Jpn. J. Protozool. Vol. 37, No. 2. (2004)

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Original
Effect of components released from bacteria on encystment in ciliatedprotozoan Colpoda sp.
Chiharu YAMASAKI, Akemi KIDA, Takahiko AKEMATSU and Tatsuomi MATSUOKA*
Institute of Biological Science, Faculty of Science, Kochi University, Kochi 780-8520, JapanSUMMARY

INTRODUCTION

Ca2+-induced encystment of Colpoda sp. wasfound to be cancelled by bacteria suspended in thesurrounding medium. Continuation of the uptake ofpolystyrene latex particles into the food vacuoles
slightly suppressed encystment. When the cells ofColpoda containing a large number of food vacuoles filled with Congo-red stained bacteria weretransferred into an encystment-inducing medium,
they encysted after promptly expelling the contentsof the food vacuoles. Supernatant obtained frombacterial suspension, commercial albumin or polypeptone markedly prevented encystment, although
free amino acid did not have much of an affect.
Boiling or dialysis (MWCO: 10,000) of the supernatant reduced the encystment-suppression effect.
These results suggest that the essential factors forthe suppression of encystment are components suchas peptides or proteins that are released from bacteria suspended in the surrounding medium.

Free-living protozoa must contend with anumber of ecological challenges, including lack offood organisms, desiccation, or pollution of thesurrounding medium. In order to survive such
hostile conditions, some of them are transformed
into resting cysts. The processes of encystment
and excystment, which involve drastic morphogenetic reconstruction of the cell (Grimes, 1973;
Matsusaka, 1979; Walker et al., 1980) that isprobably regulated by gene expression or silencing(Matsusaka, 1979; Hirukawa et al., 1998; Suizu andMatsuoka, 1998; Izquierdo et al., 2000), must firstbe preceded by the detection of environmental
signals.
Encystment of soil ciliate, Colpoda sp. is
induced by an increase in the concentrations of ionscontained in the surrounding medium (Watoh et al.,2003); among the ions, Ca2+ is the most effective(Yamaoka et al., 2004). Encystment of the cells
induced by 1 mM Ca2+ is markedly suppressed bybacteria (106 - 107 cells/ml) suspended in the surrounding medium (Watoh et al., 2003; Yamaoka etal., 2004). However, a much higher concentrationof ions invalidates the encystment-suppression
effect of bacteria (Yamaoka et al., 2004). The encystment-suppression effect of bacteria is attribut-*Corresponding author
Tel: + 81 88 844 8315
Fax: + 81 88 844 8356
E-mail: tmatsuok@cc.kochi-u.ac.jp
Received: 16 March 2004; Accepted: 29 April 2004.

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Effect of bacterial component on encystment

able to (1) the simple formation of food vacuoles,(2) the nutrient supply from the food vacuoles, and(3) the suspension of components released from thebacteria in the surrounding medium. The present
study examined three elements derived from bacteria that were suspended in the surrounding medium,and revealed that the components released frombacteria were essential for the suppression of encystment.

MATERIALS AND METHODS
Colpoda sp. was isolated from cysts adhering to dried fallen leaves in the field, and it wascultured in an infusion of dried cereal leaves(0.1% ) inoculated with bacteria (Enterobacteraerogenes) at 23°C in the dark. The bacteria werecultured 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
vegetative cells were rinsed 3 times in each test
solution and transferred using a thin glass pipette;
50-60 cells were subsequently suspended in 1.5 mlof each test solution. In order to obtain the substances released from the bacteria, the cultured
bacteria were rinsed twice in standard saline solution (1 mM CaCl2, 1 mM KCl，5 mM Tris-HCl,
pH 7.2) by sedimentation (8,000 g, 5 min), andsubsequently the bacteria were suspended in thesolution overnight at room temperature. The supernatant obtained from the bacterial suspension
was dialyzed in standard saline solution throughtwo types of membranes (MWCO, 1,000 Da or
10,000 Da; Float-A-Lyzer, Spectrum Laboratories,Inc.) at room temperature for about 48 hr. During
dialysis, the surrounding saline solution was replaced by fresh solution several times. The densityof bacteria was spectroscopically determined in adiluted bacterial suspension, the value of which
had been calibrated by comparing the cell density
obtained by counting the colonies on plates withthe value of the optical density at 600 nm (OD600).
The density of polystyrene latex particles (PLP)(1.1 µm in diameter, Aldrich Chem. Co.) was also
spectroscopically determined; its value was calibrated by comparing the density obtained by directly counting the number of particles under amicroscope with the value of the optical density at
600 nm (OD600). The rate of encystment was expressed as a percentage of the total number oftested cells (50-60 cells). The columns (points)
and attached bars shown in Figs. 2-8 (except forFig. 4a, open and closed squares) correspond to themeans of 4 identical measurements (50-60 cellsper measurement) and standard errors, the valuesof which were obtained 8 hr after encystment induction. Each series of measurements was performed using the cells obtained from each of thebatch cultures.

RESULTS AND DISCUSSIONFirst of all, it was confirmed that Colpoda
cells actually fed on living bacteria, killed (5-minboiled) bacteria or PLP (Fig. 1). When 3-day cultured Colpoda cells containing a few food vacuoles(Fig. 1a) were resuspended in bacteria-free standard
saline solution (1 mM CaCl2, 1 mM KCl, 5 mM
Tris-HCl, pH 7.2) for 1 hr, the food vacuoles completely disappeared (Fig. 1b). Such starved cells
formed a large number of food vacuoles when they
were transferred into standard saline solution withsuspensions of living bacteria (Fig. 1c), killed bacteria (Fig. 1d), and killed and Congo Red-stained
bacteria (Fig. 1e), or PLP (Fig. 1f).
As has been reported previously (Watoh etal., 2003; Yamaoka et al., 2004), the cells of Colpoda that were transferred into encystmentinduction media quickly encyst within 4-5 hr, andthe rate of encysted cells reaches a plateau level. In
the present study, therefore, the rate of encysted
cells was measured 8 hr after encystment induction.

Jpn. J. Protozool. Vol. 37, No. 2. (2004)

Fig. 1. Photomicrographs showing food vacuoles:(a) three-day cultured Colpoda that is partiallystarved; (b) a cell that was starved for 1 hr in astandard saline solution (1 mM CaCl2, 1 mM KCl, 5mM Tris-HCl pH 7.2); (c)-(f) cells that were incubated with suspension of bacteria or non-nutrient
polystyrene latex particles (PLP, 1.1 µm in diameter). The starved cells (b) were suspended for 1 hr
in standard saline solution with suspensions of living bacteria (c), killed (5-min boiled) bacteria (d),killed bacteria subsequently stained with 1% Congored (e) or PLP (f). The density of the bacteria andPLP were adjusted to 107 cells /ml and 3 x 106 particles /ml, respectively. In order to take micrographs, cells were paralyzed with 0.05 mM NiCl2(final concentration) was added to the cell suspension.

When the Colpoda was transferred into standard
saline solution, most of the cells encysted (Fig. 2).
However, the presence of living or killed (5-minboiled) bacteria (107 cells/ml) in the surrounding
medium completely cancelled any cyst formation(Fig. 2). As shown in Fig. 3, continuous uptake of
PLP into the food vacuoles caused a slight suppression of cyst formation (significantly different
from the encystment rate in the standard salinesolution; p < 0.05, Mann-Whitney test). The result
indicates that simple induction of food vacuole
formation with PLP is effective for the suppressionof encystment, although this effect is not complete,

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Fig. 2. Suppression of Ca2+-induced encystment ofColpoda by the addition of living or killed (5-minboiled) bacteria (Enterobacter aerogenes, 107 cells/ml each) in the surrounding standard saline solution.

as also suggested in the previous report by Tomaru(2002). The fact that living or killed bacteria completely cancel encystment (Fig. 2) implies thatexistence of bacteria in the surrounding medium
and/or a continuation of the nutrient supply fromfood vacuoles may be involved in the suppressionof encystment. When cells of Colpoda containing alarge number of food vacuoles that were filled withkilled bacteria, which were expected to supply asufficient nutrient source, were transferred into a
fresh standard saline solution (without bacteria),
cyst formation was only suppressed slightly (Fig.
4a). Such a slight suppression may be attributable
to the simple formation of food vacuoles as induced by PLP. The number of food vacuoles in avegetative cell quickly decreased after the inductionof encystment (Fig. 4a, open squares). In this case,the undigested contents of food vacuoles wereexpelled just prior to the transformation of the cell
into a rounded shape (Fig. 4b, arrows). As shown in
Fig. 4a (closed squares), in consequence, very few

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Effect of bacterial component on encystment

Fig. 3. Suppression of Ca2+-induced encystmentof 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 suggest that the continuation of the nutrient supply
from food vacuoles is not an essential factor for
preventing the encystment of Colpoda.
Some components released from living orkilled (5-min boiled) bacteria prominently cancelled cyst formation (Figs. 5, 6). It is likely thatthe effective components may be metabolic products released from the living bacteria and/or cellularcomponents excreted by the disrupted cells. Whenthe components obtained from the suspension ofliving bacteria were dialyzed through a 1,000 Daor 10,000 Da-cutoff membrane, the encystmentsuppressing effect decreased significantly in comparison with that of the crude sample that had notbeen dialyzed (p < 0.05, Mann-Whitney test each),although encystment-suppression activity still
remained (Fig. 7). The sample boiled for 5 min also
showed significantly reduced encystmentsuppression activity in comparison with crude bacterial products (p < 0.05, Mann-Whitney test) (Fig.

Fig. 4. Encystment of Colpoda cells containing a
number of food vacuoles and changes in the number of food vacuoles in a vegetative cell or roundedcell after the induction of encystment with Ca2+.
(a) ○, the rate of encystment of starved cells (Ref.
Fig. 1b); ●, the rate of encystment of cells containing a number of food vacuoles filled with killed (5min boiled) and then Congo red (1%)-stained bacteria (Ref. Fig. 1e); □, changes in the number offood vacuoles of vegetative cells; ■, changes in
the number of food vacuoles of encysting(rounded) cells. In order to induce food vacuole
formation, starved cells were transferred and keptfor 1 hr in standard saline solution containing
Congo red-stained bacteria (107 cells/ml). The cyst
formation was induced by suspending the starved
cells or food vacuole-bearing cells in the standard
saline solution without bacteria. The number offood vacuoles in each cell was counted and expressed as the mean and standard error obtained
from 4-10 cells. (b) Photomicrograph of encysting cells that just expelled the contents of foodvacuoles. Arrows, expelled Congo Red-stained
contents of food vacuoles.

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115Fig. 5. Suppression of Ca2+-induced encystment ofColpoda by the addition of components released
from living bacteria. The vegetative cells weretransferred into standard saline solutions containing
living bacteria (Enterobacter aerogenes, 107 cells/ml), or containing components released from different densities of bacteria (106 - 108 cells/ml).

Fig. 6. Suppression of Ca2+-induced encystment ofColpoda by the addition of products released fromkilled (5-min boiled) bacteria. The vegetative cells
were transferred into standard saline solutions
containing living or killed (boiled for 5 min) bacteria (Enterobacter aerogenes, 107 cells/ml).

7). These results suggest that not only small
molecules but also large molecules such as proteins
are involved in the suppression of encystment.
Commercial albumin (from chicken egg) andpolypepton remarkably suppressed encystment of
the cells at concentrations above 1 mg/ml (Fig. 8).
On the other hand, glycine only slightly suppressedencystment at concentrations of less than10 mM(0.75 mg/ml) (Fig. 8). These results suggest thatthe encystment-suppression effect induced by proteins or peptides may be responsible for some sidechain groups of amino acid residues or a specificconformation dependent upon a short common
sequence of amino acids. It is likely that this encystment-suppression effect may not be responsible
for the common structure of amino acids such asamino and carboxyl groups. In the measurements
shown in Fig. 8, the cells were transferred into thesolutions containing albumin or polypepton every
1.5 hr, because contaminated bacteria may prolif-

erate in these solutions; when the bacteria wereinoculated into these solutions, the number of bacteria increased twice per 4 hr in albumin solution(10 mg/ml), and twice per 1.2 hr in polypepton
solution (10 mg/ml) at room temperature. On theother hand, the inoculated bacteria did not grow inthe supernatant obtained from bacterial suspension(107 cells/ml). The mean of density of bacteria
contaminated in the test solutions with suspensionof Colpoda (50-60 cells/ml) was 13 cells/ml (n=3),
the value of which was obtained by counting thecolonies on culture plates. On the other hand, thebacterial density less than 103 cells/ml did not suppress Ca2+-induced encystment of Colpoda. In thiscase, mean values of the encystment rate (n=4)
were above 78.5% when the Colpoda cells weretransferred and kept for 8 hr in the standard saline
solutions containing less than103 bacteria/ml(61.9% at 104 bacteria/ml; 78.5% at 103 bacteria/ml; 87.9% at 102 bacteria/ml; 89.3% in case of no116

Effect of bacterial component on encystment

Fig. 7. Effects of boiling or dialysis of products
obtained from living bacteria on the Ca2+-induced
encystment of Colpoda. “Saline solution,” standard
saline solution; “Bacterial products,” supernatantobtained from suspension (standard saline solution)of living bacteria (Enterobacter aerogenes, 107cells/ml); “Boiled,” supernatant boiled for 5 min;“Dialyzed (MWCO:1000),” supernatant cutoff
below 1,000 Da; “Dialyzed (MWCO:10000),”
supernatant cutoff below 10,000 Da. In order toobtain bacterial components in the supernatant, theliving bacteria (107 cells/ml) were suspended overnight in the standard saline solution at room temperature.

addition of bacteria); there is no significant difference among the encystment rates at the bacterial
density less than 103 cells/ml (p > 0.05, KruskalWallis test). In consequence, a small number ofbacteria (10-20 cells/ml) in the test solution may notaffect the rate of encystment shown in Fig. 8.
In conclusion, one essential factor in preventing Ca2+-induced encystment of Colpoda is thepresence of molecules such as proteins, peptides orother small molecules released from living or disrupted bacteria. In addition, the simple formationof food vacuoles is apt to suppress encystment,
although it is not important whether the contents ofthe vacuoles are nutrient or non-nutrient particles.

Fig. 8. Effects of albumin from chicken egg (○),
polypepton (●), or glycine (□) on the Ca2+-induced
encystment of Colpoda. These chemicals weredissolved in standard saline solution. In order toeliminate the proliferation of bacteria in the testsolutions (standard saline solution) containing
polypepton or albumin, the cells were transferred
into fresh solution every 1.5 hr. The solution containing polypepton had been sterilized.

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