Group Title: PLoS One
Title: Heterogeneous Response to a Quorum-Sensing Signal in the Luminescence of Individual Vibrio fischeri
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Title: Heterogeneous Response to a Quorum-Sensing Signal in the Luminescence of Individual Vibrio fischeri
Series Title: PLoS One
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Creator: Pablo Delfino Pérez
Stephen J. Hagen
Publisher: Public Library of Science
Publication Date: 2010
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Resource Identifier: 10.1371/journal.pone.0015473

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OPEN ACCESS Freely available online


Heterogeneous Response to a Quorum-Sensing Signal in

the Luminescence of Individual Vibrio fischeri

Pablo Delfino Perez, Stephen J. Hagen*
Physics Department, University of Florida, Gainesville, Florida, United States of America


Abstract
The marine bacterium Vibrio fischeri regulates its bioluminescence through a quorum sensing mechanism: the bacterium
releases diffusible small molecules (autoinducers) that accumulate in the environment as the population density increases.
This accumulation of autoinducer (Al) eventually activates transcriptional regulators for bioluminescence as well as host
colonization behaviors. Although V.fischeri quorum sensing has been extensively characterized in bulk populations, far less
is known about how it performs at the level of the individual cell, where biochemical noise is likely to limit the precision of
luminescence regulation. We have measured the time-dependence and Al-dependence of light production by individual
V.fischeri cells that are immobilized in a perfusion chamber and supplied with a defined concentration of exogenous Al. We
use low-light level microscopy to record and quantify the photon emission from the cells over periods of several hours as
they respond to the introduction of Al. We observe an extremely heterogeneous response to the Al signal. Individual cells
differ widely in the onset time for their luminescence and in their resulting brightness, even in the presence of high Al
concentrations that saturate the light output from a bulk population. The observed heterogeneity shows that although a
given concentration of quorum signal may determine the average light output from a population of cells, it provides far
weaker control over the luminescence output of each individual cell.

Citation: Perez PD, Hagen SJ (2010) Heterogeneous Response to a Quorum-Sensing Signal in the Luminescence of Individual Vibrio fischeri. PLoS ONE 5(11):
el5473. doi:10.1371 /journal.pone.0015473
Editor: Michael N. Nitabach, Yale School of Medicine, United States of America
Received August 3, 2010; Accepted September 27, 2010; Published November 16, 2010
Copyright: 2010 Perez, Hagen. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: National Science Foundation award MCB 0347124 (http://www.nsf.gov). The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
E-mail: sjhagen@ufl.edu


Introduction

Numerous bacterial species use a form of chemical communi-
cation known as quorum sensing (QS) to regulate gene expression
[1]. The bacteria synthesize and release small diffusible molecules
known as autoinducers, which accumulate as the bacterial
population density grows. As their concentration rises, the
autoinducers activate transcriptional regulators that trigger
important phenotypic changes in the cells. QS therefore allows a
population-sensitive switch between different phenotypic states [1].
However, 1l ...n 1, QS is most easily interpreted as a population-
counting behavior, QS pathways are typically complex, often
employing multiple autoinducer signals and receptors. They may
also interact with other physical and biological parameters of
the organism's environment in addition to the population density
[2-5].
The complexity of these pathways raises questions about how
bacteria use QS to probe their environment and exactly what types
of information they may -,1 .. :1i ..... 1 this mechanism.
Understanding the capabilities and fundamental limitations of
QS requires detailed experimental and theoretical studies of QS
systems at the level of individual cells. The goal of this study is to
characterize the overall performance of QS at the single-cell level
in one important model organism. We aim to measure the
precision with which an individual Vibrio fischeri cell converts a
well-defined QS signal input to a bioluminescence output.
Vfischeri is a Gram-negative marine bacterium that regulates its
own bioluminescence i....._ 1. QS [6]. The luminescence is


PLoS ONE | www.plosone.org


produced by a bacterial luciferase that utilizes FMNH2, 02, and
a long-chain aldehyde as substrates. At low cell densities, as in
open seawater, the lux genes that synthesize the luciferase and
substrates are switched off and the bacterial cells are dark.
However, the bacterium also colonizes the light organs of fish and
squid species, where it attains high cell densities and the lux genes
become strongly induced. In the light organ of its symbiotic host
squid Eupjymna scolopes, Vfischeri may attain 101010 cells/cm3
and a single cell may emit -103 photons/s [7].
Studies of bulk populations of Vfischeri have revealed an
intricate molecular mechanism for this population-sensitive switch
[6,8]. The QS pathway employs three autoinducer synthases, three
corresponding autoinducers, and three cognate receptors [8]. The
full pathway integrates the separate autoinducer signals to regulate
not only the luminescence behavior but also other phenotypes
related to colonization of the symbiotic host [9]. Of the three
signal channels, the LuxI/LuxR pathway shown in Figure 1A has
been the subject of the most extensive study. It consists of an
autoinducer synthase LuxI, an autoinducer (N-3-oxohexanoyl-L-
homoserine lactone, 30C I.HHT and the transcriptional activator
LuxR, as well as the luminescence genes luxCDABEG. When the
concentration of 3OC6HSL is sufficiently high, it forms a complex
with LuxR that activates transcription of the lux operon, leading to
luciferase synthesis and bioluminescence. The other two QS
pathways (not shown in Figure 1A) detect a second homoserine
lactone autoinducer \ .... i .. I-L-homoserine lactone, C8HSL)
that is produced by a synthase AinS and a third autoinducer AI-2
(as in V. harveyi [8]) that is produced by LuxS.


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Single-Cell Luminescence of Vibrio fischeri


Cell exterior


Figure 1. Schematic of Luxl/LuxR regulation of V.fischeri
bioluminescence, and bulk response. (A) Luxl synthesizes the
autoinducer Al (N-3-oxohexanoyl-L-homoserine lactone) which binds to
LuxR, the transcriptional activator for the luminescence genes luxCDABE
[6]; (B) Luminescence response of a bulk culture of V.fischeri strain MJ11
growing in defined medium at room temperature. The points show the
response of a (bulk) population of exponential phase cells in a 48-well
plate, following addition of exogenous autoinducer (Al) at time t=0.
After 70 minutes an Al-dependent response is developing. After 130
minutes the response has reached a steady state. Data for t2>130
minutes are fit to a cooperative binding model (black dotted curve) to
give an equilibrium constant Keq=200+10 nM and Hill coefficient
n= 2.60.4. Luminescence data are normalized to the optical density at
600 nm to give the luminescence per cell, in arbitrary units.
doi:10.1371/journal.pone.0015473.g001


Because it was the first known example of a Gram-negative QS
system and remains one of the best understood, Luxl/LuxR has
been a model system for theoretical and computational studies of
the dynamics of quorum regulation. Several authors have
modelled its deterministic dynamics [10-14] as well as the
stochasticity [15-17] arising from the biochemical noise in gene
expression [18]. The deterministic models characterize the
stability of the "on" and "off' states of Luxl/LuxR luminescence
as well as the dynamics of switching and hysteresis. Experiments
on bulk cultures can provide a suitable test of such models [14].
However, bulk studies measure only average properties of the
population. They do not address stochasticity and they do not
reveal exactly what information the individual cell ii1,. in
probing its environment with a QS mechanism. In particular, the
accuracy of the QS pathway as a sensor of the individual cell's
environment and as a regulator of phenotype, and the impact of
stochasticity on QS, can only be tested by experimental
measurements on individual cells [14,19-21]. Here we ask how
accurately the autoinducer signal input to a single cell defines or
predicts the bioluminescence output from that cell.
A single-cell study of Vfischeri presents technical challenges, as
the bioluminescence emission from individual bacterial cells is
exceedingly weak and has rarely been measured quantitatively
[22-25]. The light output from one V.fischeri cell is estimated to lie
in the range from 10 2 to 104 photons/s, depending on the strain,


1- PLoS ONE | www.plosone.org


A ._.* Al


Figure 2. Individual V.fischeri imaged in dark field and
bioluminescence. (A) Dark field (externally illuminated) and (B)
bioluminescence (light emission) images of V.fischeri cells adhered to
the glass window of the perfusion chamber at 24C in the presence of
500 nM exogenous Al. The cells appear as rods (-3-5 pm long) in the
dark field image. The bioluminescence image shows in false color the
luminescent emission detected in a 16 minute exposure. Images were
collected in an inverted microscopy configuration with an intensified
CCD camera and a 100x oil immersion objective.
doi:10.1371/journal.pone.0015473.g002


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t I Cell interior





\ /
luxR CDABE


.... I . .... I ..... .

O 70 minutes ..Q
A 130 /
V 160 / /
S190 /
- .i

.l i..l f ^... ....I . ... .I .


the environment, and whether the culture is fully induced by its
multi-input QS system [7,26]. Only a fraction of this photon flux
can actually be collected, and therefore the measurable flux from
one cell is typically weaker than the signal that can be collected
from even a single molecule of a fluorescent reporter like EGFP
[27,28]. Under stable conditions and with sufficiently long
integration times, however, the luminescence from one cell can
be measured with a photomultiplier [24] or with an intensified or
cryogenically-cooled CCD camera -'-'-' We used an
intensified camera and long image exposures (10-15 minutes) to
track the bioluminescent emission from individual cells of Vfischeri
strain MJ11. The cells were immobilized on the window of an
observation chamber that was continuously perfused with medium
containing exogenous 3OC6HSL autoinducer (Al), so that each
cell was subject to a precisely defined local AI concentration.
Tracking individual cells over periods of several hours, we found
that cells differ widely in the time scale of their bioluminescence
response and in the overall intensity of that response. Hence, while
QS can coordinate and synchronize the average luminescence
output of the bacterial population, it has relatively imprecise
control over the response of an individual cell.

Results

Individual bacterial cells emit very weak bioluminescence and
the corresponding signal levels are far weaker than (e.g.) the
fluorescence that is typically collected from a cell expressing GFP.
Therefore, as explained in the Materials and Methods and Text S1,
we used several procedures to ensure that the microscopy imaging
and alignment were stable over the 3-4 hr period of luminescence
observations and that any observed heterogeneity in the light
output from individual cells was not a detection artifact. We
verified that the cells remained stationary and in focus during
imaging (Figure SI), that the observed variations in luminescent
emission were larger than our measurement uncertainties (Figure
S2), and that the camera, microscope, and images were physically
stable over periods of 4 hours or longer.
In the absence of exogenous autoinducer the Vfischeri cells in the
perfusion chamber produced no detectable luminescence. How-
ever, when at least -50 nM of autoinducer (AI, 3OC6HSL) was
provided in the flowing medium the luminescence of individual
cells was clearly resolved. Figure 2 compares dark-field (i.e.


B




c 4
O8




S2
E
3


10 100 1000
Al concentration (nM)






Single-Cell Luminescence of Vibrio fischeri


externally illuminated) and bioluminescence (i.e. luminescence
emission without external illumination) images of individual V.
fischeri cells adhering to the glass window in the presence of
500 nM AI Qualitatively the image already suggests that different
cells emit with different intensities, even at a high AI concentration
that saturates the output of the bulk population (Figure 1B). A
quantitative analysis of all data confirmed that the brightness of
the cells was heterogeneous at all autoinducer concentrations
studied (0-1000 nM AI). At 1000 nM AI we found many
individual cells i 1... -little light during a ten minute exposure,
even 1 .... 1, we observed these same cells growing and dividing
during the -4 hr duration of observation.
Studies of bulk cultures under our .. i 11 conditions established
that the shape of the luminescence versus AI response curve was
established within 2-3 hrs following introduction of AI
(Figure 1B). Therefore, an observation period of ~3-4 hrs in a
perfusion chamber should be sufficient to observe the response of
individual cells to introduction of AI Figure 3 shows the time
course of the luminescence collected from an ensemble of
individual cells. The luminescence of each cell is tracked over
time 1.1 .. ..1 a series of 10-minute camera exposures (see Materials
and Methods), following the introduction of exogenous AI at t= 0.
The initial response of the cells (0 transient increase or decrease in average luminescence, as the AI
concentration in the perfusion chamber may be greater or less
than in the starting culture. On a longer time scale (t= 150-250
minutes) the cells attain average emission levels that are consistent
with the supplied concentration of exogenous AI However a large
degree of cell-to-cell variability is apparent. The brightness of the
different cells diverges over time, with many cells luminescing at
very modest levels while a small fraction of cells emit much more
.1 ;1,11
Heterogeneity is also apparent in the time scale of response.
Figure 3 shows that, when a high AI concentration (1000 nM) is
introduced at t= 0, some cells begin to respond quickly, with 250
350 photons/minute detected after 250 minutes. Other cells
however are only beginning to respond after ~ 150 min. Figure 4
shows the progression of the brightness distribution as a group of
cells responds to the introduction of 1000 nM AI. The variability
in the time scale of response (the kinetic heterogeneity) can be
summarized by the distribution of the onset time t,/2, which we
define as the time at which the luminescence of a particular cell is
halfway between its initial (t= 0) and final (t 250 minutes) values.
Figure 5B shows that tl/2 has a very broad and flat distribution at
200 nM, and this distribution remains broad even at a saturating
AI concentration of 1000 nM.
Hence we observe several types of heterogeneity in the response
of Vfischeri to a defined AI concentration. Cells in the same
environment respond on widely 1i; -;,i time scales when AI is
introduced, and they also differ in the overall amplitude of that
response. Furthermore the individual trajectories of Figure 3
suggest that the luminescence of at least some cells occasionally
fluctuates by --20-40% on time scales of -30 minutes.
As shown in Text S1 and Figure S3, our experimental
configuration also allows us to observe other kinetic and steady
state phenomena in single-cell Vfischeri luminescence, such as the
"rich medium effect" [30-32]. However we focus here on the
heterogeneity of the QS response.

Discussion
The luminescence of Vfischeri is activated 1l1..n_.. a quorum
sensing (QS) mechanism in which the cells remain dark until their
population reaches the high densities that signify colonization of


PLoS ONE | www.plosone.org


400 Al= 50 nM

added at t= 0
200



S400OOM
100 nM
U


C 200
00
6 400 200 nM

200


0
o 400
E 1000 nM
_0
a 200-
5



control

200


0
0 50 100 150 200
time (minutes)

Figure 3. Luminescence of individual V.fischeri cells following
addition of autoinducer, and detection stability test. At each
autoinducer concentration, roughly 25-40 MJ11 cells were imaged
repeatedly over a period of -4 hrs following introduction (at t= 0) of
exogenous autoinducer Al at the indicated concentration. The light
emission from each cell was quantified through analysis of a series of
10-minute camera exposures (see Materials & Methods). The state of
induction of the initial cell culture determines the luminescence of the
cells at t=0. However, once adhered in the flow chamber and
exposed to the flow of medium (containing exogenous Al), the cells
respond by adjusting their luminescent output. This leads to a
transient increase or decrease in the emission over the next -1-2 hrs.
After -3 hrs the cells have adapted to the applied Al level. The
control shows an experimental verification of the stability and
sensitivity of microscopy and data analysis. For this measurement,
green fluorescent latex spheres were illuminated with a severely
attenuated blue light source and then imaged with the same camera
settings, magnification, 10-minute exposure time, and data analysis,
as used for the V.fischeri measurements. Image focus and excitation
intensity were not adjusted during the 4 hr measurement. Twelve
representative trajectories are shown. See Materials and Methods and
Text S1. The time-dependence of all emission versus time "trajecto-
ries" in this figure has been smoothed by a Gaussian filter with width
o= 10 minutes.
doi:10.1371/journal.pone.0015473.g003


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Single-Cell Luminescence of Vibrio fischeri


A 0.6
0.3

0.6
0.3

S0.6
8 0.3

o 0.6
0.3
0.6

0.33


0.6
0.3
0.6



0 2
Detected emissic
B 140
.2 120
S100
Ss
.| 80
E 60
0 40 3. 3
T a .,/ /1-- 2^-s,


A 0.2 AI=OnM
A= 50 nM


t = 27 min


71 min


3
100 min


4
131 min


0

0.'2
A Al= 200 nM




0=
0.2
Al = 1000 nM

1 2 5 10 20 50 100 200
Detected emission (photons/minute/cell)


159 min


186 min
ii


7
214 min

00 400
n (phot/min/cell)


7
50%


=----- 25%
50 100 150 200
time (minutes)


Figure 4. Spreading of the luminescence histogram over time.
(A) Cell brightness histograms for MJ11 cells at the indicated times,
following introduction of 1000 nM Al at t= 0. (B) Median (red curve) cell
brightness and the 25% and 75% percentiles of brightness (blue
curves). The distribution of intensities broadens as the cells response to
the exogenous Al signal. A substantial fraction of the cells emit near the
detection threshold (-10-20 photons/minute/cell) even at t=4 hrs.
doi:10.1371/journal.pone.0015473.g004


the light organ of the symbiotic host. Here we ask how ;l-11 this
QS system regulates the luminescence output of an individual cell
in response to a defined chemical signal (i.e. the 30C6HSL
autoinducer concentration). We find that an ensemble of cells
produces a distinctly heterogeneous response to the AI input, with
significant cell-to-cell variability in the overall level of emission and
in the onset time for this response, as well as indications of short
term fluctuations in brightness.
In the absence of exogenous AI the light emission from the cells
was below measureable levels. However, after ~ 150-250 minutes
in exogenous AI the individual cells were ;_ .i. i1 brighter on
average, as in a bulk culture. The addition of AI not only increases
the average brightness, but also increases the (absolute) differences
in the brightness of individual cells; hence the individual brightness
levels eventually span an order of magnitude, as shown in
Figure 5A. Similarly the luminescence onset time ti/2 shows a
broad distribution at both 200 nM and 1000 nM AI (where the
response of the bulk population in Figure 1B is seen to saturate).
As the distributions for both the individual cell intensities and the


0 20 40 60 80 100120140160180200220240260
t/2 (minutes)

Figure 5. Histograms of luminescence levels and onset times at
high autoinducer concentration. (A) Distribution of luminescence
levels detected for individual V.fischeri cells, at time t=240 minutes
after autoinducer (Al, 3OC6HSL) was introduced at concentrations
indicated. Cells emitting -10-20 photons/minute are at the measure-
ment uncertainty, i.e. are consistent with no emission. (B) Distribution of
luminescence onset times t1/2 in the presence of 200 nM and 1000 nM
Al. The onset time t1/2 is the time at which the luminescence output 1(t)
of a particular cell is halfway between its initial value I(t= 0) and its final
value l(t 250 minutes), when Al was introduced at t=0.
doi:1 0.1371/journal.pone.0015473.g005


onset times in Figure 5B are not at all clustered about the mean
values they are clearly not Gaussian (normal) distributions.
Nevertheless these single-cell data are still consistent with the
behavior of a bulk culture, as can be seen by comparing the AI
response curves of single cells and a bulk culture under the same
_i.. 11 conditions. Figure 6B shows that a nonlinear least squares
fit of a cooperative binding model to the single-cell data gives an
equilibrium constant iK, 120-20 nM and a Hill coefficient
n 2.7-0.8. By comparison, the average luminescence of a bulk
culture of the same strain (Figure 1B) gives K, 200- 10 nM and
n 2.6-0.4. The smooth AI-induced luminescence response of the
bulk population is a result of averaging over large numbers of cells;
it conceals a very heterogeneous character in the response of
individual cells in that population.
We find it remarkable that such large variations in emission
persist in a homogeneous Al environment, even several hours after
introduction of the exogenous signal. Even 1 ..... 1. we anticipate
that stochasticity will generate cell-to-cell variability, the coefficient
of variation (cv= standard deviation/mean -1) in Figure 6A
appears much greater than is expected from stochastic simulations
of the LuxI/LuxR system. For example, Cox et al. estimated the
kinetic parameters for a chemical model of the LuxI/LuxR
network [15]. Their stochastic simulations predicted relatively
modest variability in the activation of luxl as a function of AI


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- I


m


m


C ,~,,,, "


,.< ', PLoS ONE I www.plosone.org






Single-Cell Luminescence of Vibrio fischeri


Ai2
A 2






0


100
E -
E 4
10

O-
0[ j


y 0

COctrorl


*

Fit: Keq= 120 20 nM
n=2.70.8
Bulk: K,, =200 10 nM ,
n = 2.6 0.4
0 200 400 600 800 1000
Al (nM)


Figure 6. Variation and mean of luminescence levels versus
autoinducer concentration. (A) Coefficient of variation (cv=stan-
dard deviation/mean) in the luminescence of different cells. Variation is
calculated from emission levels recorded t >100 minutes after
introduction of exogenous Al; (B) Luminescence emission detected
from 188 individual cells (blue circles) after 150-250 minutes exposure
to Al. Data for each Al concentration represents a different group of
cells. Solid curve (blue) is a fit to a cooperative binding model, giving
Ke, 120+20 nM and Hill coefficient n 2.740.8. For comparison with
the expected average behavior, the dashed curve (red) shows the Al
response that is obtained from a bulk population after 150-250 minutes
in autoinducer (Figure 1).
doi:1 0.1371/journal.pone.0015473.g006

concentration. A\lilM.....1 the LuxI concentration was variable at
low (<50 nM) AI concentrations, the simulations predicted
minimal fluctuation, with a standard deviation less than ~10%
of the LuxI concentration once the AI concentration reached the
induction threshold. By contrast we find a large variation in light
output persisting across the AI induction curve. The cv of the
luminescence is near unity even at 1000 nM AI This variability is
presumably not attributable to heterogeneity in intracellular AI
concentrations, as the Aldiffuses rapidly across the cell membrane
[32] and the exogenous Al level is well-controlled by the flow of
medium.
Our emission versus time trajectories also show some evidence
for short-term fluctuations in the single-cell luminescence. The
time series data of Figure 3 suggest that the light output from
some cells occasionally fluctuates by ~20-40%. Furthermore,
while the brightness of each cell is reasonably stable on short time
scales, the brightness of one cell is poorly correlated with its
brightness ~30-60 minutes later (Text S1 and Figure S4). An
early study of the time dependence of V fischeri luminescence
found no significant oscillation or pulsing in the luminescence
output at frequencies 0.01-10 Hz, ,ll....1, it did not investigate
the low frequency behavior (~lu- Hz) studied here [24].
Whether the noise in lux gene expression does in fact have a
bursting or intermittent character under stable environmental
conditions is an intriguing question that requires further study.
However the short time scale of these fluctuations suggests that
they originate in intrinsic (i.e. purely biochemical stochastic) noise
[33]. By contrast the slower intercellular variability in the onset
times for AI response and in the overall luminescence output is
more suggestive of extrinsic noise originating in the variable
concentrations of cellular components such as ribosomes,
polymerases, or in different stages in the .,.-, 11, cycle, etc. [34].
A recent study of the QS bioluminescent emission of individual
V harveyi also found very substantial cell-to-cell variability [19].


PLoS ONE | www.plosone.org


Anetzberger et al. allowed V hareyi cells to accumulate their own
autoinducer for intervals up to 8 hours and reach quorum
conditions. This produced an approximately bimodal response,
with many cells luminescing '.i;_ll while roughly 25% of live
cells remained relatively dark, or roughly one-tenth as bright as the
more luminescent cells. Alil..... 1, the LuxI/LuxR pathway probed
here has a different structure from the lux regulatory system of V
hareyi (i.e. LuxI/LuxR does not directly include the type of
phosphorelay switch and sRNA regulation found in V.harvey),
these findings are similar to ours: after several hours in Al, roughly
25% of V fischeri cells were emitting luminescence at or below our
detection limits (Figure 4B). Our results show that this
heterogeneity occurs across a range of AI concentrations and also
extends to the kinetics of the onset of luminescence.
However another recent single-cell study of the V haroeyi QS
pathway found a more homogeneous response to autoinducer
[20]. Long et al. constructed a qrr4-gfp transcriptional fusion that
allowed them to use GFP fluorescence -rather than the native
luminescence to monitor the effect of two autoinducer signals on
the activation of the quorum-regulatory RNAs that are controlled
by the phosphorylation of LuxO. LuxO phosphorylation is in turn
regulated by the three autoinducer receptors in Vhaveyi. Long
et al. found much less variance in the response of different cells at
the same autoinducer concentrations than Anetzberger et al.
observed in the bioluminescence response, and much less than we
report here in Vfischeri luminescence. For the two different AI
receptor mutants (each responsive to a single autoinducer) that
they studied, they observed a coefficient of variation cv ~0.2-0.4
in the gfp expression, ;_;.r. i11 smaller than the cv -1 that we
observe here in Vfischeri luminescence.
To explain the observation of heterogeneity in the luminescence
(but not in the gfp reporter strains) of Vharveyi, Anetzberger et al.
suggested a possible role for positive feedback in the Vharveyi
master regulator LuxR (not homologous to Vfischeri LuxR), which
is regulated by the sRNAs and controls expression of the lux genes
for luminescence. They proposed that the absence of autoinducer
synthases in the GFP reporter strains eliminated possible feedback
loops involving AI synthesis and detection, leading to a more
homogeneous behavior in those strains. The fact that our system
defines the AI concentration exogenously -also eliminating AI
feedback -yet still exhibits heterogeneity argues against this
interpretation. However a role for feedback in the observed noise
is nevertheless plausible in LuxI/LuxR. Williams et al. recently
studied the dynamics of AI sensing by an E.coli model strain luxOl,
in which LuxR is activated by 3OC6HSL to control the expression
of gfp while the autoinducer synthase LuxI is absent [14]. Cell
cytometry studies found a bimodal response of gfp expression to the
AI signal level, with the more responsive cells exhibiting a roughly
log-normal distribution in GFP fluorescence. They argue that the
external AI concentrations feed into an autoregulatory feedback
loop for LuxR expression, and that this generates hysteresis in the
LuxI/LuxR system's response to AI. That is, its activation at any
particular Al concentration depends in part on its prior history and
initial LuxR levels. This LuxR mechanism would help to explain
some of the cell-to-cell variability that is observed in the
luminescence onset time in Figure 5B, as natural stochastic
variations in initial LuxR levels would be amplified by feedback to
give large changes in activation of the luminescence genes.
Alternatively it is possible that the heterogeneity in light output
results from some differences in the energy resources of different
cells, with some cells in bright (energy-intensive) states and others
in dark (recovering) states. However our data suggest that the
overall luminosity state 1.1 ; i I .. or darker) of a cell tends to persist
over relatively long periods of hours, comparable to the doubling


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Single-Cell Luminescence of Vibrio fischeri


time. Cycles of energy depletion and recovery would presumably
play out over shorter time scales. We also found that the output
variability was not due to a shortage of the C14 long chain
substrate needed for the luciferase reaction (see Text Si).
Furthermore, heterogeneity was not exclusive to a luminescence
reporter of the LuxI/LuxR system: under full induction of Luxl/
LuxR, the expression of a gfp reporter by Vfischeri mutant JB10
showed heterogeneity (c = 0.8) similar to that of the biolumines-
cence (Figure 7). These points suggest that cell-to-cell variability
in luminescence response is not primarily due to a deficiency of the
luminescence substrate or energy resources.
Our findings raise some interesting questions about the
performance of Vfischeri QS at the single cell level. For example,
the broad heterogeneity in the light output from the cells -which
always remained short of the estimated maximum output of
~1000 photons/s/cell [7] -raises the question of whether the
observed heterogeneity is still present in cultures emitting at
maximum brightness (e.g. within the symbiotic host). It would also
be interesting to determine whether the two other signal inputs in
Sfischeri, i.e. the C8HSL and AI-2 autoinducers, drive a similarly
noisy response or whether they improve the noise performance of
the overall system. Mehta et al. recently argued that the processing
of information by the QS system of V harveyi is limited primarily by
interference between the three input signal channels of the QS
pathway, and secondarily by noise originating within each
pathway [35]. Because noise in any one signal input channel
ultimately feeds forward into the regulated output, a well-defined
input concentration for one of the three autoinducer species will
not ensure a predictable output. In the present V.fischeri study we
have defined the 3OC6HSL level externally and also set the other
two autoinducer concentrations (C8HSL and /II -' virtually to


A
0.2

. |
30,1 |


0. 02

B


mean




1 2 3 4 5 6 7
Luminescence (normalized to median)


Fluorescence (normalized to median)


Figure 7. Heterogeneity of native luminescence versus fluores-
cence reporter for V.fischeri quorum system. (A) Histogram of
bioluminescence emission levels from 47 individual V.fischeri cells of
wildtype strain MJ11, following induction by 1000 nM Al. The
luminescence levels are normalized to the median value. (B) Histogram
of fluorescence levels for 127 individual V.fischeri cells of mutant JB10,
following induction by 1000 nM Al. The JB10 mutant contains a
chromosomal gfp insertion between luxl and luxC in the Luxl/LuxR
system. Fluorescence values are normalized to the median value. Both
luminescence and fluorescence reporters for the QS system show a
broadly heterogeneous response at full induction, although the
fluorescence shows slightly less variability (cv 0.8) than the lumines-
cence (cv 1.0).
doi:10.1371/journal.pone.0015473.g007


zero by advection; hence it appears unlikely that these additional
receptors contribute significant noise to the luminescence output.
The heterogeneity observed here may also argue against an
interpretation of the LuxI/LuxR system -or at least its regulation
of the bioluminescence genes -as allowing an individual cell to
acquire much useful information about its local microenvironment
[4,5,36]. The individual luminescence response seems to contain
little information about (i.e. it is a poor indicator of) the local AI
level, just as the AI concentration is a weak predictor of the
luminescence response. If a group of cells in a well-defined and
homogeneous environment exhibit widely divergent responses,
one cannot consider the QS system to be a reliable sensor of local
diffusion constants, for example. In a more heterogeneous natural
microenvironment one expects that the cell-to-cell variability in
response would only increase.
There are scenarios in which phenotypic variations arising from
noisy gene expression can provide a tangible benefit to the cell
[37,38]. Therefore it is intriguing to consider whether noise in
Vfischeri luminescence benefits the bacterium or influences its
symbiosis with a host animal. In the symbiotic relationship
Vfischeri is subject to a strong selective pressure to maintain bright
luminescence. For example, the squid E.scolopes does not tolerate
colonization by dark mutants of Vfischeri [39,40]. However,
11 .. ... 1 the host can select a strain for its average luminescence
output, the squid presumably cannot detect temporal or other
types of heterogeneity at the single-cell level. It may detect the
mean -but not the variance -of the cell brightness. Therefore the
individual cell is not likely to endure host pressure to minimize its
brightness fluctuations. Thus one possible interpretation of our
results is that the signal response is poorly coordinated across the
population because the host cannot apply feedback to enforce tight
coordination.
Of course, this interpretation only raises the question of
whether the uncoordinated response brings any benefit to the
bacteria. It would be interesting to determine whether cells that
emit a weak luminescence are directing more energy into other
QS-regulated behaviors, as if to divide colonization tasks across the
population. Alternatively, since bright emission is energy inten-
sive, one may speculate that a form of QS cheating occurs, with
the less luminous cells enjoying a _1.. 11. advantage. In a fully
induced cell the luminescence may require more than ~ 104 ATP
molecules per second and account for up to ~20% of the oxygen
consumption [7]. Such cheating does appear to provide a benefit
to individual bacteria, lil...,, _1 it is expected to be less pervasive
in clonal populations where kin selection favors cooperation [41].
(Cultures grown from a single colony of MJ11 were as
heterogeneous in light output as cultures grown from multiple
colonies, as described in Text S1.) Finally, a variable luminescence
output could be an optimal strategy in fluctuating environments
or when some of the autoinducer signals are weak or absent, so
that the cell's obligation to luminesce is uncertain. Noisy output
would be less advantageous in the rich, supportive environment of
the host light organ.
In summary we have observed that the luminescence response
of individual, wild-type Vfischeri cells is very imprecisely
regulated by the local quorum signal level. As QS regulation
plays an important role not just in the bioluminescence of V.
fischeri but also in colonization of the symbiotic host [9] it will be
interesting to conduct mutational studies to investigate whether
the noisy behavior observed in this particular output also
extends to other targets of QS regulation in this organism, and
how this influences the organism's ability to colonize the
heterogeneous microscopic environment within the host light
organ.


November 2010 | Volume 5 | Issue 11 | e15473


,.* ', PLoS ONE I www.plosone.org






Single-Cell Luminescence of Vibrio fischeri


Materials and Methods

Vibriofischeri strain MJ11 (NCBI Taxonomy ID: ." I. a strain
derived from the host fish Monocentrisjaponicus [42], was provided by
Prof. M. Mandel and Prof. E. Ruby. Cells were prepared in
exponential phase at 24'C in defined artificial seawater medium
[43] containing glycerol as carbon source. Approximately 15 Wl of
culture in exponential phase was deposited at the center of the lower
window of a perfusion chamber. This chamber consisted of a
cylinder (volume approximately 1.5 cm ) constructed from two
parallel, circular coverslips (25 mm diameter) spaced 5 mm apart.
The lower window was coated with poly-L-lysine to promote
adherence of the cells. The chamber was then closed and the cells
were allowed to settle and adhere to the window. After ~ 15 minutes
the chamber was then washed with approximately two chamber
volumes of defined medium from a programmable syringe pump.
This wash diluted away any autoinducer that was present in the
starting culture and removed any non-adhering cells. The chamber
was then placed on the stage of an inverted microscope and the
pump flow rate was reduced to 0.2 ml/hr in order not to disturb the
adhered cells during observation. The cells in the chamber were
primarily located within a small area (few mm2) of the window,
directly above the microscope objective, which was an infinite-
conjugate 100x plan oil immersion objective, NA 1.25. The blue/
green (near 490 nm) bioluminescence from the cells on the lower
window was collected by the objective and focused onto an
intensified CCD camera (512 x512 pixel, I-MAX-512-T operating
at -35'C, Princeton Instruments, Princeton NJ) via an achromatic
doublet lens, to give a final image scale of 0.278 pm per pixel.
The concentration of 30C6HSL autoinducer was selected by
adding exogenous autoinducer (AI, N-(3-Oxohexanoyl)-L-homo-
serine lactone, CAS 143537-62-6, No. K3007 from Sigma
Aldrich, St. Louis) to the medium flowing in the chamber. The
continuous flow of medium removed unattached (freely swimming)
cells from the chamber and maintained the AI concentration at the
selected level. AI released by the few cells adhered on the glass was
efficiently removed by diffusion into the passing flow. This was
verified in two ways. First, numerical integration of the diffusion/
advection equation for our experimental configuration gives an AI
accumulation of less than 50 pM at the window (for AI synthesis at
10-21 g/s/cell and diffusion at 100 lim2/s [44]). This concentra-
tion is insufficient to induce detectable luminescence. Second,
when cells were perfused with medium that contained no added
autoinducer, we observed that any luminescent emission from the
immobilized cells soon diminished to undetectable levels.
The doubling time for the ,.-1 11, of the cells in the chamber
was approximately 2-3 hr, operating at 24'C. This i_1. I, i.: set
a practical limit of roughly 4 hrs to our observations of individual
cell luminescence in the perfusion chamber. Once the cells on the
window had divided more than once or twice, the cells appeared
as clusters and it became difficult to distinguish the luminescence
of neighboring cells in the camera images. We studied the
luminescence of wild type strains only. Preliminary studies of
Vfischeri strain ATCC 7744 gave results similar to those presented
here for strain MJ11. A fluorescence study of gfp-reporter strain
JB10 is described below.
In most of our studies, the programmable syringe pump
supplied a flow of defined medium containing 0-1000 nM added
autoinducer to the chamber. Dni ;,._ i1.. "rich medium" study (see
Text S1) the syringe delivered commercial photobacterium
medium (No. 786230, Carolina Biological, Burlington NC) mixed
with defined medium and AI as indicated. For the tetradecanoic
acid study (see Text Sl), we prepared a 1 mM stock solution of
tetradecanoic acid (myristic acid, M3128 from Sigma Aldrich) in


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ethanol and diluted this 1000 x into the defined medium, to give a
final concentration of 1 gM.
After placing the perfusion chamber on the microscope stage
and starting the flow of medium + AI, we used dark field images
(i.e. externally illuminated images with brief exposure times) to
locate and focus on individual cells. We then disconnected the
illumination source and collected a bioluminescence image (i.e.
collecting only bioluminescent emission) with an exposure time of
(typically) ten minutes, and then collected another dark field image
for comparison. Figure 2 and Figure SI show sample images.
We repeated this process over a period of ~4 hrs for each group of
cells (at a fixed AI concentration), collecting alternately both dark
field and bioluminescence images at regular intervals. Compari-
sons of successive dark-field images provided a running check of
the physical and optical stability of the cells and the scene being
imaged.
To quantify the emission levels of individual cells in the
bioluminescence images, we first used the dark field images to
obtain the pixel coordinates of individual cells that had remained
immobile during the experiment. We then defined a small
rectangular region surrounding each cell. We binned (2 x2, to
improve SNR) the pixels of the corresponding region within the
dark-subtracted luminescence image, generated the brightness
histogram of the pixels in that region, and fit the lower portion
(only) of that histogram to a Gaussian distribution. This
distribution accurately models the background intensity distribu-
tion in cell-free regions of the image. We then subtracted the fit
Gaussian from the actual histogram and summed the residual.
This provided a satisfactorily robust count of the luminescence
emission of a single cell, typically 10-100 photons/minute/cell.
We confirmed that the luminescence emission count from a single
cell was insensitive to the precise size of the rectangular image
region used to estimate that count. Thermally generated
background (e.g. dark noise) in the CCD image contributes some
uncertainty to this emission count. By applying the above data
analysis to several image regions that contained no cells, we
estimated the magnitude of this uncertainty as ~20 photons/
minute peak-to-peak per cell per image frame. This defines a
baseline noise level, prior to Gaussian filtering of the emission level
versus time record ("trajectory") of a cell. The image intensifier
itself also contributes some noise, which is best characterized by
imaging a stable light source, as discussed below. Camera readout
noise and photon shot noise were smaller than either of the above
noise sources.
We typically detected ~10-100 photons/minute/cell from
Vfischeri strain MJ11 in our flow chamber, even in the presence
of an AI concentration (1000 nM) that would saturate the output
of a bulk culture. Therefore, our single-cell luminescence
measurements involved signal levels that were drastically lower
than are commonly obtained in gene regulation studies using
fluorescent proteins like GFP. For this reason it was important to
verify that the detected signals and their variations were not due to
experimental or analysis artifacts. Text S1 provides further detail
on measures that we took to ensure the stability of the optical
configuration, with minimal drift in the focus and minimal
movement in the cells adhered to the glass. These included
collecting and comparing a series of dark-field images (i.e. one
externally-illuminated dark-field image between each pair of
luminescence images) to check that cells under observation
remained in focus and had not physically moved.
Text S1 also describes control experiments to verify the stability
and sensitivity of our detection system. That is, we verified that the
observed variations in the light output from individual Vfischeri
cells were representative of cellular emission, and were not


November 2010 | Volume 5 | Issue 11 | e15473







Single-Cell Luminescence of Vibrio fischeri


generated within the image intensifier or due to uncertainty in our
detection or analysis. A suitable control must be a micron-sized
light source that is comparable in size to the Vfischer cells, feebly
luminescent (no brighter than the weak luminescence of a single
Vfischeri cell), and absolutely stable in its output. For this purpose
we used micron-sized green fluorescent latex spheres (FluoSpheres,
Invitrogen Inc.) dispersed at low density onto the lower window of
the perfusion chamber and illuminated with a heavily attenuated
blue LED excitation source. Under exceedingly faint excitation the
fluorescence from these spheres in a ten minute camera exposure
was comparable in magnitude to the emission detected from
individual Vfischeri (i.e. -100 photons/minute/particle) and it
remained stable for extended periods. We imaged these spheres
with exactly the same instrumentation parameters (camera gain
and temperature, exposure time, magnification, etc.) as used for
the Vfischeri cells. Performing the same image analysis as used for
the live cell images, we obtained a highly stable and consistent
photon count from the spheres. Figure 3 shows that the emission
detected from the control spheres remained stable 1,1...1. more
than four hours of observation, without any manual adjustment of
microscope focus. After Gaussian filtering (width a = 10 minutes)
of all emission versus time trajectories, the noise level (standard
deviation) for the emission from the individual particles was 10-12
photons/minute. Figure S2 shows that the emission from
different spheres in the same image was closely similar as expected
(standard deviation/mean 0.12). These results show that the
microscopy system and the data analysis were sufficiently sensitive
and stable for resolving heterogeneity in the luminescent emission
from different Vfischeri cells.
We also used fluorescence microscopy to measure GFP levels in
individual cells of Vfischeri strain JB10, which was provided by
Prof. E. Stabb. In theJB10 mutant a chromosomal gfp reporter is
placed under the control of the LuxI/LuxR system by insertion
between luxl and luxC, i.e. luxlgfp-luxCDABEG, so as to express
GFP when the LuxI/LuxR system is activated by 30C6HSL [45].
Cells were grown overnight in the same defined medium used for
the luminescence experiments and then transferred to fresh
medium containing 1000 nM exogenous AI. After incubating the
cells with shaking for -2 hrs we dispersed the cells on a coverslip
and measured the fluorescence of 127 individual cells, using an
inverted microscope with a 60 x oil immersion objective and a
cooled CCD camera (Micromax, Princeton Instruments).

Supporting Information

Figure S1 Sequential dark field and luminescence
images for one V.fischeri cell. (A) Dark field and (B)
bioluminescence images of an individual cell adhered to the
window of the perfusion chamber, and (C) luminescence levels
extracted from these images. (The luminescence trajectory has not
been Gaussian filtered.) Images were collected at the numbered
time points indicated in (C). (TIF)
Figure S2 Variability in signal levels for V.fischeri cells
and for reference particles. Histograms comparing the
luminescent emission from individual Vfischeri cells (A) to the
fluorescent emission under weak excitation of a control sample of


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Text S1 (DOC)

Acknowledgments

We thank Prof. MarkJ. Mandel, Prof. Edward G. Ruby, and Prof. Eric V.
Stabb for providing Vfischeri strains MJ11 (MJ.M., E.G.R.) and JB10
(E.V.S.) used in this study, and for their helpful advice and suggestions. We
also thank Leslie Pelakh for assistance with data collection.

Author Contributions
Conceived and designed the experiments: SJH PDP. Performed the
experiments: PDP. Analyzed the data: SJH PDP. Wrote the paper: SJH.



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