Report

Cyanobacteria Maintain Constant Protein

Concentration despite Genome Copy-Number
Variation
Graphical Abstract Authors
Xiao-yu Zheng, Erin K. OShea

Correspondence
erin_oshea@harvard.edu

In Brief
Zheng and OShea demonstrate that
S. elongatus cells with different genome
copy numbers maintain a relatively
constant protein concentration. The cell
volume and total protein amount both
positively, linearly correlated with
genome copy number, suggesting
changes in cell volume play an important
role in buffering variance in genome copy
number.

Highlights
d S. elongatus protein concentration is constant despite
genome copy-number variance

d Cell volume is positively, linearly correlated with genome

copy number

d Total protein amount is positively, linearly correlated with

genome copy number

d Cell volume changes serve as an important compensation

mechanism

Zheng & OShea, 2017, Cell Reports 19, 497504

April 18, 2017 2017 The Author(s).
http://dx.doi.org/10.1016/j.celrep.2017.03.067
Cell Reports
Report
Cyanobacteria Maintain Constant Protein
Concentration despite Genome Copy-Number Variation
Xiao-yu Zheng1,2,4 and Erin K. OShea1,2,3,5,*
1Howard Hughes Medical Institute, Harvard University Faculty of Arts and Sciences Center for Systems Biology
2Department of Molecular and Cellular Biology
3Department of Chemistry and Chemical Biology
Harvard University, Cambridge, MA 02138, USA
4Present address: Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
5Lead Contact
*Correspondence: erin_oshea@harvard.edu
http://dx.doi.org/10.1016/j.celrep.2017.03.067
SUMMARY
The cyanobacterium Synechococcus elongatus PCC
7942 has multiple copies of its single chromosome,
and the copy number varies in individual cells,
providing an ideal system to study the effect of
genome copy-number variation on cell size and
gene expression. Using single-cell fluorescence im-
aging, we found that protein concentration remained
constant across individual cells regardless of genome
copy number. Cell volume and the total protein
amount from a single gene were both positively, line-
arly correlated with genome copy number, suggest-
ing that changes in cell volume play an important
role in buffering genome copy-number variance.
This study provides a quantitative examination of
gene expression regulation in cells with variable
genome copies and sheds light on the compensation
mechanisms for variance in genome copy number.
INTRODUCTION
Since the rates of biochemical reactions are determined by the
concentrations of gene expression products, maintaining these
concentrations at proper levels is essential for cellular functions.
Single-cell studies in Bacillus subtilis, Saccharomyces cerevi-
siae, and mammalian cells have demonstrated that for isogenic
cells grown in the same condition, the concentration of any tran-
script or protein is centered on one value, that is, largely homo-
geneous across individual cells (Cookson et al., 2010; Kempe
et al., 2015; Newman et al., 2006; Ozbudak et al., 2002; Pado-
van-Merhar et al., 2015). Except for one study of replicating
mammalian cells, the populations of cells examined in the
abovementioned studies contain the same DNA dosage (i.e.,
genome copy number). However, genome copy numbers with
larger than 2-fold variances are common in cells that contain
multiple and variable sets of their chromosomes, such as skel-
etal muscle cells, plant epidermal cells, and cyanobacteria
(Comai, 2005; Griese et al., 2011; Taylor, 2002). These cells natu-
Cell Reports 19, 497504, April 18, 2017 2017 The Author(s). 497
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
rally exhibit large genome copy-number variance within a popu-
lation; the genome copy number of the cyanobacterium Syne-
chococcus elongatus can range from one to ten within a
population (Griese et al., 2011). In these cells, though an increase
in genome copy number is generally associated with an increase
in cell volume (Comai, 2005; Melaragno et al., 1993), quantitative
studies of the correlation between cell size and genome copy
number are scarce. It is also not known how changes in genome
copy number affect the total protein level. Most importantly, the
concentration of protein or transcript has not been examined and
correlated with genome copy number at the single-cell level.
Therefore, it remains an open question whether cells that natu-
rally have variable genome copy-number maintain a constant
protein concentration. Here, we addressed these questions by
conducting single-cell measurements of genome copy number,
cell volume, and protein levels in the cyanobacterium Synecho-
coccus elongatus PCC 7942 (S. elongatus).
S. elongatus is a rod-shaped cyanobacterium with a chromo-
some size of
2.7 Mb. Early flow cytometric analysis has re-
vealed the DNA amount across a population of cells to be a
multipeak distribution, indicating that there is heterogeneity in
genome copy number in the population (Mori et al., 1996). Visu-
alization of individual chromosomes in single cells further illus-
trated that S. elongatus cells possess variable numbers of full
copies of its chromosome, with each chromosome copy occu-
pying a discrete space along the long axis of the cell (Chen
et al., 2012; Jain et al., 2012). With such significant and measur-
able variation in genome copy number within a population,
S. elongatus is an ideal system to examine the correlation be-
tween genome copy number and cell size as well as protein con-
centration at the single-cell level.
In this work, we quantified protein concentration in individual
S. elongatus cells using YFP as a marker protein. While the pro-
tein concentration varies in cells grown in different growth condi-
tions and at different times of day under the control of a circadian
promoter, for cells in the same growth condition and at the same
time of day, the protein concentration is relatively homogeneous
in individual cells regardless of chromosome copy number.
Furthermore, this homogeneity of protein concentration is main-
tained in mutants with increased variance in chromosome copy
number. In all conditions tested, cell volume and total protein
Figure 1. Protein Concentration Is Constant in Cells with Different Chromosome Copy Numbers
(A) Each chromosome was labeled with mTurquoise2-tagged tetracycline repressor protein (TetR-mTurq2) and tetracycline operator arrays (tetO). The YFP
marker protein was integrated into each chromosome.
(B) Using a fluorescence microscope, chromosomes can be visualized and YFP fluorescence can be measured. A single z section of representative images in
each channel is shown.
(C) Computational analysis enabled automatic cell segmentation and chromosome foci identification. Green lines outline the cells segmented by the Oufti
software, overlaid with a single z section of the image from the YFP channel (top). Red dots show chromosomes identified by custom-written scripts, overlaid with
the maximal projection from the z stack image from the CFP channel (bottom).
(D) YFP fluorescence intensity concentration in arbitrary units per cubic micrometer (A.U./mm3) for each cell is plotted against chromosome copy number. Data
shown are from the strain with BbbJ23119::YFP. Each blue point represents one single-cell measurement. Violin plots represent distributions of single-cell data.
Yellow diamonds represent the sample mean. Error bars represent SD. Top panel bar graph shows the coefficient of variation from cells with the same chro-
mosome copy number. Bottom histogram shows the distribution of chromosome copy numbers. N shows the total sample size. Data from one biological replicate
is shown, but similar results were obtained in three biological replicates.
(E) Mean YFP concentrations from groups of cells with the same chromosome copy number for the pkaiBC::YFP strain are shown. Blue and red colors represent
data obtained at the peak phase and trough phase of the circadian cycle, respectively. The corresponding distributions of chromosome copy numbers are
presented in the top and bottom histograms. Error bars represent SD. Data from one biological replicate is shown, but similar results were obtained in two
biological replicates.
See also Figure S1.

amount both exhibit a positive, linear correlation with genome RESULTS

copy number, which explains the homogeneity of protein con-
centration and suggests changes in cell volume as an important
compensation mechanism buffering variance in genome copy
number. Our investigation not only reveals basic biological prop-
erties of S. elongatus but also sheds light on the fundamental
biological mechanisms regulating the concentration of gene
expression products in cells that exhibit variation in genome
copy number.
The Homogeneity of Protein Concentration Is
Maintained from Cell to Cell despite Genome
Copy-Number Variation
To simultaneously measure cell volume, chromosome copy
number, and protein amount, we constructed S. elongatus
strains for fluorescence-based imaging (Figure 1A). We visual-
ized individual chromosomes using a previously developed

498 Cell Reports 19, 497504, April 18, 2017

fluorescent repressor-operator system (Jain et al., 2012). Specif-
ically, tetracycline operator arrays and the fluorescently tagged
tetracycline repressor (TetR-mTurq2) were integrated into the
S. elongatus genome. TetR-mTurq2 binds to the operator arrays
and forms distinct fluorescent foci at each chromosome (Fig-
ure 1B) (Chen et al., 2012; Jain et al., 2012), which allows chro-
mosome copy number to be counted in each cell (Figure 1C).
We used YFP driven by the constitutive artificial promoter
BbbJ23119 (Lucks et al., 2011) as a marker protein to monitor
global regulation of protein levels.
In agreement with previous reports (Chen et al., 2012; Jain
et al., 2012), exponentially growing cells had a distribution of
chromosome copy numbers ranging from 1 to 8 with a median
of 3 or 4 (Figure S1). Since a small sample size would have low
statistical power, we grouped cells based on their chromosome
copy numbers and excluded data from groups whose sample
sizes were less than 20. For each cell, YFP concentration was
calculated as the sum of YFP fluorescence signals over the entire
cell divided by the cell volume. YFP concentration was generally
the same in cells with different chromosome copy numbers (Fig-
ure 1D). Groups of cells with different chromosome copy
numbers exhibited similar coefficients of variation (Figure 1D,
top). Thus, protein concentration is maintained at a similar level
in individual cells despite variation in genome copy number.
The BbbJ23119 constitutively active promoter is not known to
be subject to temporal or spatial regulation, thus representing
the simplest situation similar to a housekeeping gene. How- and genome copy number is not specific to constitutive
ever, most endogenous genes exhibit condition-specific expres-
sion patterns, originating from specific transcriptional controls
imposed by their promoters. For example, the circadian clock
of S. elongatus drives temporal oscillations in gene expression
corresponding to the 24 hr day/night cycle (Gordon et al.,
1997). To investigate whether the homogeneity of protein con-
centration across individual cells is affected by these pro-
moter-specific transcriptional controls, we examined YFP
expression under the control of the S. elongatus endogenous
kaiBC promoter, which drives the circadian expression of the
KaiB and KaiC proteins (Ishiura et al., 1998). In cells entrained
to 12 hr light/12 hr dark cycles, YFP driven by the kaiBC promoter
shows synchronous circadian oscillations in its fluorescence in-
tensity, correlated with the oscillation of endogenous kaiC mRNA
concentration with a phase delay (Chabot et al., 2007). We
imaged synchronized cells and found that YFP concentration
at the peak phase of the oscillation was about two fold higher
than that at the trough phase, in accordance with previously pub-
lished results (Chabot et al., 2007). At either the peak phase or
the trough phase, however, YFP concentration did not differ in
cells with different chromosome copy numbers (Figure 1E).
These results illustrated that while protein concentration can
exhibit circadian oscillations under the regulation of circadian
clock, it is maintained at a homogeneous level across different
cells at any single time. The homogeneity of protein concentra-
tion across cells is not specific to constitutive promoters.
Cell Volume Is Positively, Linearly Correlated with
Genome Copy Number
Since S. elongatus is rod shaped and its cell length is linearly
correlated with chromosome copy number (Chen et al., 2012;
Jain et al., 2012), cell volume is likely correlated with chromo-
some copy number. Indeed, our measurements showed that in
both the BbbJ23119::YFP strain and the pkaiBC::YFP strain,
cell volume (V) and genome copy number (N) exhibited a positive
correlation that fits a linear model V = aN + b (Figures 2A and
S2; Table S1). We observed that the intercept of the best-fitted
model is nonzero, indicating the existence of a basal, chromo-
some-independent component of cell volume. The cell volume
does not double as chromosome copy number doubles; how-
ever, every unit increase in chromosome copy number is accom-
panied by the same volume increment.
Total Protein Level from a Single Gene and Chromosome
Copy Number Exhibit a Positive, Linear Correlation
We further examined the changes in total YFP protein amount,
represented by total YFP fluorescence intensity, correlated
with variation in chromosome copy number. We found that to-
tal YFP amount (Y) was also positively, linearly correlated with
chromosome copy number, which is best fitted by a linear
model Y = cN + d with a non-zero intercept (Figure 2B; Table
S1). Therefore, the increment in total YFP amount correspond-
ing to a unit increase in genome copy number does not
vary as the chromosome copy number changes and stays
proportional to the increment in cell volume. Data from the
pkaiBC::YFP strain support the same conclusions (Figure S2),
demonstrating that the correlation between total YFP level
promoters.
Despite the correlation between genome copy number and
cell volume, we observe heterogeneity in cell volumes across in-
dividual cells with the same genome copy number (Figure 2A).
Such variation in cell size has been observed in mammalian cells
with the same genome copy number and was shown to be
compensated for by a general regulation of transcription burst
size to ensure transcript concentration homogeneity (Padovan-
Merhar et al., 2015). Interestingly, we also found a similar phe-
nomenon in the protein level in S. elongatus: among cells with
the same genome copy number, those with a larger volume
generally had a higher total YFP level (Figure 2C).
The Maintenance of Protein Concentration
Homogeneity and the Correlation between Cell
Volume and Genome Copy Number Are Independent
of Growth Rate
The concentration of a gene expression product is commonly
dependent on the cell growth rate (Klumpp and Hwa, 2014;
Klumpp et al., 2009; Scott et al., 2010). To verify that the mainte-
nance of protein concentration homogeneity is independent of
growth rate, we examined S. elongatus populations grown
under different light intensities, the change of which dramatically
affects growth rate (Kuan et al., 2015). We examined the
S. elongatus strain with BbbJ23119::YFP grown under constant
illumination at 9 mE m2s1 (low light intensity) or at 60 mE m2s1
(high light intensity), in which conditions cell growth rates
were 0.02 h1 and 0.05 h1, respectively. Different from the
laboratory constant illumination condition, S. elongatus in
natural environments experiences darkness at night. As dark-
ness can cause dramatic physiological changes in S. elongatus
Cell Reports 19, 497504, April 18, 2017 499

Figure 2. Cell Volume and Total YFP Amount, Respectively, Are
Positively, Linearly Correlated with Chromosome Copy Number
(A and B) Data of cell volume (A) and total YFP amount (B) from the S. elongatus
strain with BbbJ23119::YFP is presented. Each blue point represents one
single-cell measurement. Violin plots represent distributions of single-cell
data. Yellow diamonds represent the sample mean. Error bars represent SD.
The histogram shows the distribution of chromosome copy numbers. N shows
the total sample size. The red line shows the best linear fit, the parameters of
which are listed in Table S1.
500 Cell Reports 19, 497504, April 18, 2017
(Binder and Chisholm, 1990; Mori et al., 1996) but is also a phys-
iologically relevant growth condition, we also tested whether the
homogeneity of protein concentration in a population is main-
tained after a 24-hr period of darkness exposure. In all growth
conditions examined, YFP concentration was homogeneous
across individual cells regardless of chromosome copy number,
and cell volume remained positively, linearly correlated with
chromosome copy number. Total YFP levels and chromosome
copy number also exhibited a positive linear correlation (Figures
3 and S3; Table S1). Therefore, the homogeneity of protein
concentration among cells with various genome copy numbers
is independent of growth rate.
The Homogeneity of Protein Concentration Is
Maintained in Mutants with Increased Variation in
Genome Copy Number Induced by Disruption of Cell
Division Septum Placement
Wild-type S. elongatus cells divide in the middle and daughter
cells inherit approximately half of the chromosome copies due
to their evenly spaced distribution along the long axis of the
cell (Figure 4A, left). The correct placement of the division
septum is dependent on MinD (Synpcc7942_0896), and a strain
lacking minD (DminD) exhibits incorrect and random placement
of division septum (Jain et al., 2012). As a result, very small or
very large cells that are rarely observed in a wild-type population
can be found at higher frequencies in a DminD population (Fig-
ures S4A and S4B). Since the even spacing of chromosomes is
not affected in the DminD mutant, the number of chromosome
copies inherited by each daughter cell is determined by the po-
sition of division septum (Figure 4A, right) (Jain et al., 2012). To
investigate whether protein concentration stays homogeneous
across individual cells with larger variances in cell volume and
genome copy number, we performed the same single-cell mea-
surements on the DminD strain. As expected, the DminD mutant
population exhibited wider distributions of cell volumes and
chromosome copy numbers compared to wild-type populations
(Figures S1 and S4). In addition, the frequencies of extreme chro-
mosome copy numbers such as 1 and 8 were higher compared
to a wild-type population (Figures S1A and S4C). We observed
that the DminD mutant population still maintained YFP concen-
tration homogeneity across individual cells with chromosome
copy number ranging from 1 to 11 (Figure 4B). Chromosome
copy number was still positively, linearly correlated with both
cell volume and total YFP level (Figures 4C and 4D; Table S1).
These results further support that protein concentration homo-
geneity is well maintained in S. elongatus despite the large vari-
ation in genome copy number.
(C) Total YFP fluorescence intensity was plotted against cell volume. Each
point represents a single-cell measurement. Green, blue, and red represent
cells with two, four, and six chromosome copies, respectively. Histograms
show the corresponding volume distributions. For chromosome copy number
2 and 6, data presented are from all 185 cells and 184 cells measured,
respectively. For chromosome copy number 4, a random 180-cell subset of
876 cells is shown. The black line shows the best linear fit of total YFP intensity
versus cell volume based on data from all 2,575 cells.
For all subfigures, data are from the same biological replicate as that shown in
Figure 1D. Similar results were obtained from three biological replicates. See
also Figure S2 and Table S1.
Figure 3. Cells Grown in Low Light Intensity or Darkness Maintain the Linear Correlation between Cell Volume and Genome Copy Number
and Maintain Homogeneous Protein Concentration Despite Genome Copy-Number Variation
(A) Cells (the BbbJ23119::YFP strain) were grown under constant illumination at low light intensity (9 mE m2s1).
(B) Cells (the BbbJ23119::YFP strain) were first grown in constant light (30 mE m2s1) until reaching exponential growth, then shifted to darkness for 24 hr.
The histogram shows the distribution of chromosome copy numbers. Data from cells with a chromosome copy number frequency less than 20 are not shown.
N shows the total sample size presented. Each blue point represents one single-cell measurement. Violin plots represent distributions of single-cell data.
Yellow diamonds represent the sample mean. Error bars represent SD. The red line shows the best linear fit, the parameters of which are listed in Table S1. Data
shown are from one biological replicate. Similar results were obtained in two biological replicates for the low light intensity growth condition. See also Figure S3
and Table S1.

DISCUSSION
Here, we conducted single-cell measurements of protein con-
centration, genome copy number, and cell volume in
S. elongatus, a bacterium that naturally exhibits large variation
in genome copy number within a population. Our data demon-
strate that protein concentration is constant in cells with different
genome copy numbers. We also revealed that cell volume and
the total protein amount positively, linearly correlated with chro-
mosome copy number.
How is the protein concentration maintained constant? In the
linear models fitted to our data (cell volume V = aN + b, total pro-
tein amount Y = cN + d), since the intercepts d and b respectively
represent chromosome-independent components of total pro-
tein amount and cell volume, one hypothesis is that cells have
a basal component, which has a volume b and a protein amount
d, and grow by proportional increases in cell volume and protein
amount, which correspond to each unit increment in chromo-
some copy number. The basal component could be a residual
from mother cells, which would have a similar protein to volume
ratio d=b, that is, concentration, to that c=a of the growing
portion. Indeed, linear models fitted to our data have similar
values of d=b and c=a (Table S1). By this hypothesis, a
change in cell volume alone accounts for the change in the
total amount of gene expression products, compensating for
variance in genome copy number. Another hypothesis considers
all proteins in the cell originate from an equal contribution of
all chromosomes, and thus, protein production per genome
Y=N = cN + d=N = c + d=N decreases as genome copy
number increases. By this hypothesis, variance in genome
copy number is compensated for by a combination of changes
in cell volume and protein production per genome. Though our
data do not distinguish between these two hypotheses, our ob-
servations suggest that cell volume changes are essential for
buffering variance in genome copy number. Our quantitative
data also provide a foundation for future modeling analysis eval-
uating these compensation hypotheses.
Using YFP as a marker protein, we concluded that steady-
state protein level is positively, linearly correlated with
genome copy number. This steady-state protein level reflects
the net result of regulation of transcription and/or translation.
Though it has been shown that YFP fluorescence correlates

Cell Reports 19, 497504, April 18, 2017 501


well with RNA levels under the control of kaiBC promoter
(Chabot et al., 2007), it would be interesting to further study
whether the transcript level exhibits a similar correlation with
genome copy number at the single-cell level by implementing
single RNA fluorescence in situ hybridization (FISH) in
S. elongatus. Although we showed that the linear correlation
between YFP protein level and chromosome copy number is
not specific to constitutive promoters, we cannot exclude
that there may be specific regulation targeting certain genes.
Further investigation of this topic would require the generation
and measurement of specific functional fluorescence-tagged
proteins.
Though the correlation between cell volume and genome copy
number is well supported by our data, we observed variation
from the mean cell volume in cells with the same genome copy
502 Cell Reports 19, 497504, April 18, 2017
Figure 4. The DminD Mutant that Is Defec-
tive in the Correct Placement of the Division
Septum Still Maintains Homogeneous Pro-
tein Concentration in Cells with Different
Chromosome Copy Numbers
(A) In wild-type cells, the division septum is placed
in the middle of the cell and chromosome copies
are evenly distributed along the long axis of the
cell; thus, daughter cells inherit approximately
equal numbers of chromosome copies from the
mother cell. In the DminD mutant, the division
septum is randomly placed but the chromosome
spatial ordering maintains; thus, daughter cells
inherit unequal numbers of chromosome copies.
(BD) For the DminD mutant strain with
BbbJ23119::YFP, YFP fluorescence concentra-
tion (B), cell volume (C), and total YFP intensity (D)
were plotted against chromosome copy number,
respectively. The histogram shows the distribution
of chromosome copy numbers. N shows the total
sample size presented. Each blue point repre-
sents one single-cell measurement. Violin plots
represent distributions of single-cell data. Yellow
diamonds represent the sample mean. Error bars
represent SD. The red line shows the best linear fit,
the parameters of which are listed in Table S1.
Similar results were obtained in three biological
replicates.
See also Figure S4 and Table S1.
number. In addition to technical noise,
this variation could come from stochastic
events in gene expression that influence
cell growth and size. This variation could
also originate from the fact that DNA
replication is asynchronous among indi-
vidual cells in a population. Since cell
growth is continuous while DNA replica-
tion is not (Chen et al., 2012; Jain et al.,
2012; Mori et al., 1996; Watanabe et al.,
2012), variations in cell volume with the
same genome copy number might be ex-
pected between cells that have just un-
dergone DNA replication and cells that
are about to trigger DNA replication.
The correlation between genome copy number and cell vol-
ume is an interesting phenomenon that has been observed in
other cells like S. cerevisiae and plant epidermal cells (Melaragno
et al., 1993; Mundkur, 1953). However, it is unclear how this cor-
relation is achieved and maintained in these systems. It could
originate from the intrinsic biophysical constraints such as the
size of DNA and protein molecules, the diffusion rates of pro-
teins, and the cell surface-to-volume ratio (Dill et al., 2011). For
example, the size and specific arrangement of DNA could deter-
mine the physical volume it occupies (Gregory, 2001). The sizes
and diffusion rates of proteins from one chromosome copy may
determine the amount of cell growth and cell volume that a single
chromosome copy can support (Dill et al., 2011). In addition, this
correlation could come from active molecular mechanisms
coupling cell growth and DNA replication. For example, a
sensor could actively monitor cell growth and trigger DNA
replication when cell growth reaches a certain threshold (Robert,
2015; Turner et al., 2012). Future studies employing both phys-
ical and biological principles will be needed to gain further in-
sights about this proportionality between the genome copy
number and cell volume.
Finally, our data suggest that S. elongatus cell size could be
regulated by a mechanism similar to the adder mechanism
describing bacterial cell size homeostasis, according to which
cells grow by a constant volume between two division events
regardless of their original cell sizes (Campos et al., 2014;
Taheri-Araghi et al., 2015). We observe a linear correlation be-
tween cell size and genome copy number, indicating that cell
size increases by a constant amount corresponding to a unit in-
crease in chromosome copy number. This constant amount is
unrelated to the original cell size and is comparable to the notion
of the adder. By this hypothesis, our data further suggest that the
adder is tightly coupled to DNA replication. Future time-course
experiments monitoring dynamic changes in genome copy num-
ber and cell size would help to evaluate this hypothesis.
EXPERIMENTAL PROCEDURES
Strain Construction and Growth Conditions
Relevant plasmids and strains are presented in Tables S2 and S3, respectively.
S. elongatus was grown in BG11 medium at 30 C under constant illumination
with cool fluorescent light. Unless otherwise noted, light intensity was 30 mE
m2s1. During the construction of S. elongatus strains, TetR-mTurq2 was
always transformed last and anhydrotetracycline (aTC) was added to the
transformation selection plate at a final concentration of 2.5 mg/mL to alleviate
the growth defect caused by interactions between the repressor and the oper-
ator arrays. After transformants were patched on new plates, starter cultures
were prepared from single colonies and used to inoculate final cultures after
2 days. The cells were imaged in exponential growth. For the pkaiBC::YFP
strain, the final culture was entrained following inoculation with two 12 hr
light/12 hr dark cycles and then grown in constant light condition. Cells were
taken at ZT 6 (zeitgeber time) (trough phase) and ZT 18 (peak phase) for imag-
ing. Since TetR-mTurq2 was driven by the Ptrc promoter (Huang et al., 2010),
its basal expression level was low enough so that the cells could propagate
healthily without loss of tetracycline operator arrays for at least 2 weeks
without added aTC. Therefore, no aTC was added to liquid cultures to achieve
a better signal-to-noise ratio in imaging. However, since operator arrays were
unstable in cells stored for longer periods of time, fresh transformants were
made for each imaging experiment. PCR-based genotyping was performed
before and after every imaging experiment to confirm all insertions were pre-
sent in all chromosome copies and all operator arrays were intact.
Microscopy
Exponentially growing cells were collected from liquid culture and resus-
pended in filtered BG11 medium. An aliquot of cells was placed on Lab-Tek
II chamber coverglass (Electron Microscopy Sciences) and overlaid with a
2% (weight/volume) ultrapure agarose (Invitrogen) pad. Samples were imaged
with a 633/1.4 oil objective on an inverted Zeiss Axio Observer equipped with
a Hamamatsu Flash 4.0 v2 sCMOS camera. z stack (250 nm step size) images
were acquired in bright-field, YFP, CFP (TetR-mTurq2), and Cy5 (autofluores-
cence) channels. For each experiment, wild-type cells grown in the same
conditions were also imaged to calculate background fluorescence.
Image Analysis
Images were first analyzed using Oufti software (Paintdakhi et al., 2016) to
perform cell segmentation. Cell volumes were automatically calculated by
the Oufti software, which treated each cell as a sum of segments along the
long axis and estimated volumes of each segment by assuming the height of
the segment is equal to its width (Paintdakhi et al., 2016). With the cell outlines
identified by Oufti, custom-written scripts in MATLAB (The MathWorks) were
then used to extract YFP fluorescence intensity and identify chromosome
foci in each cell. Details are provided in Supplemental Experimental Proced-
ures. All correlation analysis was performed using the Curve Fitting Toolbox
in MATLAB.
All single-cell data are available in Data S1. Raw images for all the experi-
ments presented in the figures are available from the Dryad Digital Repository
at http://dx.doi.org/10.5061/dryad.ps319.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, three tables, and one data file and can be found with this informa-
tion online at http://dx.doi.org/10.1016/j.celrep.2017.03.067.
AUTHOR CONTRIBUTIONS
X.-Y.Z. and E.K.O. designed and wrote the paper. X.-Y.Z. performed experi-
ments and analyzed data.
ACKNOWLEDGMENTS
We thank Drs. V. Vijayan and I.H. Jain and members of the E.K.O. laboratory for
discussions and critical reading of the manuscript and the Harvard Center for
Biological Imaging for infrastructure and support. This work was funded by the
Howard Hughes Medical Institute (HHMI Investigator Award 010050).
Received: January 31, 2017
Revised: March 22, 2017
Accepted: March 23, 2017
Published: April 18, 2017
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