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Plant Biology ISSN 1435-8603

RESEARCH PAPER

Observation of polyphosphate bodies and DNA during the cell

division cycle of Synechococcus elongatus PCC 7942
Y. Seki1, K. Nitta2 & Y. Kaneko3
1 Graduate School of Science and Engineering, Saitama University, Saitama, Kanagawa, Japan
2 Analysis Technology Center, Research and Developmental Management Headquarters, Fuji Photo Film Co. Ltd., Minamiashigara, Kanagawa, Japan
3 Biology Section in the Faculty of Education; Graduate School of Science and Engineering; Institute for Environmental Science and Technology, Saitama
University, Saitama, Japan

Keywords ABSTRACT
Cyanobacteria; electron microscopy; Hoechst
33342; Neisser staining. Although most cyanobacterial cells contain prominent polyphosphate bodies in the
central cytoplasmic area enclosed by the peripheral thylakoid membranes, their roles
Correspondence: Yasuko Kaneko, Saitama are not fully understood. Storing phosphate for nucleotide production might be one
University, Shimo-okubo 255, Sakura-ku, of their important roles in survival of the cells. As a step towards identifying a possible
Saitama 338-8570, Japan. contribution of the polyphosphate bodies to DNA synthesis, the relationship between
E-mail: yakaneko@mail.saitama-u.ac.jp polyphosphate bodies and DNA throughout cell division cycle of Synechococcus elong-
atus PCC 7942 cells cultured under light/dark cycles was investigated with light and
Editor electron microscopy. During the dark period, the average size of polyphosphate bodies
S. Wick increased gradually without significant change in their number and distribution.
However, during the light period, the number of polyphosphate bodies increased,
Received: 16 September 2012; Accepted: 21 while the size of each polyphosphate body decreased and cells elongated until the end
December 2012 of the light period, when most cells divided. The ratio of the content of polyphosphate
bodies to cell length increased gradually during the dark period and decreased during
doi:10.1111/plb.12008
the light period. Hoechst 33342-stained DNA appeared diffuse during the dark period,
but in the light period it became condensed and eventually formed a wavy, rope-like
structure prior to cell division. Close association between fibres containing DNA and
polyphosphate bodies was demonstrated by TEM using DNA-specific staining and
BrdU labelling. These regular coordinated changes of polyphosphate bodies and DNA
shape during the cell division cycle, together with their intimate interaction, imply a
role of polyphosphate bodies in supplying material for DNA.

copy and/or electron tomography. Carboxysomes and

INTRODUCTION
polyphosphate bodies were observed in close contact in the
Cyanobacteria are photosynthetic prokaryotes that have had an central cytoplasm of unicellular cyanobacteria (Nierzwicki-
enormous influence on the living environment on Earth since Bauer et al. 1983; Liberton et al. 2011); but in Synechocystis sp.
their appearance. Cyanobacterial cells share many characteris- PCC6803, only a few polyphosphate bodies were found in elec-
tics with the chloroplasts of green plants and are considered to tron tomograms of whole cells (Van de Meene et al. 2006).
be their origin. Several conspicuous structures are evident in It is now known that every living cell contains polyphos-
cells of the rod-shaped cyanobacterium Synechococcus elongatus phate, which is a linear polymer of several to hundreds of
PCC 7942, such as thylakoid membranes with phycobilisomes, orthophosphate residues linked by high-energy bonds (Brown
carboxysomes consisting of Rubisco molecules and large poly- & Kornberg 2004). For a long time the role of polyphosphate
phosphate bodies. During the cell division cycle, these struc- in cell physiology was neglected and it was called a ‘molecular
tures are segregated evenly into the two daughter cells, fossil’ (Manganelli 2007); however, more recently various
resulting in a population of cells with more-or-less similar physiological roles have been associated with it, such as adjust-
appearance. Mechanisms that regulate the position and num- ment of metabolism and regulation of genes and enzymes
ber of each structure within each cell must exist, but no such (Brown & Kornberg 2008; Achbergerova & Nahalka 2011).
mechanisms have yet been satisfactorily elucidated. Stored polyphosphate may also supply material for phosphate-
Although polyphosphate bodies are among the most promi- containing molecules such as DNA and RNA. There have been
nent structures in S. elongatus PCC 7942 cells, thorough obser- several reports pointing to a close association between fibrous
vation of the bodies with conventional electron microscopy is structures and polyphosphate bodies in the nucleoid region of
difficult to accomplish. Because of poor penetration of resin cyanobacterial cells (Voelz et al. 1966; Lawry & Jensen 1979).
into polyphosphate bodies, large polyphosphate bodies tend to Furthermore, newly synthesised DNA labelled with BrdU
be lost during ultra-thin sectioning (Nitta et al. 2009). There incorporation was imaged near polyphosphate bodies through
have been attempts to obtain the three-dimensional ultrastruc- observation of whole ice-embedded S. elongatus PCC 7942 cells
ture of cyanobacterial cells using high-voltage electron micros- with phase contrast transmission electron microscopy (TEM;

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Seki, Nitta & Kaneko Polyphosphate bodies and DNA in cyanobacterial cells

Nitta et al. 2009). It is known that Synechococcus sp. cells con- 200% on a PC display, and diameters of polyphosphate bodies
tain two to five copies of genomic DNA (Binder & Chisholm and cell lengths were measured with a ruler.
1990, 1995; Mori et al. 1996) and that the cell division of the
species is controlled by light/dark cycles. It has been shown that
Transmission electron microscopy and energy dispersive
the chromosomal DNA of the cells becomes compact during
X-ray (EDX) analysis
the light period, whereas it takes on a diffuse appearance dur-
ing the dark period (Smith & Williams 2006). DNA synthesis For conventional electron microscopy, collected cells were fixed
seems to occur during the light part of the cycle (Binder & in 2% glutaraldehyde in 0.05 M potassium phosphate buffer
Chisholm 1990). (pH 7.0) for 2 h at room temperature and in a refrigerator
In this paper, the distribution and size changes of polyphos- overnight. After rinsing in buffer, the cells were post-fixed in
phate bodies were observed, along with the prominent struc- 2% OsO4 in 0.05 M potassium phosphate buffer (pH 7.0) for
tural changes of fluorescently labelled DNA in rapidly 2 h at room temperature. Cells were then dehydrated in an ace-
proliferating cells cultured under light/dark cycles. The signifi- tone series and embedded in Spurr’s resin. Ultra-thin sections
cance of the coordinated changes of polyphosphate bodies and (ca. 90-nm thick, estimated from the silver–gold interference
DNA and their relationship is discussed. colour) were cut with a diamond knife on a Sorvall MT-2B
microtome (Sorvall, Norwalk, CT, USA). After staining with
uranyl acetate for 5 min and lead citrate for 1 min, sections
MATERIAL AND METHODS were observed with a Hitachi H-7500 TEM (Hitachi, Tokyo,
Japan) at an accelerating voltage of 100 kV.
Cyanobacterial strain and culture conditions For EDX analysis, post-fixation with OsO4 was omitted from
Synechococcus elongatus PCC 7942 cells were cultured at 23 °C the chemical fixation. Semi-thin sections (ca. 120-nm thick,
under a light intensity of 50 lE
m 2 s 1 with 12-h light and estimated from the gold–purple interference colour) were
12-h dark cycles on BG 11 plates containing 1.5% (wt/vol) agar made from dehydrated and resin-embedded samples and
and 0.3% (wt/vol) sodium thiosulphate (Rippka et al. 1979). mounted on copper grids. They were analysed with a JEOL
The cells were transferred to fresh media every 1–2 weeks. The JEM-2100F equipped with a JEOL JED-2300T EDX system
fifth day culture was used for observation of polyphosphate (JEOL, Tokyo, Japan).
bodies and DNA.
For BrdU incorporation, the cells were cultured in liquid Localisation of DNA with osmium ammine
BG-11 medium under continuous light (50 lE
m 2
s 1) at
23 °C. The cells were transferred to fresh media every 7 days Conventionally prepared, resin-embedded ultra-thin sections
and the second day culture was used for BrdU incorporation. were treated with osmium ammine-SO2, which is known to be
BrdU (Sigma, St. Louis, MO, USA) was added to the medium DNA specific (Levin-Zaidman et al. 2000). Sections mounted
to a final concentration of 10 lM. After 24 h of culture in on nickel grids were incubated in 5 M HCl for 30 min at room
BrdU-containing medium, cells were collected by centrifuga- temperature, rinsed with distilled water, and treated with 0.2%
tion and processed for electron microscopy. osmium ammine-B (Polysciences) in 8 M acetic acid/40 mM
sodium metabisulphite for 1 h at 37 °C. Sections were thor-
oughly rinsed with distilled water and dried, then examined
Staining of polyphosphate bodies and DNA without additional staining with a Hitachi H-7500 TEM.
for light microscopy
Polyphosphate bodies were visualised with the Nisser staining RESULTS
method (Pelezar et al. 1957). Collected cells were stained for Under 12-h light and 12-h dark culture conditions, most cells
20 s with a solution composed of two parts of solution A displayed similar structural changes of polyphosphate bodies
(0.1 g methylene blue, 2 ml ethanol, 5 ml acetic acid, 95 ml and DNA (Fig. 1). During the light part of the cycle, the num-
distilled water), and one part of solution B (0.1 g crystal violet, ber of large polyphosphate bodies detectable with light micros-
1 ml ethanol, 30 ml distilled water), and rinsed with water. copy increased from one or two per cell to three or four per
Cells were then stained with 0.3% chrysoidin solution for sev- cell as the cells elongated (Fig. 1a–c). During the dark part of
eral minutes and rinsed with water. Stained cells were dried on the cycle, most of the cells contained one or two polyphosphate
a microscope slide and examined with a Nikon ECLIPSE 50i bodies (Fig. 1d). Fluorescence-stained DNA changed shape
light microscope (Nikon, Tokyo, Japan). For fluorescence dramatically during the cycle (Fig. 1e–h); whereas stained
labelling of DNA, collected cells were stained with Hoechst DNA diffused throughout the cells at L2 (Fig. 1e), a condensed
33342 at a final concentration of 0.1 lg
ml 1 for 10 min in the structure was observed at L6 (Fig. 1f), and at L10, the con-
dark. Stained cells were observed with a Nikon ECLIPSE 50i densed DNA appeared in the form of undulating rope-like
fluorescence microscope (Nikon) using a V-2A filter. shapes in most cells (Fig. 1g). During the dark part of the cycle,
diffuse DNA was observed throughout the cells (Fig. 1h).
In order to estimate the relationship between the content
Measurement of cell size and polyphosphate bodies
(diameter 9 number) of polyphosphate bodies and cell size,
Light microscopic images observed with an oil immersion the number and size of polyphosphate bodies per cell was
1009 objective lens (N.A. 1.30) were acquired with a Nikon counted and cell length measured every 2 h during cell cycles
digital camera DXM 1200C at the highest resolution (Fig. 2). We note that smaller polyphosphate bodies beyond
(4116 9 3072 pixels). The acquired images were enlarged to the range of light microscope resolution, which are very likely

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Polyphosphate bodies and DNA in cyanobacterial cells Seki, Nitta & Kaneko

a b c d

e f g h

Fig. 1. Light microscopic images of cyanobacterial polyphosphate bodies visualised with Neisser staining (a–d), and fluorescence microscopic images of Hoe-
chst 33342-stained DNA (e–h) throughout the light/dark cycle. The number of polyphosphate bodies per cell increased during the light period (a–c) until cell
division at the end of the light period. Condensed DNA (f) took on a wavy rope-like shape (g) prior to cell division. Observations made every 4 h and three time
points during the light period (L2, L6, L10) and one time point during the dark period (D6) are shown. No significant change was observed during the dark per-
iod. Scale bars = 1 lm.

present in the cells, are not included in the analysis shown in stained intensely, indicating the presence of DNA (Fig. 4a). No
Fig. 2. Average cell length increased from ca. 1.9 lm to 3.4 lm similar staining was observed on the sections without osmium
during the light period and suddenly decreased at the end of ammine-SO2 treatment (Fig. 4b).
the light period, whereas average cell length stayed almost con- An EDX analysis was conducted on the semi-thin sections of
stant during the dark part of the cycle (Fig. 2a). The average cells cultured in medium containing BrdU (Fig. 5). Elements
diameter of polyphosphate bodies gradually decreased during at the centre point of each circle were analysed. The data in the
the light period and increased towards the end of the dark per- top panels of Fig. 5 indicate that the polyphosphate body in
iod (Fig. 2b). The ratios of cells containing one, two, three or the cell contains Ca and Br (asterisk) as well as P; however, in
four polyphosphate bodies at each time point were calculated the area in the cytoplasm shown in the bottom panels only a
and are presented in Fig. 2c. Cells containing three or four much smaller peak of P and a small peak of Cl were observed,
polyphosphate bodies gradually increased during the light per- but no Ca and Br (asterisk) could be detected. Br was also
iod, while those containing one or two bodies decreased. In detected at structures presumed to be clumps of DNA, to the
contrast, most of the cells contained one or two polyphosphate right of the circled polyphosphate body in Fig. 5 (data not
bodies throughout the dark part of the cycle. These data were shown).
used to calculate the ratio between the content of polyphos-
phate bodies (average diameter X average number) of each cell
DISCUSSION
and cell length (Fig. 2d). This calculation made clear the trend
that the relative content of polyphosphate bodies per cell grad- Regular coordinated changes of polyphosphate bodies and
ually increased during the dark and then decreased, especially DNA shape during the cell division cycle of S. elongatus PCC
at the start and towards the end, during the light part of the 7942 were clearly observed. In the present study, Hoechst
cycle (Fig. 2d). 33342 was used to visualise DNA in the cells with a fluores-
In order to illustrate the different sizes of polyphosphate cence microscope. The structural changes of DNA during the
bodies contained in the cells, ultra-thin sections of the cells at light period were more striking with Hoechst 33342 than with
L2 and L10 are shown in Fig. 3. Large holes were frequently DAPI staining, possibly because the former dye penetrates bet-
observed in the sections of L2 cells, indicating that large poly- ter into living cells. In contrast to the spot-like appearance of
phosphate bodies were lost during the sectioning process DNA stained with DAPI (Nitta et al. 2009), Hoechst 33342-
(Fig. 3a). In the case of L10 samples, polyphosphate bodies stained DNA was more or less continuous within the cells and
with diameters <90 nm were often retained in the ca. 90-nm exhibited distinct structural changes. The condensed rope-like
thick sections (Fig. 3b). In either case, fibrous structures were structure of DNA observed prior to cell division may have
closely associated with the polyphosphate bodies. In order to some significance in duplication and segregation of DNA for
characterise these fibres, ultra-thin sections were treated with cell division. Diffusion and compaction of chromosomal DNA
osmium ammine-SO2. Fibrous structures within the cells, under light/dark cycles has been reported previously in
including those associated with the polyphosphate body S. elongatus PCC 7942 (Smith & Williams 2006). The number

260 Plant Biology 16 (2014) 258–263 © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands
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Seki, Nitta & Kaneko Polyphosphate bodies and DNA in cyanobacterial cells

a a b

Fig. 3. TEM image of cyanobacterial cells at the onset of the light period (a)
and towards the end of the light period (b). Scale bars = 100 nm. (a) Large
c polyphosphate bodies were poorly embedded in the resin and were lost dur-
ing ultra-thin sectioning, leaving holes. (b) Relatively smaller polyphosphate
bodies were retained in the ultra-thin sections. P: polyphosphate bodies or
holes left by polyphosphate bodies.

Fig. 2. Relationships between cell length and size and number of polyphos-
phate bodies per cell. (a) Average length of cells during light/dark cycle.
About 30 cells were measured at each time point. Cells gradually elongate
during the light period until the end of the light period, at which point most
of the cells divide. Bars indicate standard error. (b) Average diameter of poly-
phosphate bodies during light/dark cycle. About 50 polyphosphate bodies
were measured at each time point. Diameter of polyphosphate bodies
decreased gradually during light period and increased during dark period. Fig. 4. TEM image of cyanobacterial cells stained with osmium ammine-
Bars indicate standard error. (c) Ratio of cells with one, two, three or four SO2 (a) and cells before staining (b). Scale bars = 100 nm. (a) Fibrous struc-
polyphosphate bodies among cell populations at each time point during ture containing DNA stained intensely (white arrows). Stained fibres were
light/dark cycle. The number of polyphosphate bodies was counted in about often attached to a polyphosphate body (P). (b) No staining of DNA is dis-
20 cells at each time point. The number represented by each colour pattern cernible. P: hole left by polyphosphate body.
is: plain blue: 1, dotted red: 2, checked green: 3, lined purple: 4. Ratio of
cells with three or four polyphosphate bodies increased during the light per-
iod. The majority of the cells contained one or two polyphosphate bodies
always contained one or two polyphosphate bodies. There must
throughout the dark period. (d) Ratio between the content of polyphos-
be an unknown mechanism that controls the occurrence and
phate bodies (average diameter x average number) of each cell and cell
distribution of the observable polyphosphate bodies within the
length. The value increased gradually during the dark period and decreased
during the light period.
cells.
The most obvious function that can be imputed to poly-
phosphate bodies is the storage of phosphate. Since the size of
of large polyphosphate bodies per cell increased along with cell polyphosphate bodies gradually increased during the dark
elongation during the light cycle. At subsequent cell division, period when no cell division was observed, inorganic phos-
they were distributed in such a fashion that each daughter cell phate from the media is presumably continuously taken up by

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Polyphosphate bodies and DNA in cyanobacterial cells Seki, Nitta & Kaneko

Fig. 5. EDX analysis of cyanobacterial cells cultured with

BrdU. Central point of each circle was analysed. At a
point on a small polyphosphate body, small peaks of Br
(asterisk) and Ca were discernible, besides P (top). In a
point in the cytoplasmic area only a small peak of P was
detected (bottom). Scale bars = 100 nm.

the cells and accumulated in the polyphosphate bodies. Incor- The compound BrdU has been widely used in cell biological
poration of phosphate into polyphosphate bodies also research to label newly synthesised DNA in vivo (Ngwenya
appeared to occur during the light period, based on the obser- et al. 2005). Br was detected with EDX analysis at clumps of
vation that the number of polyphosphate bodies per cell DNA and polyphosphate bodies only after the cells were
increased during this period. However, during the light period, cultured in medium containing BrdU. Br was not detected in
phosphate from the polyphosphate bodies must be constantly other portions of the cells cultured in BrdU-containing
used for some cellular activities during active growth of the medium (Fig. 5). The existence of Br should be an indication
cells and preparation for cell division, which mostly takes place for localisation of newly synthesised DNA incorporating
during the light period. The presumed high consumption of BrdU. Localisation of BrdU in DNA after cells were cultured in
phosphate during the light period may be the cause of the BrdU-containing medium was previously confirmed with flu-
gradual decrease in size of the polyphosphate bodies. Accumu- orescently labelled anti-BrdU antibody (Nitta et al. 2009). The
lation and vigorous consumption of phosphate in the poly- Br localisation associated with polyphosphate bodies is not
phosphate bodies seem to occur simultaneously during the likely a result of random incorporation of Br into polyphos-
light period. The consumption seemed to be higher at the phate bodies. Uptake of various metals, such as Mg, Ba, Mn, by
beginning of the light cycle and towards the end, during which polyphosphate bodies in cyanobacteria was detected using X-
time cellular activity that consumes much phosphate is pre- ray microanalysis in the cyanobacterium Plectonema boryanum
sumably proceeding. (Baxter & Jensen 1980). Incorporated metals were all positively
According to a study that estimated the phosphate content in charged, whereas negatively charged Cl was not incorporated,
each cell component of batch-cultured Synechococcus, more than although the metals were applied as chloride salts (Baxter &
50% of the phosphate was contained in RNA, about 10% in Jensen 1980). In our analysis, Br was detected at a polyphos-
polyphosphate bodies and 7% in DNA (Grillo & Gibson 1979). phate body along with Ca but not with Cl, although Cl was
The origin of phosphate used for DNA and RNA synthesis has detected in the cytoplasm. This result implies that negatively
been examined in Chlorella, which were first grown in P32 med- charged Br was incorporated through biological activity; the
ium and then transferred to a cold medium (Miyachi & Tamiya presence of Br in the vicinity of polyphosphate bodies of the
1961; reviewed in Harold 1966). In the dark, no synthesis of cells cultured in a BrdU-containing medium most likely indi-
DNA and RNA occurred and all the polyphosphate fractions cates that newly synthesised DNA was located nearby (Fig. 5).
remained constant. However, in the light, in an inorganic phos- Large areas of empty space surrounding holes left by lost
phate-containing medium, phosphate in lipids and in RNA was polyphosphate bodies or surrounding remaining small poly-
obtained from inorganic phosphate in the medium, whereas phosphate bodies were frequently observed (Fig. 3). Fibrous
phosphate in DNA was derived from the polyphosphate pool. structures containing DNA appear to stretch through this space
Although Synechococcus is a prokaryote, and may thus differ to the surface of the polyphosphate bodies (Fig. 4). These
from Chlorella in the way it uses phosphate, there may be a simi- observations suggest that polyphosphate bodies have shrunk
lar mechanism for use of polyphosphate for synthesis of DNA. during the steps of chemical fixation, dehydration and resin
Synechococcus sp. cells are known to contain two to five copies of embedding prior to ultra-thin sectioning. They also suggest
chromosomal DNA, and DNA synthesis seems to occur during that DNA fibres may be firmly attached to the surface of poly-
the light period (Binder & Chisholm 1990). We have shown that phosphate bodies. We occasionally observed delineating mem-
the fibrous structures associated with polyphosphate bodies, branous structures surrounding holes left by the lost
which have been observed repeatedly in Synechococcus (Voelz polyphosphate bodies, although the existence of the membrane
et al. 1966; Lawry & Jensen 1979; Nitta et al. 2009), do contain has to be further clarified through rapid freezing and freeze
DNA through staining with osmium ammine-SO2. substitution. It is possible that the polyphosphate bodies are

262 Plant Biology 16 (2014) 258–263 © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands
 14388677, 2014, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/plb.12008 by Shizuoka University, Wiley Online Library on [17/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Seki, Nitta & Kaneko Polyphosphate bodies and DNA in cyanobacterial cells

surrounded with a membranous cover to which DNA fibres are to further investigate the nature of this relationship close to
attached; proteins that bind both DNA and membranes are the native state, studies applying rapid freezing, followed by
known in chloroplasts (Jeong et al. 2003; Kabeya et al. 2010). techniques such as electron tomography, would seem to be
There have been several reports indicating the existence of promising.
membranes surrounding polyphosphate bodies or volutin
granules, which are now known as acidocalcisomes, and simi-
larities between vacuoles and these structures have been ACKNOWLEDGEMENT
pointed out (Seufferheld et al. 2003, 2011; Brock et al. 2012).
We would like to thank Dr Kuniaki Nagayama for inspiring
This study strongly suggests a close relationship in cyano-
discussion.
bacteria between polyphosphate bodies and DNA. In order

protein has a conserved function related to the dis- Nitta K., Nagayama K., Danev R., Kaneko Y. (2009)
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