FEMS Microbiology Letters 208 (2002) 275^279
www.fems-microbiology.org
Active and energy-dependent rapid formation of cell aggregates in
the thermophilic photosynthetic bacterium Chloro£exus aggregans
Satoshi Hanada
b
a;b;
, Keizo Shimada a , Katsumi Matsuura
a
a
Department of Biology, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachioji, Tokyo 192-0397, Japan
Research Institute of Biological Resources, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6,
1-1-1 Higashi, Tsukuba 305-8566, Japan
Received 18 October 2001; received in revised form 14 January 2002; accepted 14 January 2002
First published online 12 February 2002
Abstract
The thermophilic filamentous phototroph Chloroflexus aggregans was able to form a bacterial mat-like dense cell aggregate rapidly. The
aggregate formation, which was observed in growing cells in a liquid medium in a bottle, occurred every time within 20^30 min after the
cells were dispersed by shaking. The aggregation depended on the energy supplied by photosynthesis or respiration. Cells aggregated most
rapidly under temperature and pH conditions that support maximum growth. The aggregation was also accelerated by the addition of
3-isobutyl-1-methylxanthine that inhibits cyclic 3P,5P-AMP phosphodiesterase. Microscopic observation revealed that the bacterium has a
fast gliding mobility (1^3 Wm s31 ). The distinctive cell aggregation of C. aggregans was due to this rapid gliding movement. ß 2002
Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Anoxygenic ¢lamentous phototroph; Thermophile; Cell aggregation ; Gliding motility; Cyclic 3P,5P-AMP; Chloro£exus aggregans
1. Introduction
Thermophilic ¢lamentous photosynthetic bacteria belonging to the genus Chloro£exus occur in hot springs [1^3]. They
usually form dense bacterial mats with or without thermophilic cyanobacteria in natural hot springs and grow photoheterotrophically using substrates excreted from cyanobacteria or autotrophically using sul¢de and carbon dioxide
[1,4]. The formation of mats may be supported by their ¢lamentous morphology and gliding motility. The gliding motility is a type of movement in contact with a solid or semisolid surface without £agella-like propulsive organs [5]. It is
a smooth movement somewhat resembling the progress of a
snail and typically observed among multicellular ¢lamentous
bacteria, such as cyanobacteria and Chloro£exus species.
The mechanism of gliding has not been clari¢ed. However,
electron microscopic studies are endeavouring to identify
motor structures common among some gliders [6,7].
Chloro£exus aggregans which is able to rapidly form bacterial mat-like dense aggregates was isolated from a hot
spring [8]. The rapid aggregate formation observed in growing C. aggregans cells in a liquid medium has not been
observed in any strains of the other species in this genus,
i.e. Chloro£exus aurantiacus. Such a rapid cell aggregation
has also been reported in several ¢lamentous cyanobacteria: Anabaena cylindrica produced a clump of cells in a
liquid medium [9], and an aggregation observed in thermophilic cyanobacterium, Oscillatoria terebriformis, was particularly rapid [10,11]. Walsby [9] concluded that the aggregation was due to their rapid gliding motility. In addition,
Ohmori et al. found that a cell suspension of a ¢lamentous
cyanobacterium (Spirulena platenis) rapidly began to aggregate when cyclic 3P,5P-AMP (cAMP) was added [12]. cAMP
enhanced the respiration of the cells accompanying the activation of cellular movement in the cyanobacterium.
In this paper, we describe the rapid cell aggregation that
is the distinct characteristic of C. aggregans and show the
e¡ect of cAMP on this aggregation.
2. Materials and methods
* Corresponding author. Tel. : +81 (298) 61 6590;
Fax : +81 (298) 61 6587.
E-mail address : [email protected] (S. Hanada).
2.1. Bacterial strain and growth conditions
C. aggregans strain MD-66T (T = type strain, DSM
0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 4 8 0 - 9
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S. Hanada et al. / FEMS Microbiology Letters 208 (2002) 275^279
9485) was grown in PE medium [8]. The initial pH of the
medium was adjusted to 7.5. The cultures were incubated
in screw-capped bottles at 55‡C under incandescent light
(30 W m32 ). C. aurantiacus strains J-10-£T (DSM 635) and
OK-70-£ (DSM 636) were used as reference strains for
the physiological comparison [13]. These strains were
also grown in PE medium under the same conditions as
C. aggregans.
2.2. Measurement of gliding rates
Cell aggregates of C. aggregans grown photoheterotrophically were suspended in fresh PE medium (pH
7.5). A depression in the middle of a microscope slide
was ¢lled with the cell suspension. Then, a glass cover
was put on the depression slide, and sealed up with silicon
grease. Observation was done at 55‡C under additional
incandescent light via a glass ¢ber (Nikon Corporation,
Tokyo, Japan) under a microscope (Nikon Corporation,
Tokyo, Japan) ¢tted with a TV camera (Hamamatsu Photonics, Hamamatsu, Japan). Thermal conditions were
achieved by blowing hot air and monitored with tapes
which change color depending on the temperature (Nichiyugiken Industry, Saitama, Japan). The movement of
¢laments was monitored with a time-lapse video recorder
(Panasonic, Tokyo, Japan). The gliding movements of 10
¢laments were traced on a TV screen to obtain the rate of
gliding.
2.3. Aggregation rates under various conditions
When cell density (OD620 ) was approximately 1.0 after
vigorous shaking of the culture, which was the middle of
the exponential phase on a growth curve of the bacterium
(OD620 was approximately 1.8, when a batch culture had
fully grown), cells were harvested and suspended in fresh
PE medium at the same concentration (OD620 = 1.0). Each
3-ml or 10-ml portion of the cell suspension was placed in
a cuvette or a Petri dish with a diameter of 35 mm, respectively. Anaerobic conditions were achieved by gasphase substitution with argon. PIPES and Tricine (Wako
chemicals, Osaka, Japan) were used as bu¡ers at a ¢nal
concentration of 10 mM in the experiments of pH dependence. The rates of aggregation were determined by measuring the diameter of the cell aggregates at appropriate
time intervals [11].
by measuring the diameter of aggregates monitored with a
time-lapse video recorder.
3. Results and discussion
3.1. Active aggregate formation
C. aggregans strain MD-66T grew in liquid media and
formed cell aggregates that resembled green balls [8]. The
aggregates were observed during the exponential phase
growth. These dense aggregates formed every time after
the bottle was shaken to make a uniform suspension,
and its formation was rapid within 20^30 min (the edge
of the aggregate drew together at a typical speed of about
20 Wm s31 ). Such an active and rapid aggregation has not
been observed in any strains in C. aurantiacus, another
species in this genus. The type strain of C. aurantiacus
(J-10-£T ) usually grows as a uniform suspension. Although
some strains of C. aurantiacus, e.g. strain OK-70-£, grow
by secreting mucilage and forming aggregates, this aggregation has an irregular shape and does not occur again
rapidly after dispersing cells.
Fig. 1 displays sequential photographs showing the aggregation of the cell suspension of C. aggregans in a
200-ml bottle of PE medium. After the C. aggregans cells
(grown photoheterotrophically) were dispersed by vigorous shaking and the uniform cell suspension was placed
at 55‡C under illumination (30 W m32 ), the cells in the
uniform suspension drew together. Within 15^30 min, they
formed a dense aggregate with a diameter of approximately 20 mm in a bottle with a diameter of 60 mm.
The aggregation of C. aggregans cells seems to be due to
their gliding motility. Microscopic observation at 55‡C
revealed that the bacterium has rapid gliding motility.
The ¢laments (approx. 1.5 Wm wide; 200^300 Wm long)
of C. aggregans actively glided on a glass plate along their
long axis. By tracing 10 ¢laments, it was shown that the
gliding rates of C. aggregans were 1^3 Wm s31 under the
conditions tested, while those of C. aurantiacus strains
J-10-£T and OK-70-£ measured under the same conditions
were 0.01^0.04 Wm s31 . The gliding rate of ¢laments of
C. aggregans was approximately 100U greater than that
of C. aurantiacus strains. The considerable di¡erence of
gliding rates between the two species is probably the reason for the di¡erence in their respective ability of rapid
aggregation.
2.4. Dependence on cAMP
3.2. Aggregation under various conditions
10 ml of cell suspension (OD620 = 1.0) was placed in a
Petri dish with a diameter of 35 mm. The incubation was
carried out semi-aerobically at 55‡C under incandescent
light (30 W m32 ). The e¡ects of various concentrations
of cAMP (Sigma, St. Louis, MO, USA) and 3-isobutyl1-methylxanthine (IBMX; Sigma), the inhibitor of phosphodiesterase, on the rate of aggregation were determined
FEMSLE 10370 10-4-02
The aggregation in C. aggregans cells was dependent on
the illumination or the oxygen supply (Table 1). Under
anaerobic conditions in the experiment in a 3-ml cuvette,
the edge of the cell aggregate drew together at a rate of 17
Wm s31 in a light intensity of 30 W m32 , the rate being
slower in lower light (5 W m32 ). No aggregation occurred
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S. Hanada et al. / FEMS Microbiology Letters 208 (2002) 275^279
277
Fig. 1. Aggregation of cell suspension of C. aggregans in a bottle of PE medium at 55‡C under anaerobic and light conditions. A dense cell aggregate
was formed rapidly within around 20 min.
under anaerobic conditions in the dark. Under semi-aerobic conditions, the aggregation rates were slightly greater
than those under anaerobic conditions, and the cells also
aggregated without illumination at a reduced rate. These
observations indicate that the aggregation is dependent on
the energy supplied by photosynthesis and/or oxygen respiration.
The dependence on the temperature and pH of aggregation was also investigated (Fig. 2). The aggregation was
most rapid at 55‡C, and the rate at 45‡C was less than
one-¢fth of the maximum. Cells aggregated at a pH between 7.0 and 8.5, and no aggregation was observed at pH
6.5 or 9.0. The optimum temperature for the growth of
C. aggregans was around 55‡C. The organism was able to
grow at a pH range from 7.0 up to 9.0, and little growth
occurred below pH 7.0 or above pH 9.0 [8]. The dependence on temperature and pH in the aggregation indicates
that C. aggregans ¢laments aggregate most rapidly under
conditions supporting maximum cell growth.
The ability of rapid aggregation may be advantageous
for the organism in the bacterial mat in a £owing stream
of natural hot springs, because the cells can rapidly gather
together in a mat and grow well under the conditions
without being carried away by water £ow.
3.3. E¡ects of cAMP on cell aggregation
It has been reported that the ¢lamentous cyanobacterium S. platenis began to aggregate when cAMP was
Table 1
Rates of cell aggregation (Wm s31 ) under anaerobic and semi-aerobic
conditions with di¡erent light intensity
Conditions
Anaerobic
Semi-aerobic
Light intensity (W m32 )
30
5
0
17
22
13
22
No aggregation
8
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Fig. 2. The dependence on temperature (A) and pH (B) of aggregation
rate in C. aggregans measured in PE medium under illumination (30 W
m32 ). PIPES and Tricine (10 mM) were used as pH bu¡ers.
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S. Hanada et al. / FEMS Microbiology Letters 208 (2002) 275^279
added to a suspension of cells [12]. IBMX also showed a
stimulatory e¡ect on the aggregation of this cyanobacterium [14]. Since IBMX is an inhibitor of cAMP phosphodiesterase [15], its addition presumably increases the intracellular level of cAMP.
When a cell suspension of C. aggregans in a Petri dish
(diameter = 35 mm) was incubated under illumination at
55‡C, cells rapidly gathered and began to form a diskshaped aggregate which became smaller with time. Fig.
3A shows changes in the diameter of the disks of aggregates after the additions of cAMP. The diameter of a disk
added with a low concentration of cAMP (the ¢nal concentration, 1 WM) decreased at the same rate as that of a
control sample. However, supplementation with a higher
concentration (100 WM) stimulated cell aggregation. The
acceleration of aggregation was shown more clearly in the
presence of IBMX (Fig. 3B). The addition of IBMX at a
¢nal concentration of 1 mM approximately tripled the rate
of aggregate formation.
It is noteworthy that even though C. aggregans and
cyanobacteria are phylogenetically distant to each other,
the acceleration of cell aggregation by cAMP was observed in both of them. This ¢nding is also interesting
from an ecological point of view. In a natural environment, C. aggregans usually forms bacterial mats together
with ¢lamentous cyanobacteria in the streams of hot
springs. Cyanobacteria contain relatively large amounts
of cAMP [16^19], and the excretion of cAMP by cyanobacterial cells into their surrounding medium has also
been reported [20]. In hot springs, ¢lamentous cyanobacteria may excrete cAMP as they grow in the form of mats,
which may a¡ect the aggregation of ¢laments of C. aggregans closely associated with cyanobacteria.
3.4. Rapid gliding movement of C. aggregans
The gliding rate of C. aggregans (1^3 Wm s31 ) was approximately 100U higher than that of C. aurantiacus
strains and about 10U more than that of the related ¢lamentous photosynthetic bacterium, Heliothrix oregonensis
[21]. The rate was also much larger than those of other
non-photosynthetic gliding bacteria, e.g. Flexibacteria and
Myxobacteria, which were less than 1 Wm s31 [5]. In oxygenic cyanobacteria, a member of Oscillatoriaceae glided
at a rate of 2^11 Wm s31 [5]. The diameters of the cells of
the cyanobacterium, however, were 5^10U wider than
those of the cells of C. aggregans. Although the correlation between cell size and gliding force is not clear, the
gliding activity of C. aggregans may be comparable to
those of Oscillatoriaceae. Thus, C. aggregans should be
one of the most rapid gliders among all gliding bacteria,
and the bacterium may be useful for the studies of the
mechanism of gliding movement which has not yet been
clari¢ed.
Acknowledgements
This work was supported by grants-in-aid from the
Ministry of Education, Science and Culture, Japan and a
special grant (1999) from Tokyo Metropolitan University.
References
Fig. 3. E¡ects of cAMP and an inhibitor of cAMP phosphodiesterase
on the cell aggregates. A: Changes in diameter of cell aggregate of
C. aggregans after addition of cAMP. cAMP was added at concentrations of 0 WM (closed circles), 1 WM (open circles), and 100 WM (open
squares). B: Changes in diameter of cell aggregate of C. aggregans after
addition of IBMX. IBMX was added at concentrations of 0 WM (closed
circles), 100 WM (open circles), and 1000 WM (open squares). These experiments were performed at 55‡C under 30 W m32 .
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