Effect of components released from bacteria on encystment in ciliated protozoan Colpoda sp.

Jpn. J. Protozool. Vol. 37, No. 2. (2004)
111
Original
Effect of components released from bacteria on encystment in ciliated
protozoan Colpoda sp.

Chiharu YAMASAKI, Akemi KIDA, Takahiko AKEMATSU and Tatsuomi MATSUOKA*

Institute of Biological Science, Faculty of Science, Kochi University, Kochi 780-8520, Japan
SUMMARY
INTRODUCTION


Ca2+-induced encystment of Colpoda sp. was
Free-living protozoa must contend with a
found to be cancelled by bacteria suspended in the number of ecological challenges, including lack of
surrounding medium. Continuation of the uptake of food organisms, desiccation, or pollution of the
polystyrene latex particles into the food vacuoles surrounding medium. In order to survive such
slightly suppressed encystment. When the cells of hostile conditions, some of them are transformed
Colpoda containing a large number of food vacu-
into resting cysts. The processes of encystment
oles filled with Congo-red stained bacteria were and excystment, which involve drastic morphoge-
transferred into an encystment-inducing medium, netic reconstruction of the cell (Grimes, 1973;
they encysted after promptly expelling the contents Matsusaka, 1979; Walker et al., 1980) that is
of the food vacuoles. Supernatant obtained from probably regulated by gene expression or silencing
bacterial suspension, commercial albumin or poly-
(Matsusaka, 1979; Hirukawa et al., 1998; Suizu and
peptone markedly prevented encystment, although Matsuoka, 1998; Izquierdo et al., 2000), must first
free amino acid did not have much of an affect. be preceded by the detection of environmental
Boiling or dialysis (MWCO: 10,000) of the super-
signals.
natant reduced the encystment-suppression effect.
Encystment of soil ciliate, Colpoda sp. is
These results suggest that the essential factors for induced by an increase in the concentrations of ions
the suppression of encystment are components such contained in the surrounding medium (Watoh et al.,
as peptides or proteins that are released from bac-
2003); among the ions, Ca2+ is the most effective
teria suspended in the surrounding medium.
(Yamaoka et al., 2004). Encystment of the cells
induced by 1 mM Ca2+ is markedly suppressed by
bacteria (106 - 107 cells/ml) suspended in the sur-
*Corresponding author
rounding medium (Watoh et al., 2003; Yamaoka et
Tel: + 81 88 844 8315
al., 2004). However, a much higher concentration
Fax: + 81 88 844 8356
of ions invalidates the encystment-suppression
E-mail: tmatsuok@cc.kochi-u.ac.jp
effect of bacteria (Yamaoka et al., 2004). The en-
Received: 16 March 2004; Accepted: 29 April 2004. cystment-suppression effect of bacteria is attribut-

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112
Effect of bacterial component on encystment
able to (1) the simple formation of food vacuoles, the value of the optical density at 600 nm (OD600).
(2) the nutrient supply from the food vacuoles, and The density of polystyrene latex particles (PLP)
(3) the suspension of components released from the (1.1 µm in diameter, Aldrich Chem. Co.) was also
bacteria in the surrounding medium. The present spectroscopically determined; its value was cali-
study examined three elements derived from bacte-
brated by comparing the density obtained by di-
ria that were suspended in the surrounding medium, rectly counting the number of particles under a
and revealed that the components released from microscope with the value of the optical density at
bacteria were essential for the suppression of en-
600 nm (OD600). The rate of encystment was ex-
cystment.
pressed as a percentage of the total number of

tested cells (50-60 cells). The columns (points)

and attached bars shown in Figs. 2-8 (except for
MATERIALS AND METHODS
Fig. 4a, open and closed squares) correspond to the

means of 4 identical measurements (50-60 cells
Colpoda sp. was isolated from cysts adher-
per measurement) and standard errors, the values
ing to dried fallen leaves in the field, and it was of which were obtained 8 hr after encystment in-
cultured in an infusion of dried cereal leaves duction. Each series of measurements was per-
(0.1% ) inoculated with bacteria (Enterobacter formed using the cells obtained from each of the
aerogenes) at 23°C in the dark. The bacteria were batch cultures.
cultured on agar plates containing 1.5% agar, 0.5%

polypeptone, 1% meat extract and 0.5% NaCl. For
encystment induction, two- or three-day cultured RESULTS AND DISCUSSION
vegetative cells were rinsed 3 times in each test

solution and transferred using a thin glass pipette;
First of all, it was confirmed that Colpoda
50-60 cells were subsequently suspended in 1.5 ml cells actually fed on living bacteria, killed (5-min
of each test solution. In order to obtain the sub-
boiled) bacteria or PLP (Fig. 1). When 3-day cul-
stances released from the bacteria, the cultured tured Colpoda cells containing a few food vacuoles
bacteria were rinsed twice in standard saline solu-
(Fig. 1a) were resuspended in bacteria-free standard
tion (1 mM CaCl2, 1 mM KCl?5 mM Tris-HCl, saline solution (1 mM CaCl2, 1 mM KCl, 5 mM
pH 7.2) by sedimentation (8,000 g, 5 min), and Tris-HCl, pH 7.2) for 1 hr, the food vacuoles com-
subsequently the bacteria were suspended in the pletely disappeared (Fig. 1b). Such starved cells
solution overnight at room temperature. The su-
formed a large number of food vacuoles when they
pernatant obtained from the bacterial suspension were transferred into standard saline solution with
was dialyzed in standard saline solution through suspensions of living bacteria (Fig. 1c), killed bac-
two types of membranes (MWCO, 1,000 Da or teria (Fig. 1d), and killed and Congo Red-stained
10,000 Da; Float-A-Lyzer, Spectrum Laboratories, bacteria (Fig. 1e), or PLP (Fig. 1f).
Inc.) at room temperature for about 48 hr. During
As has been reported previously (Watoh et
dialysis, the surrounding saline solution was re-
al., 2003; Yamaoka et al., 2004), the cells of Col-
placed by fresh solution several times. The density poda that were transferred into encystment-
of bacteria was spectroscopically determined in a induction media quickly encyst within 4-5 hr, and
diluted bacterial suspension, the value of which the rate of encysted cells reaches a plateau level. In
had been calibrated by comparing the cell density the present study, therefore, the rate of encysted
obtained by counting the colonies on plates with cells was measured 8 hr after encystment induction.

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Jpn. J. Protozool. Vol. 37, No. 2. (2004)
113
Fig. 1. Photomicrographs showing food vacuoles:
(a) three-day cultured Colpoda that is partially
starved; (b) a cell that was starved for 1 hr in a
Fig. 2. Suppression of Ca2+-induced encystment of
standard saline solution (1 mM CaCl2, 1 mM KCl, 5
Colpoda by the addition of living or killed (5-min
mM Tris-HCl pH 7.2); (c)-(f) cells that were incu-
boiled) bacteria (Enterobacter aerogenes, 107 cells/
bated with suspension of bacteria or non-nutrient
ml each) in the surrounding standard saline solu-
polystyrene latex particles (PLP, 1.1 µm in diame-
tion.
ter). The starved cells (b) were suspended for 1 hr
in standard saline solution with suspensions of liv-
ing bacteria (c), killed (5-min boiled) bacteria (d),
killed bacteria subsequently stained with 1% Congo
red (e) or PLP (f). The density of the bacteria and
as also suggested in the previous report by Tomaru
PLP were adjusted to 107 cells /ml and 3 x 106 par-
(2002). The fact that living or killed bacteria com-
ticles /ml, respectively. In order to take micro-
pletely cancel encystment (Fig. 2) implies that
graphs, cells were paralyzed with 0.05 mM NiCl2
existence of bacteria in the surrounding medium
(final concentration) was added to the cell suspen-
and/or a continuation of the nutrient supply from
sion.
food vacuoles may be involved in the suppression
of encystment. When cells of Colpoda containing a
large number of food vacuoles that were filled with
When the Colpoda was transferred into standard killed bacteria, which were expected to supply a
saline solution, most of the cells encysted (Fig. 2). sufficient nutrient source, were transferred into a
However, the presence of living or killed (5-min fresh standard saline solution (without bacteria),
boiled) bacteria (107 cells/ml) in the surrounding cyst formation was only suppressed slightly (Fig.
medium completely cancelled any cyst formation 4a). Such a slight suppression may be attributable
(Fig. 2). As shown in Fig. 3, continuous uptake of to the simple formation of food vacuoles as in-
PLP into the food vacuoles caused a slight sup-
duced by PLP. The number of food vacuoles in a
pression of cyst formation (significantly different vegetative cell quickly decreased after the induction
from the encystment rate in the standard saline of encystment (Fig. 4a, open squares). In this case,
solution; p < 0.05, Mann-Whitney test). The result the undigested contents of food vacuoles were
indicates that simple induction of food vacuole expelled just prior to the transformation of the cell
formation with PLP is effective for the suppression into a rounded shape (Fig. 4b, arrows). As shown in
of encystment, although this effect is not complete, Fig. 4a (closed squares), in consequence, very few

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114
Effect of bacterial component on encystment
Fig. 3. Suppression of Ca2+-induced encystment
of Colpoda by the addition of polystyrene latex
particles (PLP) in the surrounding standard saline
solution. The density of the bacteria and PLP
were adjusted to 3 × 106 cells (particles) /ml.
food vacuoles were observed in the rounded cells
(early stage of cyst formation). These results sug-
Fig. 4. Encystment of Colpoda cells containing a
gest that the continuation of the nutrient supply
number of food vacuoles and changes in the num-
from food vacuoles is not an essential factor for
ber of food vacuoles in a vegetative cell or rounded
preventing the encystment of Colpoda.
cell after the induction of encystment with Ca2+.
Some components released from living or
(a) ?, the rate of encystment of starved cells (Ref.
Fig. 1b); ?, the rate of encystment of cells contain-
killed (5-min boiled) bacteria prominently can-
ing a number of food vacuoles filled with killed (5-
celled cyst formation (Figs. 5, 6). It is likely that
min boiled) and then Congo red (1%)-stained bac-
the effective components may be metabolic prod-
teria (Ref. Fig. 1e); ?, changes in the number of
ucts released from the living bacteria and/or cellular
food vacuoles of vegetative cells; ?, changes in
components excreted by the disrupted cells. When
the number of food vacuoles of encysting
the components obtained from the suspension of
(rounded) cells. In order to induce food vacuole
formation, starved cells were transferred and kept
living bacteria were dialyzed through a 1,000 Da-
for 1 hr in standard saline solution containing
or 10,000 Da-cutoff membrane, the encystment-
Congo red-stained bacteria (107 cells/ml). The cyst
suppressing effect decreased significantly in com-
formation was induced by suspending the starved
parison with that of the crude sample that had not
cells or food vacuole-bearing cells in the standard
been dialyzed (p < 0.05, Mann-Whitney test each),
saline solution without bacteria. The number of
although encystment-suppression activity still
food vacuoles in each cell was counted and ex-
pressed as the mean and standard error obtained
remained (Fig. 7). The sample boiled for 5 min also
from 4-10 cells. (b) Photomicrograph of encyst-
showed significantly reduced encystment-
ing cells that just expelled the contents of food
suppression activity in comparison with crude bac-
vacuoles. Arrows, expelled Congo Red-stained
terial products (p < 0.05, Mann-Whitney test) (Fig.
contents of food vacuoles.

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Jpn. J. Protozool. Vol. 37, No. 2. (2004)
115
Fig. 5. Suppression of Ca2+-induced encystment of
Fig. 6. Suppression of Ca2+-induced encystment of
Colpoda by the addition of components released
Colpoda by the addition of products released from
from living bacteria. The vegetative cells were
killed (5-min boiled) bacteria. The vegetative cells
transferred into standard saline solutions containing
were transferred into standard saline solutions
living bacteria (Enterobacter aerogenes, 107 cells/
containing living or killed (boiled for 5 min) bacte-
ml), or containing components released from dif-
ria (Enterobacter aerogenes, 107 cells/ml).
ferent densities of bacteria (106 - 108 cells/ml).
7). These results suggest that not only small erate in these solutions; when the bacteria were
molecules but also large molecules such as proteins inoculated into these solutions, the number of bac-
are involved in the suppression of encystment.
teria increased twice per 4 hr in albumin solution
Commercial albumin (from chicken egg) and (10 mg/ml), and twice per 1.2 hr in polypepton
polypepton remarkably suppressed encystment of solution (10 mg/ml) at room temperature. On the
the cells at concentrations above 1 mg/ml (Fig. 8). other hand, the inoculated bacteria did not grow in
On the other hand, glycine only slightly suppressed the supernatant obtained from bacterial suspension
encystment at concentrations of less than10 mM (107 cells/ml). The mean of density of bacteria
(0.75 mg/ml) (Fig. 8). These results suggest that contaminated in the test solutions with suspension
the encystment-suppression effect induced by pro-
of Colpoda (50-60 cells/ml) was 13 cells/ml (n=3),
teins or peptides may be responsible for some side-
the value of which was obtained by counting the
chain groups of amino acid residues or a specific colonies on culture plates. On the other hand, the
conformation dependent upon a short common bacterial density less than 103 cells/ml did not sup-
sequence of amino acids. It is likely that this en-
press Ca2+-induced encystment of Colpoda. In this
cystment-suppression effect may not be responsible case, mean values of the encystment rate (n=4)
for the common structure of amino acids such as were above 78.5% when the Colpoda cells were
amino and carboxyl groups. In the measurements transferred and kept for 8 hr in the standard saline
shown in Fig. 8, the cells were transferred into the solutions containing less than103 bacteria/ml
solutions containing albumin or polypepton every (61.9% at 104 bacteria/ml; 78.5% at 103 bacteria/
1.5 hr, because contaminated bacteria may prolif-
ml; 87.9% at 102 bacteria/ml; 89.3% in case of no

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Effect of bacterial component on encystment
Fig. 7. Effects of boiling or dialysis of products
obtained from living bacteria on the Ca2+-induced
Fig. 8. Effects of albumin from chicken egg (?),
encystment of Colpoda. “Saline solution,” standard
polypepton (?), or glycine (?) on the Ca2+-induced
saline solution; “Bacterial products,” supernatant
encystment of Colpoda. These chemicals were
obtained from suspension (standard saline solution)
dissolved in standard saline solution. In order to
of living bacteria (Enterobacter aerogenes, 107
eliminate the proliferation of bacteria in the test
cells/ml); “Boiled,” supernatant boiled for 5 min;
solutions (standard saline solution) containing
“Dialyzed (MWCO:1000),” supernatant cutoff
polypepton or albumin, the cells were transferred
below 1,000 Da; “Dialyzed (MWCO:10000),”
into fresh solution every 1.5 hr. The solution con-
supernatant cutoff below 10,000 Da. In order to
taining polypepton had been sterilized.
obtain bacterial components in the supernatant, the
living bacteria (107 cells/ml) were suspended over-
night in the standard saline solution at room tem-
perature.
addition of bacteria); there is no significant differ-
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