 min

2 3
2 3

4
4

60 min 90 min

1 1

2 3 2 3

4 4

1
(b) (c)

Fig. 6. Microscopy of persister regrowth. (a) Time-lapse light microscopy of persister regrowth. Snapshots are from Video S1, taken
every 30 min. Arrow 1 shows the tip of an elongated single cell dividing twice; arrow 2 points to the division of the same cell near its
centre; arrows 3 and 4 point to other cells dividing near their tips. (b, c) Infrastructure of regrown persisters surviving ciprofloxacin.
Cells were imaged by electron microscopy. Representative cells containing condensed or diffuse nucleoid (panels b and c, respec-
tively). Cell features: 1, condensed nucleoid; 2, widened periplasmic space. Bars, 10 µm (a) and 1 µm (b).

By contrast, MazF-expressing persister cells were morpho-
logically unique. Their lengths were increased, while their
width remained similar to control cells. Many cells
appeared stalled in septum formation, suggesting that
MazF inhibits cell division. Their nucleoids were com-
pacted and had glass-like translucent regions where the
chromosomal DNA appeared as electron-dense thread-like
structures [38]. The extreme compactness of DNA chro-
mosomes suggests that their genes are largely inaccessible
to transcription and translation. After exposure to ampicil-
lin, cell structures did not change significantly. It was only
after cells resumed growth that they became susceptible to
lysis and their nucleoids unravelled. Ribosomes were
observed outside of the nucleoid region, suggesting that
translation is largely shut down in MazF-expressing per-
sisters [20].
MazF and ciprofloxacin inhibit cell division during
persister regrowth
Normal cell growth entails a balance between elongation
and division. E. coli cells add a constant volume before
dividing [39]. MazF-expressing persister cells suppress both

318
 Cho et al., Microbiology 2017;163:308–321

(a) (b)
–iii
1.4 +KAN
+MazF +KAN
1.2

1.0 i
+MazF –KAN
Optical density

0.8

0.6 ii ii
KAN
0.4
+MazF
0.2

0.0

Time (min) 0 100 200 300 400 500 0 100 200 300 400 500 600

(c) (i) (ii) (iii)

(d) (e)
100
7
6 90
Cell length (µm)

Cell viability

5
4 80
3
2 70
1
0 60
KAN + + KAN + +
MazF + MazF +
MazE + MazE +
Sample i ii iii Sample i ii iii

Fig. 7. Formation and regrowth of persisters surviving kanamycin treatment. (a) Growth curves. E. coli SE28/pSC3326/pSC228 was
grown in LB, MazF was induced and kanamycin (KAN) was added at the indicated time points. Error bars represent ±standard devia-
tions of three biologically independent cultures for each growth curve. (b) Regrowth of persisters. Cell sample b was washed, MazE
was induced and the sample was regrown in fresh LB. (c) Cells imaged by fluorescence light microscopy. Cell samples [(i), (ii) and (iii)]
were removed from cultures in panels (a) and (b). Bar, 20 µm. (d) Cell lengths. Mean values (±standard deviations) are plotted from
data of cell population distributions (Fig. S2). (e) Cell viability. Plotted are the average viabilities (±standard deviations) from the per-
centage of live (green) cells by the Live/Dead assay.

elongation and division (Fig. 1). After removing extracellu- remarkably long cells, which were mostly viable. This exper-
lar ciprofloxacin and neutralizing intracellular MazF with iment demonstrates that cell elongation can continue with-
MazE, we found that persisters regrew to become out cell division. After nutrients were exhausted, the long

319
 Cho et al., Microbiology 2017;163:308–321
cells divided multiple times, preferentially at the cell tips,
into normal-length cells. At this later stationary phase, the
cells appear to have completely recovered their normal phe-
notype. The extreme filamentation was specific to ciproflox-
acin, suggesting that how persisters recover depends on
their prior antibiotic exposure.
E. coli can grow remarkably long, to 750 µm or more [30],
and remain viable. During recovery, persisters must repair
damage to RNA molecules caused by MazF before cell elon-
gation can occur, as well as damage to chromosomal DNA
caused by ciprofloxacin before cell division is activated. The
exceedingly long cells imply that cell division stays sup-
pressed while cell elongation occurs. Previous studies have
shown that ciprofloxacin-treated cells are somewhat elon-
gated and largely dead [31]. Ciprofloxacin inhibits cell divi-
sion indirectly through the SOS response pathway (Fig. 1)
[40]. During the transition into stationary growth phase, a
cascade of regulators leads to cell division, from ppGpp to
RpoS to FtsZ [41], which could explain the transition from
cell elongation to division in the recovering persisters.
In conclusion, we have shown that MazF-expressing per-
sister cells remain largely viable with altered cell morpholo-
gies and internal structures. These cells effectively tolerate
different bactericidal antibiotics targeting different cellular
machineries. MazE induced the regrowth of ciprofloxacin-
treated persister cells, which activated cell elongation fol-
lowed by cell division.
Funding information
This work was financed through grants from the National Institute of
Food and Agriculture (NIFA Hatch grant OKL02914 to K. S. W.) and the
Oklahoma Center for the Advancement of Science and Technology
(OCAST HR14–146 grant to K. S. W.).
Acknowledgements
We are grateful to Kenn Gerdes (University of Copenhagen) for provid-
ing us with plasmids and E. coli strains, the Biochemistry and Molecu-
lar Biology Array and Bioinformatics Core Facility at Oklahoma State
University for helping with DNA sequencing and analysis, and two
anonymous referees for their constructive comments on the manu-
script before its publication.
Conflicts of interest
The authors declare that there are no conflicts of interest.
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