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DOI: 10.1126/science.

1229858
, 91 (2013); 339 Science
et al. Yuichi Wakamoto
Dynamic Persistence of Antibiotic-Stressed Mycobacteria
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, 19 of which can be accessed free: cites 42 articles This article
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5-amino-1-(5-phospho-D-ribosyl)imidazole-4-
carboxamide (AICAR) as chemically stable, pre-
viously validated metabolic surrogates of these
pathways (Fig. 3A and fig. S5) (15, 16). Studies
in Escherichia coli have supported a view of
folate biosynthesis in which inhibition at vari-
ous points in the folate pathway leads to a gen-
eralized depletion of folate intermediates and
folate-dependent products downstream of the
site of inhibition (15). In contrast, treatment with
PASresulted in selective increases in serine, homo-
cysteine, AICAR, and dUMP levels that kineti-
cally followed the accumulation of PABA(Fig. 3,
A and B). These changes differed from those ob-
served with sulfamethoxazole, sulfanilamide, and
dapsone (Fig. 3A), which inhibit M. tuberculosis
DHPS, and also those reported for E. coli treated
with an inhibitor of its dihydrofolate reductase
(15, 16). Moreover, co-incubation of M. tubercu-
losis with exogenous PABA reversed the changes
seen with PAS but did not affect the uptake of
PAS or basal levels of the monitored folate-
dependent reporters (Fig. 3C). Thus, PAS exerts
its antimycobacterial activity through its effects
on M. tuberculosis folate metabolismdownstream
of DHPS.
The specific site (or sites) of PAS action on
M. tuberculosis folate pathway that result in
growth inhibition remain to be elucidated. How-
ever, studies of bacterial and human thymidylate
synthases, including those of Mtb, and AICAR
transformylases have reported potent inhibition by
dihydrofolate- and methotrexate-related species
highly similar to the PAS-derived folate analogs
identified here (1721).
Enzyme inhibition is a cornerstone of ra-
tional drug development, but observing growth
inhibition upon exposure of a cell to an enzyme
inhibitor does not suffice to reveal the compounds
mode of action. Without the ability to monitor
directly the accumulation of a drug, its biotrans-
formation, and its intracellular effects, a full under-
standing of mode of action cannot be achieved.
The metabolomic approach used here showed
that PAS, despite its in vitro activity as a competi-
tive inhibitor of DHPS, did not inhibit growth of
M. tuberculosis by inhibiting DHPS. Sulfona-
mides that are more potent DHPS inhibitors than
PAS but poor antimycobacterial agents do not
owe their lack of efficacy to inadequate uptake,
as had been inferred. Instead, they are inactivated
by bacterial metabolism and inhibit DHPS only
weakly in situ. PAS poisons folate-dependent path-
ways not only by serving as a replacement sub-
strate for DHPS but also by the products of that
reaction serving as replacement substrates and/or
inhibitors of subsequent enzymes.
This discovery revises our understanding of
an old drug (PAS) and old target (DHPS) and
underscores that catalysis by, rather than inhibi-
tion of, an enzyme can be exploited for drug
development.
References and Notes
1. M. Raviglione et al., Lancet 379, 1902 (2012).
2. E. R. Long, The Chemistry and Chemotherapy of
Tuberculosis (Williams & Wilkins, Baltimore, MD, 1958),
pp. 347352.
3. G. B. Elion, G. H. Hitchings, Adv. Chemother. 2, 91 (1965).
4. C. Nathan, Cell Host Microbe 5, 220 (2009).
5. J. Rengarajan et al., Mol. Microbiol. 53, 275 (2004).
6. V. Mathys et al., Antimicrob. Agents Chemother. 53,
2100 (2009).
7. V. Nopponpunth, W. Sirawaraporn, P. J. Greene,
D. V. Santi, J. Bacteriol. 181, 6814 (1999).
8. L. P. de Carvalho et al., Chem. Biol. 17, 1122 (2010).
9. T. S. Huang et al., J. Antimicrob. Chemother. 67, 633
(2012).
10. D. L. Schuessler, T. Parish, PLoS ONE 7, e34471
(2012).
11. K. G. Rosdahl, Svensk Kemisk Tidskrift 60, 12 (1948).
12. M. Gengenbacher, T. Xu, P. Niyomrattanakit, G. Spraggon,
T. Dick, FEMS Microbiol. Lett. 287, 128 (2008).
13. S. T. Cole et al., Nature 393, 537 (1998).
14. W. Lu, Y. K. Kwon, J. D. Rabinowitz, J. Am. Soc. Mass
Spectrom. 18, 898 (2007).
15. Y. K. Kwon, M. B. Higgins, J. D. Rabinowitz, ACS Chem.
Biol. 5, 787 (2010).
16. J. R. Wei et al., Proc. Natl. Acad. Sci. U.S.A. 108,
4176 (2011).
17. C. J. Allegra, J. C. Drake, J. Jolivet, B. A. Chabner,
Proc. Natl. Acad. Sci. U.S.A. 82, 4881 (1985).
18. C. J. Allegra, R. L. Fine, J. C. Drake, B. A. Chabner, J. Biol.
Chem. 261, 6478 (1986).
19. H. T. Spencer, J. E. Villafranca, J. R. Appleman,
Biochemistry 36, 4212 (1997).
20. A. S. Fivian-Hughes, J. Houghton, E. O. Davis,
Microbiology 158, 308 (2012).
21. J. H. Hunter, R. Gujjar, C. K. Pang, P. K. Rathod,
PLoS ONE 3, e2237 (2008).
22. M. J. Brauer et al., Proc. Natl. Acad. Sci. U.S.A. 103,
19302 (2006).
Acknowledgments: We thank C. Nathan for critical discussions
and reading of the manuscript, D. Garboczi for assistance
in preparing purified FolP1 and FolC, R. Lee and M. Serono for
the generous gifts of pure folate intermediates and pteroate
analogs, S. Fischer for expert mass spectrometric support, a
Burroughs Wellcome Career Award in the Biomedical Sciences
to K.Y.R., the William Randolph Hearst Foundation, and the Bill
and Melinda Gates TB Drug Accelerator Program (OPP1024050)
for support. This work was supported in part by the Intramural
Research Program of NIAID, NIH. Primary data are deposited in
Dryad; the provisional doi is 10.5061/dryad.vh36n.
Supplementary Materials
www.sciencemag.org/cgi/content/full/339/6115/88/DC1
Materials and Methods
Figs. S1 to S5
Table S1
References (2326)
17 August 2012; accepted 18 October 2012
Published online 1 November 2012;
10.1126/science.1228980
Dynamic Persistence of
Antibiotic-Stressed Mycobacteria
Yuichi Wakamoto,
1
* Neeraj Dhar,
1
* Remy Chait,
2
Katrin Schneider,
1
Franois Signorino-Gelo,
1
Stanislas Leibler,
2,3
John D. McKinney
1

Exposure of an isogenic bacterial population to a cidal antibiotic typically fails to eliminate a small
fraction of refractory cells. Historically, fractional killing has been attributed to infrequently
dividing or nondividing persisters. Using microfluidic cultures and time-lapse microscopy, we
found that Mycobacterium smegmatis persists by dividing in the presence of the drug isoniazid
(INH). Although persistence in these studies was characterized by stable numbers of cells, this
apparent stability was actually a dynamic state of balanced division and death. Single cells
expressed catalase-peroxidase (KatG), which activates INH, in stochastic pulses that were negatively
correlated with cell survival. These behaviors may reflect epigenetic effects, because KatG pulsing
and death were correlated between sibling cells. Selection of lineages characterized by infrequent
KatG pulsing could allow nonresponsive adaptation during prolonged drug exposure.
M
utations and genetic exchange are im-
portant drivers of microbial diversity,
but these events are rare. Often cells
display phenotypic diversity that is not due to
genetic variations (1, 2). This diversity is critical
for microbial persistence in fluctuating environ-
ments because it ensures that some individuals
may survive a lethal stress that would otherwise
extinguish the population. For example, fraction-
al killing of bacterial populations by antibiotics
is attributed to phenotypic variants called per-
sisters (35). Recent work has also uncovered
a similar role for persisters in fractional killing
of cancer cells by cytotoxic drugs (6, 7).
Microbial persistence was identified by Bigger
in early studies on penicillin, which inhibits bac-
terial cell wall biogenesis (8). The ineffectiveness
of penicillin against stationary-phase bacteria
(9, 10) suggested that persisters might constitute
a subpopulation of nondividing cells. This con-
cept was confirmed when preexisting subpop-
ulations of infrequently dividing or nondividing
1
School of Life Sciences, Swiss Federal Institute of Technology
in Lausanne (EPFL), 1015 Lausanne, Switzerland.
2
Laboratory
of Living Matter, The Rockefeller University, New York, NY
10021, USA.
3
Simons Center for Systems Biology and School
of Natural Sciences, Institute for Advanced Study, Princeton,
NJ 08540, USA.
*These authors contributed equally to this work.
Present address: Research Center for Complex Systems Biol-
ogy, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo
153-8902, Japan, and PRESTO, Japan Science and Technology
Agency ( JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012,
Japan.
Present address: Institute of Science and Technology Austria,
A-3400 Klosterneuberg, Austria.
To whom correspondence should be addressed. E-mail:
john.mckinney@epfl.ch
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Escherichia coli cells were shown to be refractory
to ampicillin (11). Stochastic growth rate switch-
ing might be an evolved strategy for survival in
fluctuating environments (12).
Reversible adoption of a slowly growing or
nongrowing state is regarded as a general strategy
for persistence. This assumption has been the ra-
tionale for persister-enrichment methods (13, 14),
evaluation of antibiotic therapies (15), evolutionary
models of genetic resistance (16) and social be-
haviors (17), and drug discovery strategies (18, 19).
Here, we show that persistence is not correlated
with single-cell growth rates in Mycobacterium
smegmatis exposed to isoniazid (INH), which in-
hibits cell wall biogenesis (20). Instead, cell fate is
linked to single-cell dynamics of the INH-activating
enzyme catalase-peroxidase (KatG), which is ex-
pressed in stochastic pulses that are negatively
correlated with survival. KatG pulsing and death
are positively correlated between sibling cells,
which may reflect epigenetic effects.
Fractional killing is characterized by multi-
phasic kinetics of population decay. We confirmed
that INHkilling of M. smegmatis was multiphasic,
comprising delay (d), killing (k), and persistence
( p) phases (fig. S1). We measured INH killing
kinetics at the single-cell level by culturing the
bacteria in a microfluidic device (fig. S2) and track-
ing individual cells by time-lapse microscopy. In
antibiotic-free medium, cells grew and divided to
formmicrocolonies (movie S1). Within ~30 min of
adding INH to the flow medium, all cells mark-
edly slowed their rates of growth and division.
Cell lysis began after ~6 hours and continued
throughout 144 hours of INH exposure (Fig. 1A
and movie S2). When INH was withdrawn,
one of the intact cells in Fig. 1A resumed rapid
growth and division; a second exposure to INH
killed the progeny with kinetics similar to those
of the first exposure (Fig. 1A and movie S2). We
conclude that persistence was due to reversible
phenotypic tolerance rather than stable genetic
mutations.
We tracked the fates of individual lineages
by generating pedigrees for 153 progenitor cells
that were present at the time of INH addition
(Fig. 1B and fig. S3A). After 72 or 144 hours of
INHexposure, 114 or 136 (respectively) of the 153
lineages were extinguished because the founder
cell and all of its progeny were lysed. Unexpect-
edly, divisions continued under INH exposure;
129 of 153 progenitor cells divided at least once.
Figure 1C depicts representative trajectories of
an individual that was killed and an individual
that continued dividing throughout the entire
period of INH exposure. In separate experiments
we confirmed that growth, division, and death
Fig. 1. Multiphasic dynam-
ics of INH-mediated killing.
Bacteria expressing GFP were
imaged on fluorescence and
phase channels for 212hours
at 15-min intervals and ex-
posed to INH (50 mg/ml) at
12 to 156 hours and at 188
to 212 hours. This experi-
ment was repeated10times.
(A) Representative image se-
ries. Numbers indicate hours
elapsed. Scalebar, 5mm. 7H9,
Middlebrook 7H9 medium.
(B) Pedigree tree of cells de-
scended from a single pro-
genitor. Broken lines indicate
INH addition (12 hours) or
withdrawal (156 hours). Bi-
furcations indicate divisions.
End points indicate deaths.
Grayscale in the background
corresponds to slope of pop-
ulation decay curve shown
in (D). (C) Cell size was mea-
sured at 15-min intervals:
(i) nonpersistent lineage, (ii)
persistent lineage. (D to F)
Population dynamics re-
constructed from single-
cell measurements. Broken
lines indicate INH addition
(12hours). (D) Summednum-
bers of surviving cells. (E)
Summed sizes (areas) of sur-
viving cells in arbitrary units.
(F) Mean cell size (black line)
and SD (red lines).
0
2
4
6
8
10
2
4
6
8
0
10
0 20 40 60 80 100 120 140 160
10
100
1000
0 50 100 150
0
2
4
6
8
10
12
0 50 100 150
10
100
1000
0 50 100 150
Time (h) Time (h) Time (h)
M
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(

m
2
)
S
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(
a
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.
)
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Time (h) Time (h)
0 20 40 60 80 100 120 140 160
C
e
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s
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(

m
2
)
(i) Non-persistent lineage
(ii) Persistent lineage
B C
D E F
(d)
(k)
(p)
(d)
(k)
(p)
0 h 12 h
7H9 7H9INH
24 h
INH
36 h
INH
72 h
INH INH7H9
156 h
7H9INH
188 h
INH
212 h
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continued for at least 240 hours under INH ex-
posure (fig. S3B).
Reconstruction of population behavior from
single-cell data revealed that INH-mediated kill-
ing of microfluidic cultures followed first-order
kinetics [y = a exp(bt), where a is the initial cell
number of each phase and y is the cell number
at time t after entering each phase], with rate
constants b = 3.50 (T0.02) 10
2
hour
1
during
the k phase and b = 0.38 (T0.02) 10
2
hour
1
during the p phase (Fig. 1D). During the d phase,
cell numbers increased (Fig. 1D) while aggregate
cell area stayed the same (Fig. 1E), indicating that
INH inhibits growth more rapidly than it inhibits
division. Reductive divisions caused a sharp but
transient decline in mean cell size (Fig. 1F, black
line). Single-cell size variation increased with time
(Fig. 1F, red lines), which suggested that INH
might perturb mechanisms coupling division to
cell size.
We tested the hypothesis that persisters are
preexisting, either slowly growing or nongrowing,
individuals by comparing the pre-INHgrowth rates
of single cells. We found that single-cell growth
was size-dependent, such that larger cells tended
to growfaster (Fig. 2A). The frequency distribution
of cell elongation rates was unimodal and could
be viewed as a Gaussian distribution with an addi-
tional left tail (P=0.28, Kolmogorov-Smirnov test
for the Gaussian null hypothesis) (Fig. 2B). For
cells exposed to INH for 72 or 144 hours, the
mean pre-INH elongation rates of persistent and
nonpersistent individuals were not significantly
different (P = 0.17 or 0.20, respectively, Welchs t
test) (Fig. 2C). We conclude that single-cell growth
rates and cell fates are not correlated in INH-
stressed M. smegmatis, in contrast to ampicillin-
stressed E. coli (11).
Single-cell analysis revealed that the transi-
tion from fast (k phase) to slow ( p phase) killing
was due to a decreasing death rate rather than an
increasing division rate (Fig. 2D). Consequently,
the p phase is a dynamic state in which the rates
of cell division and death are balanced, resulting
in a deceptively static number of viable cells.
When bacteria divide, each newborn cell in-
herits a new pole from the last division and an
old pole from a previous division (Fig. 3A). Ac-
cording to a recent report, old-pole siblings elongate
faster and are more susceptible to antibiotics than
new-pole siblings (21). Although we did not ob-
serve a significant difference in elongation rates
of old- and new-pole siblings (P= 0.16, Welchs t
test) (Fig. 3B), pole age could potentially influ-
ence survival for other reasons.
We tested whether the fates of sibling cells
were correlated by counting the number of obser-
vations of each of the four possible combinations
of cell fates for 1764 sibling pairs (Fig. 3C). A
cell died if it underwent lysis and survived if
it divided to produce two daughter cells. The num-
bers of events in each category (Fig. 3D) were used
to calculate the conditional survival probabilities
of old- and new-pole cells depending on the fate
of the paired sibling. We found that the survival
of sibling pairs was positively correlated (P= 2
10
7
, c
2
test) (Fig. 3E), suggesting the possible
contribution of epigenetic effects. This analysis
also revealed a weak positive association between
survival and inheritance of the old pole (P= 0.027,
c
2
test) (Fig. 3E); this association is the opposite
of that reported by Aldridge et al. (21).
INH is a prodrug that requires activation by
bacterial catalase-peroxidase (KatG) (fig. S4A)
(20). We investigated the relationship between
KatG levels and INH killing with the use of a
strain that expressed katG from an anhydrote-
tracycline (ATc)inducible promoter. Cells grown
with increasing concentrations of ATc expressed
increasing levels of katG mRNA (fig. S4B) and
protein (fig. S4C). ATc-induced cultures were
exposed to INH and the fraction of surviving
colony-forming units was measured. These exper-
iments revealed a sensitive scaling relationship
between KatG levels and INH killing, such that
a factor of ~2 increase in KatG(fig. S4C) resulted
in a factor of ~1000 enhancement of killing at
48 hours (fig. S4D). This effect was not apparent
at 24 hours, indicating that overexpression of
KatG enhanced killing only during the p phase
(fig. S4D).
The sensitivity of INHkilling to small changes
in KatG expression suggested that cell-to-cell
variation in KatG levels might influence cell
fate under INHexposure. We tested this hypothe-
sis in a strain in which chromosomal katG was
replaced by a katG::dsRed2 fusion gene. The
wild-type and katG::dsRed2 strains were equally
sensitive to INH (fig. S5). Unexpectedly, single
cells expressed KatG-DsRed2 in short-lived sto-
chastic pulses (Fig. 4A and movie S3), separated
by intervals in which KatG-DsRed2 fluorescence
barely exceeded the detection threshold (Fig. 4B).
KatG-DsRed2 pulsing was independent of INH,
although cells were brighter in the presence of
the drug (movie S3). Native KatG protein was
less stable than green fluorescent protein (GFP),
which we used as an internal control (Fig. 4C).
These results suggest that active degradation
contributed to the sharp decline in KatG-DsRed2
fluorescence after peak intensity in pulsing cells.
We hypothesized that pulsing cells might be
more vulnerable to INH killing as a consequence
0
0.02
0.04
0.06
0.08
0 20 40 60 80 100 120 140
Death rate
Division rate
D
0
0.1
0.2
0.3
0.4
0.5
0 0.2 0.4
B
0
0.5
1
1.5
2
0 2 4 6 8
0
0.2
0.4
0.1 0.2 0.3 0
0
0.1
0.2
0.3
0.4
0.5
C 72 hours INH exposure 144 hours INH exposure
F
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A
(
t
)

(

m
2
)
F
r
e
q
u
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n
c
y
Frequency
Elongation rate (h
-1
)
Cell size A(t) (m
2
)
P
r
e
-
e
x
p
o
s
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e
l
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a
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i
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a
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(
h
-
1
)
Non-persistent
n = 179
Persistent
n = 41
Non-persistent
n = 112
Persistent
n = 14
R
a
t
e

(
h
-
1
)
Time under INH (h)
n = 312
n = 312
A
P = 0.17 P = 0.20
k p d
Fig. 2. Persistence of single cells under INH exposure. (A) Relationship between cell size A(t) and size
increment DA(t), where Dt = 1 hour. Circles, single-cell measurements; squares, means T SE of DA(t) in
size windows n A(t) < (n + 1) mm
2
, where n is a positive integer. Histograms are frequency distributions
of A(t) (top) and DA(t) (right). (B) Single-cell elongation rates were calculated by measuring cell length
every 15 min from birth until next division and fitting to exponential curves. (C) Single-cell elongation
rates and cell fates under INH exposure. A persistent cell produced one or more progeny that survived; a
nonpersistent cell failed to produce any surviving progeny. Circles, single-cell elongation rates during
the 1-hour interval before INH exposure; squares, means T SD. (D) Rates of division and death under INH
exposure. Divisions (squares) and deaths (circles) were counted at 15-min intervals, normalized by total
cell number, and averaged over 10 hours T SE (see supplementary materials).
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of increased activation of INH. We tested this
idea by comparing the survival probabilities of
pulsing (K = 1) and nonpulsing (K = 0) cells. This
analysis confirmed that pulsing cells were less
likely to survive (P=7.9 10
5
, c
2
test) (Fig. 4D).
We also found that KatG-DsRed2 pulsing was
positively correlated between sibling pairs (P =
7.2 10
8
, c
2
test) (Fig. 4E), which might explain
why cell fate was also correlated between siblings
(Fig. 3E).
Historically, fractional killing has been attri-
buted to subpopulations of nonreplicating bacte-
ria on the assumption that antibiotics targeting
growth processes should not damage growth-
arrested cells (8). Recently, this interpretation has
been challenged by the observation that growth
arrest per se is not sufficient to explain the re-
fractoriness of stationary-phase Pseudomonas
bacteria to antibiotics (22). In populations of ex-
ponentially growing mycobacteria, we found no
correlation between single-cell growth rates and
cell fates, inasmuch as slowly growing individ-
uals were as likely to die (or persist) as rapidly
growing individuals. These observations do not
rule out the possibility that in other systems, per-
sistence may be linked to single-cell growth rates,
as in E. coli exposed to ampicillin (11).
Although bacterial numbers are relatively
constant during the p phase of INH killing, we
found that this stability masks a dynamic state
of balanced division and death. Ongoing division
could promote the emergence of resistant genetic
variants because the rate of spontaneous mutation
increases in proportion to the division rate (23).
It has been argued that resistant mutants are un-
likely to arise de novo under antibiotic exposure
on the assumption that persistent cells are non-
replicating (16, 24). Our discovery that persistent
cells may grow and divide in the presence of an
antibiotic necessitates a reexamination of how
persistence might enhance the generation of re-
sistant mutants.
The link between stochastic expression of katG
and the fate of single cells under antibiotic stress
might provide insight into the generality of per-
sistence. In principle, stochastic expression of any
factor that facilitates or opposes the action of an
antibiotic could influence the fate of single cells.
The additive contribution of independently fluc-
tuating factors might explain why the link be-
tween KatG-DsRed2 pulsing and death, although
significant, was not absolute. Consistent with
this view, a recent study showed that expression
of E. coli HipA toxin from a noisy promoter
generates supra-threshold fluctuations that pro-
tect a fraction of cells from killing by ampicillin
(25). These observations raise the possibility that
natural cell-to-cell variation in HipAlevels might
influence survival probability under antibiotic
pressure.
Because KatG activates INH, the observed
low frequency of KatG pulsing should increase
cell-to-cell variation of INH activation (fig. S6A)
relative to other modes of KatG expression, such
as high-frequency pulsing (fig. S6B) or constitu-
tive expression (fig. S6C). This prediction is con-
sistent with our observation that pulsing and
nonpulsing individuals have different survival prob-
abilities. Because low-frequency, high-amplitude
pulsing should broaden the single-cell frequen-
cy distribution of activated INH concentrations
(fig. S6D), this regime is more likely to generate
lineages that persist because they fail to activate
INH to lethal levels.
Although the mechanistic basis of KatG
pulsing is unknown, we envisage several possi-
bilities. First, recent work in E. coli has estab-
lished that single-cell gene expression is inherently
stochastic and bursty (26). Burst frequency and
amplitude can be tuned by nucleotide changes
within the nucleic acid sequences that specify
the efficiencies of transcription and translation
(27). Thus, KatGpulsing may simply reflect low-
frequency, high-amplitude stochastic bursting.
Second, recent work in Bacillus subtilis demon-
strated that stochastic pulses of transcription are
generated by a network architecture comprising
a noise-triggered ultrasensitive switch coupled
to mixed (positive and negative) feedback loops
operating on different time scales (28). Similarly,
KatG pulsing might reflect the operation of net-
work motifs that randomly switch the katG pro-
moter between on and off states. Third, in
many bacteria, transcription of katG is induced
by oxidative stress (29). Thus, stochastic produc-
tion of reactive oxygen species during respiratory
metabolism (30) could trigger transient pulses of
katG transcription. In each of these hypothetical
scenarios, instability of KatG would contribute
to a sharp rise of KatG levels at the initiation of
a transcriptional pulse and a sharp fall of KatG
levels at the termination of such a pulse.
INH-mediated selection of cell lineages char-
acterized by infrequent pulsing of KatG could
result in gradual adaptation of the population as
less-fit phenotypic variants are eliminated. It is
also plausible that antibiotics or other extrinsic
stimuli might trigger adaptive responses; indeed,
environmental factors have been shown to mod-
ify the efficiency of antibiotic killing in several
systems (3138). However, sense-and-respond
adaptive strategies are expensive because the
requisite molecular machinery must be expressed
constitutively (39). In contrast, nonresponsive,
selection-mediated adaptation could provide a
simple mechanism for cell populations to adapt
to stresses that they had never encountered in
A
C D
E
? 0 0 1 0 1 0 2 0 0 2 1 0 0 1 ?
0 0 1 ? 0 1 0 ?
0 ? 0 ?
? ?
4
3
2
1
G
e
n
e
r
a
t
i
o
n
Progenitor
B
0
0.1
0.2
0.3
0.4
0.5
P(S
o
=1 | S
n
=0)
0.32 0.03
P(S
n
=1 | S
o
=0)
0.28 0.03
P(S
o
=1 | S
n
=1)
0.44 0.04
P(S
n
=1 | S
o
=1)
0.40 0.04
P(S
o
=1)
0.360.02
Mother
cell
S
n
=0 S
o
=1
P(S
n
=1)
0.320.03
Mother
cell
S
o
=0
P(S
n
=1)
0.320.03
Mother
cell
Old-pole New-pole
E
l
o
n
g
a
t
i
o
n

r
a
t
e

(
h
-
1
)
S
n
=1
(Survival)
S
n
=0
(Death)
S
o
=1
(Survival)
S
o
=0
(Death)
Old-pole
New-pole
Total
Total
252 316
378 818
568
1196
630 1134 1764 (S
o
,S
n
)=(1,1)
Old New
(S
o
,S
n
)=(1,0)
Old New
(S
o
,S
n
)=(0,1)
Old New
(S
o
,S
n
)=(0,0)
Old New
Mother
cell
P(S
o
=1)
0.360.02
S
n
=1
Fig. 3. Positively correlated survival of sibling pairs. (A) Each individual inherits a new pole from the
most recent division (age 0) and an old pole from a previous division (age 1). In time-lapse
experiments, pole ages can be assigned from the third generation onward (white cells), excluding the
poles of unknown age inherited fromthe progenitor cell (gray cells). (B) Elongation rates of old- and new-
pole siblings. Circles, elongation rates of single cells pre-INH; squares, means T SD, which were 0.22 T
0.06 hour
1
for old-pole cells (n = 112) and 0.21 T 0.07 hour
1
for new-pole cells (n = 112). (C) Four
possible fates for a sibling pair. S = 1 if the cell survives until next division; S = 0 if the cell dies before
dividing; S
o
, old-pole sibling; S
n
, new-pole sibling. (D) Number of single-cell observations of the four fate
outcomes (n = 1764 cells). (E) Sibling pair correlation of cell fates. P(S = 1), total survival probability;
P(S
o
= 1), survival probability of old-pole cells; P(S
n
= 1), survival probability of new-pole cells. Numbers
indicate survival probabilities T 95% confidence intervals.
4 JANUARY 2013 VOL 339 SCIENCE www.sciencemag.org 94
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their evolutionary histories, or stresses that were
encountered too infrequently to repay the invest-
ment in responsive mechanisms.
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Acknowledgments: Supported by fellowships from the
Charles H. Revson Foundation and the Heiser Program for
Research in Leprosy and Tuberculosis of the New York
Community Trust (N.D.); a Harvey L. Karp Discovery Fellowship
and the JST PRESTO Program (Y.W.); and the Bill and
Melinda Gates Foundation, NIH grant HL088906, and Swiss
National Science Foundation grant 310030_135639 ( J.D.M.).
Supplementary Materials
www.sciencemag.org/cgi/content/full/339/6115/91/DC1
Materials and Methods
Figs. S1 to S6
Movies S1 to S3
References (4043)
6 September 2012; accepted 15 November 2012
10.1126/science.1229858
Fig. 4. Single-cell KatG
pulsing and cell fate. Bacte-
ria expressing KatG-DsRed
were imaged on fluores-
cence and phase channels
for 88 hours at 15-min in-
tervals and exposed to INH
(50mg/ml) at 16to88hours.
This experiment was repeated
five times. (A) Representa-
tive image series. Numbers
indicate hours under INHex-
posure. Scale bar, 5 mm. (B)
Single-cell pedigrees (upper
panels) and time traces of
KatG-DsRed fluorescence in
arbitrary units (lower pan-
els) for nonpersistent (left)
and persistent (right) line-
ages. Black points in upper
panels indicate pulse peaks.
(C) KatGstability. Cells were
pulse-labeled with [
35
S]Met
and [
35
S]Cys and chased
A
2.0 h 4.0 h 5.5 h 7.0 h
B
E
P(K
o
=1 | K
n
=0)
0.43 0.11
n = 84
P(K
n
=1 | K
o
=0)
0.41 0.11
n = 81
P(K
o
=1 | K
n
=1)
0.78 0.07
n = 149
P(K
n
=1 | K
o
=1)
0.76 0.07
n = 152
P(K
o
=1)
0.650.06
Mother
cell
K
n
=0 K
o
=1
P(K
n
=1)
0.640.06
Mother
cell
K
o
=0
P(K
n
=1)
0.640.06
Mother
cell
Mother
cell
P(K
o
=1)
0.650.06
K
n
=1
D
0 0.5 1.0
Chase time (h)
C
62
49
28
Survival
probability
Conditional
survival probability
P(S=1)
0.21 0.04
n = 488
P(S=1 | K=1)
0.16 0.04
P(S=1 | K=0)
0.31 0.07
Yes
K=1
n = 313
No
K=0
n = 175
Pulse
Non-persistent lineage Persistent lineage
L
a
b
e
l
e
d

p
r
o
t
e
i
n

(
%
)
Chase time (h)
INH INH INH INH
0
2
4
6
8
0 10 20 30 40 50
Time under INH (h)
0 10 20 30 40 50
Time under INH (h)
with unlabeled Met and Cys. Left: Pull-downs of
cell lysates with antibody to KatG (upper panel) or
GFP-Trap (lower panel) were analyzed by SDS
polyacrylamide gel electrophoresis. Right: Band
intensities were quantified by scanning auto-
radiograms. Half-lives were ~0.7 hours for KatG
versus ~5.7 hours for GFP. This experiment was re-
peated twice. (D) Negatively correlated KatG-DsRed
pulsing and cell survival. K = 1, pulse; K = 0, no
pulse. Values are survival probabilities T 95%con-
fidence intervals. (E) Sibling pair correlation of
KatG-DsRed pulsing. K
o
= 1, old-pole cell pulsed;
K
n
= 1, new-pole cell pulsed (pulsing indicated
by red outlining). Values are pulse probabilities T
95% confidence intervals.
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