New Zealand Journal of Botany

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Intra-colony motility of Microcystis wesenbergii cells

BTM Mulling, SA Wood & DP Hamilton

To cite this article: BTM Mulling, SA Wood & DP Hamilton (2014) Intra-colony motility
of Microcystis wesenbergii cells, New Zealand Journal of Botany, 52:1, 153-159, DOI:
10.1080/0028825X.2013.861856

To link to this article: https://doi.org/10.1080/0028825X.2013.861856

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New Zealand Journal of Botany, 2014
Vol. 52, No. 1, 153–159, http://dx.doi.org/10.1080/0028825X.2013.861856

SHORT COMMUNICATION
Intra-colony motility of Microcystis wesenbergii cells
BTM Mullinga, SA Wooda,b* and DP Hamiltona
a
The University of Waikato, Hamilton, New Zealand; bCawthron Institute, Nelson, New Zealand
(Received 9 September 2013; accepted 17 October 2013)

Microcystis is a widely distributed genus of cyanobacteria that often forms surface blooms. The ability
of Microcystis colonies to regulate buoyancy in response to variations in nutrients and light, and in the
presence of low rates of turbulent mixing, is well described. Here, we report on the movement of
individual cells within Microcystis wesenbergii colonies. Cells actively rearranged themselves within
colonies, but randomly relative to other cells. Of the known types of cyanobacterial movement, gliding
or twitching mediated by type IV pili was deemed the most likely causative mechanism. Transmission
electron microscopy showed, however, no pilus-like structures on M. wesenbergii cell surfaces. We
speculate that intra-colony movement may optimize colony fitness by helping individual cells to
meet specific physiological requirements that may differ between the core and outer parts of the colony.
This may increase fitness of cells and explain the recent dominance of M. wesenbergii in some
New Zealand lakes.
Keywords: colony; cyanobacteria; motility; Microcystis; type IV pilus

Introduction been documented. Filamentous species can exhibit

Cyanobacteria are phototrophic prokaryotes that
show considerable morphological and develop-
mental diversity. They range from simple unicells
to complex filaments, and may occur singly or in
colonies (Whitton 2012). Cyanobacterial colonies
are well known for passive movements caused by
differences in density between cellular constituents
and the surrounding liquid which may be induced
by altering cellular gas vacuole content or chang‐
ing photosynthetic carbohydrate stores (Brookes &
Ganf 2001). The additional presence of low turbu‐
lent mixing velocities can substantially alter rates of
ascent or descent of colonies, potentially leading to
the well-known trait of surface bloom formation in
many cyanobacteria taxa (Oliver et al. 2012).
Many cyanobacterial species are also actively
motile, which is usually the result of phototaxis.
Three different mechanisms of movement have
gliding motility throughout all or part of their de‐
velopmental cycle. Gliding occurs without obvious
locomotory organelles or changes in cell shape,
and takes place only when in contact with a semi-
solid substrate. The mechanisms of gliding are
unknown, but two main hypotheses have been
proposed. One is that filaments are pushed forward
and rotated by proteinaceous contractile fibrils
located between the peptidoglycan layer and the
outer membrane. The second is that filaments are
propelled by the extrusion of mucilage through
pores surrounding each cell septum (Hoiczyk &
Baumeister 1998; Hoiczyk 2000; Read et al.
2007). Some unicellular cyanobacteria also exhibit
gliding or ‘twitching’ when in contact with another
surface. This type of motility has been studied ex‐
tensively in Synechocystis PCC6803 (Burriesci &
Bhaya 2008). The outer cell wall possesses thick

*Corresponding author. Email: susie.wood@cawthron.org.nz

Supplementary data available online at www.tandfonline.com/10.1080/0028825X.2013.861856
Supplementary file: Video S1. Cell movement in Microcystis wesenbergii.
© 2014 The Royal Society of New Zealand
154 BTM Mulling et al.
(6–8 nm) type IV pili (Tfp). Bhaya et al. (2000)
inactivated key genes implicated in pilus biogen-
esis and function, and demonstrated that the
motility of Synechocystis PCC6803 is dependent
on the presence of Tfp on the cell surface.
Waterbury et al. (1985) observed in unicellular
marine Synechococcus, a third form of motility,
termed ‘swimming’. This discovery was considered
remarkable because of the ability of Synechococcus
to swim in liquids without visual external organelles
for movement, such as flagella (Brahamsha 1996).
Several polypeptides and an extracellular protein,
SwmA, have been linked with swimming motility
(Brahamsha 1996). Recently, Ehlers & Oster (2012)
proposed that the movement results from a conti‐
nuous looped helical track on which proteins,
driven by proton-motive force, move along, thus
propelling the cell forward.
Microcystis is a common bloom-forming genus
that is found worldwide in eutrophic freshwater
ecosystems (Šejnohová & Maršálek 2012). Its
ability to produce compounds that are toxic to
humans and animals has seen it receive consider-
able scientific attention (Codd et al. 2005; Šejno-
hová & Maršálek 2012). Microcystis cells lack
individual sheaths, but under natural environmental
conditions are usually organized into colonies that
may consist of many thousands of cells, surrounded
by a common mucilage (Komárek & Anagnostidis
1999). Here, we report on the movement of indivi‐
dual cells within Microcystis wesenbergii colonies.
To our knowledge, intra-colony movement has never
been documented in Chroococcales. We speculate
on the ecological role that intra-colony movement
might play in the fitness of this species.
Materials and methods
Batch cultures of two colonial Microcystis spe‐
cies (CAWBG11 Microcystis sp. and CAWBG07
M. wesenbergii) were sourced from the Cawthron
Institute Culture Collection of Micro-algae (CICCM;
cultures.cawthron.org.nz). Cultures were maintained
in 70 mL plastic bottles (Labserv, Biolab, Auckland,
New Zealand) containing MLA-medium (Bolsch &
Blackburn 1996). Growth conditions were 20 ± 1 °C,
12:12 h light : dark (13.4 µmol photons m−2 s−1).
All other Microcystis spp. within the culture col‐
lection no longer form colonies, a common occur‐
rence when cultured for extended periods (Zhang
et al. 2007). Surface samples (500 mL) were also
collected from three lakes containing Microcy‐
stis; Kainui (37°40′S, 175°13′E, M. wesenbergii),
Knighton (37°47′S, 175°18′E, M. flos-aquae) and
Okaro (38°17′S, 176°23′E, Microcystis sp.). Sam-
ples were stored in glass bottles at 4 °C until
analysis.
Sub-samples (990 µL) were applied to a
microscope slide specifically designed to minimize
water convection in the sample fluid and reduce
capillary movement of water between the objec‐
tive slide and the cover slip (Fig. 1). The slide con‐
sisted of two slides, each 0.25 mm thick, mounted
on top of each other. The top slide contained a
hole (2.5 mm diameter) surrounded by a barrier
ring of 2 mm width filled with paraffin oil. A cover
slip was placed over the hole and barrier ring.
Samples were viewed at ×100 and ×200 with a
confocal microscope consisting of an Olympus
IX81 inverted microscope connected to an Olym-
pus Fluoview FV1000. Samples, containing at
least one Microcystis colony, were scanned 100
times, every 15 s, resulting in a total analysis
time of 25 min. Cell movement was observed only
in M. wesenbergii and a single colony from
CAWBG07 was selected for further analyses.
The scans (every 15 s) for this sample were
combined to make a 5-s time-sequence video.
Three reference points were assigned on the edge
of the mucilage of a single colony and 120 indi‐
vidual cells were selected for directional tracking
analysis. The reference points and individual cells
were tracked manually frame by frame (Image-
Pro Plus, V6, Media Cybernetics, Rockville, MD,
USA).
To investigate the presence of Tfp, CAWBG11,
CAWBG07 and M. aeruginosa CAWBG16 (also
from the CICCM) were grown in batch culture as
described above. Aliquots of each culture were
dropped onto 200 copper mesh grids which were
air-dried and stained with 1% phosphotungstic acid
(pH 7.0) for 30 s. Grids were allowed to air dry
before being observed under a BIRU-Tecnai™ G2
 Intra-colony motility of Microcystis wesenbergii cells
Figure 1 Aerial schematic view of the specifically designed microscope slide. The slide consisted of two slides of
0.25 mm thickness, mounted on top of each other. The top slide contained a hole (2.5 mm diameter) surrounded by a
barrier ring of 2 mm width filled with paraffin oil.
Spirit Twin transmission electron microscope
(TEM) at 120 kV.
Results
Individual cell movement within colonies was
observed in M. wesenbergii in both the culture
(CAWBG07) and field sample (Lake Kainui). No
cell movement was observed in other Microcystis
species. The scans (every 15 s) from one of the
image time series from CAWBG07 were combined
to make a 5-s sequence video (Supplementary File
1). During 15 of the 100 scans, the M. wesenbergii
colony remained stationary and during the other
scans the entire colony moved at low velocity
(average velocity ± SD, 0.066 ± 0.035 µm s−1).
At each time interval when there was no
colony movement, an average of 72.4 ± 7.9% of
the tracked cells were moving in the x−y dimen-
sion. Over the total time (25 min), individual cells
travelled an average accumulated distance of
140.9 ± 26.3 µm (min 95.2 µm; max 263.6 µm).
Individual cells moved at an average speed of
0.124 ± 0.018 µm s−1 (max 1.224 µm s−1). In
addition, cells moved in and out of the field, indi‐
cating the presence of vertical movement, although
we had no means of quantifying this. The direc-
tional analysis of four cells is shown (Figs. 2A–D).
Each followed a unique path, often changing
155
direction and crossing tracks. Velocities varied
randomly throughout the 25 min (Figs. 2A–D). Eight
directional categories were assigned and the dir-
ection moved (excluding periods of colony move-
ment) was found to be relatively evenly distributed
(average 9.1 ± 2.8% of time spent moving in each
direction).
TEM observations of M. aeruginosa CAWBG16
cells led to the identification of numerous pilus-
like structures on the cell surface (Fig. 3A). By
contrast, no pilus-like structures were observed on
the cell surfaces of Microcystis sp. CAWBG11 or
M. wesenbergii CAWBG07 (Figs. 3B–D). Bacteria
associated with the CAWBG07 appeared to have
very thin pilus-like structures on their cell surface
(Figs. 3C, 3D).
Discussion
To our knowledge, movement of Microcystis cells
inside a colony has not been documented pre-
viously. Motility was present in both cultured and
field samples of M. wesenbergii, but was not
observed in other Microcystis species. Movement
in M. wesenbergii occurs in the absence of any
obvious organs of locomotion (e.g. flagella) or
change in cell shape. The motility of M. wesenbergii
cells inside the colony is unlikely to occur via the
same mechanism as gliding because extracellular
156 BTM Mulling et al.

Figure 2 Movement paths of four Microcystis wesenbergii (CAWBG07) cells within a colony over a 25-min period.
Dots indicate the location of cells every minute and arrows indicate the direction of movement.

mucilage, the only available substrate inside the
colony, is unlikely to be sufficiently viscous to
facilitate gliding. Tfp-mediated movement seemed
a probable explanation. Under this scenario, pili
would extend, attach to polysaccharide or nearby
cells and retract, resulting in the observed cell move‐
ments within the colony. Four putative Tfp genes
and pilus-like appendages have been identified in
M. aeruginosa PCC7806 (Nakasugi & Neilan 2005)
suggesting that this warranted further investigation.
TEM examination of CAWBG07 showed no pilus-
like structures on the cell surface. By contrast, pilus-
like structures were present on the cell surface of
CAWBG16. This strain was not examined for
movement because it no longer forms colonies in
the culture collection. Fine pilus-like structures
were observed on bacterial cells closely associated
with CAWBG07. Given the low abundance and
size (< 1 µm dia.) of the bacteria cells relative to
M. wesenbergii cells, we suggest that it is unlikely
that these bacteria play a role in M. wesenbergii
cell movement. Attempts to create axenic strains of
CAWBG07 to facilitate further investigations of
motility were not successful. Although the speed
of movement is slower in M. wesenbergii (max
1.22 µm s−1; this study) than in Synechococcus
(5–25 µm s−1; Waterbury et al. 1985), it seems
plausible that the motility might occur via similar
processes. The S-layer glycoprotein SwmA has been
shown to be involved in Synechococcus motility
(Brahamsha 1996). A novel surface-exposed glyco-
protein has been identified in Microcystis (Zilliges
 Intra-colony motility of Microcystis wesenbergii cells 157

Figure 3 Transmission electron micrographs. A, Microcystis aeruginosa (CAWBG16) displaying pilus-like structures.
B, Microcystis sp. (CAWBG11) showing lack of pilus-like structures. C, Microcystis wesenbergii (CAWBG07) pilus-
like structures. D, CAWBG07 cells and associated bacteria which appear to have fine pilus-like structures associated
with them.

et al. 2008) demonstrating that similar compounds,
which might have similar functions, occur in this
genus.
Colony formation may be advantageous to
Microcystis and relates to potential for reduced
predation (Yang et al. 2006.) and increased rates of
ascent and descent (Wallace et al. 2000). At a
cellular level, however, there is potential for a
heterogeneous array of conditions within the
colony, with motility providing a mechanism to
actively regulate cellular function. Outer cells may
provide protection from photo-inhibitory irradi-
ance to cells at the centre, but may be subject to
higher predatory pressure. Conversely, cells situ-
ated near the middle of a colony may receive
reduced light and access to nutrients but have
lower rates of predatory attrition. For example, the
downwelling light intensity at a given distance
from the colony wall inside a colony (Lz) can by
calculated by:
Lz ¼ Lo eðkd zÞ
where Lo is the light intensity entering the colony,
kd is the light attenuation coefficient through the
colony and z is the distance from the colony from
the periphery of the colony. Using a general phy‐
toplankton community attenuation coefficient of
0.015 m2 (mg chlorophyll a)−1 (Krause-Jensen &
Sand-Jensen 1998), and specific measurements
typical for M. wesenbergii of a cell diameter
6 µm and a colony diameter of 100 µm, and 200
individual cells per colony (Wood et al. 2004)
158 BTM Mulling et al.
(3.8 × 108 cells mL−1, chlorophyll a quota of 0.17
pg cell−1; Long et al. 2001), a cell at the centre of
the colony would experience 4.6% less light than
a cell located on the outside, assuming that cells
are distributed evenly throughout the colony. Such
variations may be important in altering, for exam‐
ple, photosynthetic carbohydrate accumulation and
might therefore produce some heterogeneity of
buoyancy of cells within the colony.
The ability of an individual cell to regulate its
position within a colony would be advantageous,
to balance both the positive and negative attri‐
butes of colony formation. Although cell motility
presumably has an energy cost, it may enable in‐
dividual cells to minimize the negative effects
of colony formation, thus benefiting the colony
collectively. Microcystis wesenbergii has only
recently been recorded in New Zealand (Wood
et al. 2004). Since its discovery, its geographic
range has expanded and it is now a common
constituent of blooms in the central and upper
North Island (Wood, unpubl. data). Molecular
characterization of CAWBG07 (Rueckert et al.
2007) showed high (99%) 16S rRNA gene seq‐
uence similarity to other M. wesenbergii (Lyra
et al. 2001). Future work will explore motility in
strains from diverse localities and the mechanisms
which enable this cell motility.
Supplementary file
Video S1. Cell movement in Microcystis wesen-
bergii. Images were taken every 15 seconds and
combined to make this five second time-sequence
video.
Acknowledgements
The authors thank Barry O’Brien (University of Wai-
kato) for technical assistance and Hilary Holloway
(Auckland University) for the TEM. This research was
supported by the New Zealand Ministry of Business,
Innovation and Employment (UOWX0505; Lake Bio-
diversity Restoration).
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