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Ammonia and the induction of microcyst differentiation in wild-type and mutant strains of the cellular slime mold Polysphondylium pallidum
DEVELOPMENTAL
BIOLOGY
g&356-364
(1982)
Ammonia and the Induction of Microcyst Differentiation in Wild-Type and Mutant Strains of the Cellular Slime Mold Polysphondylium pallidurn A. H. C. CHOI AND D. H. O’DAY Department
of Zoology, Erindale
Campus, University
of Toronto, Mississauga,
Received December 11, 1981; accepted in revised form
March
Ontario
L5L lC6, Canada
15, 1982
Although low levels (0.16 rmole/107 cells/ml) of ammonia are present in vegetative, germinating, and young encystment cultures of wild-type (WS-320) cells, a 12-fold increase in extracellular ammonia occurs during microcyst differentiation in the cellular slime mold Polysphondylium pallidurn. When WS-320 amoebae were placed in conditioned medium from other wild-type encystment cultures, microcyst formation was initiated earlier than in control (120 mM KCI) cultures. Isoosmotic solutions containing NH&l and KC1 also caused WS-320 cells to encyst earlier. Similar results were obtained with mutant strains. In 120 mM KCl, strains mic-I and mic-2 produce 21 and 64% microcysts, respectively, while strains PN582 and PN651 do not encyst. The mutant mic-1 secretes wild-type levels of ammonia, while mic-2, PN582, and PN651 all secrete much lower amounts. Conditioned medium and solutions containing NH&l and KC1 increased the rate of development and the number of microcysts produced by mic-2 and induced microcyst development in the cystless mutant PN582. Taken together these data indicate that ammonia acts as an inducer of microcyst differentiation. INTRODUCTION
The amoebae of the cellular slime mold Polysphondylium pallidurn can follow either the sorocarp, macrocyst, or microcyst developmental pathway (Blaskovits and Raper, 1957). Of these three alternative pathways, the microcyst cycle has the unique feature that cells encyst to form a homogeneous population of unicellular microcysts, each of which can subsequently germinate to release a single amoeba. Some of the ultrastructural events (Hohl et al., 1970; Erdos and Hohl, 1980) and biochemical changes (O’Day, 1973a,b, 1974, 1976, 1977; O’Day and Francis, 1973; O’Day et al., 1976; Ennis et al., 1978; Gwynne and O’Day, 1978; O’Day and Paterno, 1979) that accompany the encystment-germination cycle have been detailed. Encystment is induced when amoebae are exposed to solutions of high osmotic pressure. After testing the effectiveness of over a dozen osmotically active agents, Toama and Raper (1967) concluded that KCl, glucose, and sucrose were especially effective in inducing encystment; and Lonski (1967) has shown that cells of P. pallidurn switch from sorocarp development of microcyst differentiation when cultures are exposed to high concentrations of NH,Cl. Ammonia is also an important morphogen during sorocarp development in P. pallidurn (Lonski, 1976), P. violaceum (Thadani et al., 1977), Dictyostelium mucoroides (Thadani et al., 1977), and D. discoideum (Schindler and Sussman, 1977a,b). In submerged cultures of P. pallidurn (Paterno and O’Day,
unpublished data) and D. discoideum (Sternfeld and David, 1979), ammonia is secreted and is required for spore and stalk differentiation. In the sorocarp pathway, local accumulations of excreted ammonia can regulate territory size (Feit, 1969; Lonski, 1976) and determine whether a newly formed aggregate immediately constructs a fruiting body or transforms into a migrating slug (Schindler and Sussman, 1977a,b). Apparently, ammonia can also interact with cyclic-AMP, another important morphogen in the development of Dictyostelium species (Schindler and Sussman, 1977a,b; Thadani et al., 1977). Because of the importance of ammonia in cellular slime mold development, we decided to critically examine its role as an inducer of microcyst development. Although Lonski (1976) had measured the levels of ammonia produced by fruiting cultures, data were not provided on the amount of ammonia produced during encystment. We have extended the preliminary work of Lonski (1976) by quantifying the levels of ammonia produced during vegetative growth, encystment, and germination in strain WS-320 of P. pallidurn and during encystment in four mutant strains. These data have enabled us to correlate the release of ammonia with the processes of encystment and germination. In addition, by culturing wild-type and mutant cells in conditioned medium from wild-type encystment cultures and in isoosmotic solutions containing NHICl and KCl, we have been able to show that ammonia induces microcyst formation. 356
0012-1606/82/080356-09$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
Microcysts
357
of P. pallidurn
taining 10 g proteose peptonejliter of distilled water (hereafter called 10 g MSM; O’Day and Francis, 1973). During log phase (1 to 3 X lo6 cells/ml), the amoebae were resuspended in 10 mM phosphate buffer (pH 6.5). Sterile NTG was added to give a final concentration of 1 mg/ml at a cell density of 1 X lo6 cells/ml. After shaking (200 rpm for 30 min at 22 * l”C), the cells were washed three times in phosphate buffer and then resuspended in 10 g MSM for 4 hr. The cell density was readjusted so that each well of the multitest trays (Model IS-MRC-96, Linbro Scientific, Inc., Hamden, Conn.) received, on the average, 0.7 cell/well. When growth was detected after shaking (200 rpm at 22 * l”C), replicates were made into new wells containing 15 g MSM which contains 15 g proteose peptone/liter of distilled water (O’Day, 1974). Under these conditions, strain WS-320 formed 72 to 85% microcysts. Clones that formed few microcysts were isolated for further studies.
FIG. 1. Phase-contrast micrographs of P. pallidurn cells. (A) WS320 amoebae at 0 hr and (B) WS-320 microcysts at 26 hr in KC]. Amoebae and microcysts of the mutant strains (C) mic-1 and (D) mic2 after 26 hr in KCI. (E) Cells of PN582 after 26 hr in 57 mM NH,Cl and 60 mM KCI. x700. MATERIALS
)-
AND METHODS
Stock cultures. Five strains of P. pallidum were used in this study. These included the wild type (WS-320) and two mutant strains (mic-1 and mid?) that were isolated after nitrosoguanidine (NTG) mutagenesis of WS-320 amoebae. In addition, two mutant strains (PN582 and PN651) isolated and kindly supplied by D. Francis (University of Delaware, Newark, Del.) were employed. Stock cultures of all strains were maintained on Escherichia coli (strain B/r) growing on LPP agar plates (0.1% lactose, 0.1% proteose peptone, and 1.5% agar). Mutant isolation. The method for isolating the mutant strains, mic-1 and mic-2, was modified from that of Dimond et al. (1973). Vegetative amoebae were grown axenically in modified Sussman’s (1963) medium con-
IO
22 16 HOURS
28
FIG. 2. Encystment profiles of the WS-320 and mutant strains of P. pallidurn. The WS-320 (A), mic-I, (m), mic-2 (V), PN582 (0) and PN651 (e) cells were cultured in 120 mM KC1 at pH 6.5 and 22 k 1°C as detailed under Materials and Methods. The vertical bars represent standard deviations.
358
DEVELOPMENTALBIOLOGY TABLE
VOL~JME 92, 1982 1
CHARACTERISTICSOFTHEP. pallidum
STRAINSUSED
Fruiting
structure”
Percentage microcysts at 26 hr in KC]*
Aggregation
Stalk
Spore
WS-320
Normal streaming in aggregates, normal size of aggregation territories
A main stalk with about 10 whorls and about 5 branches radiating from each whorl
Normal ellipsoid-shaped spores in spore masses at the tips of stalk and branches
93.0 f 1.9
mic-1’
Few, small aggregation centers
Few short stalks with no branches
No spores
20.3 f 2.2
mic-&
Aggregation centers break up into several smaller clumps
Short stalks of irregular thickness and few or no branches
Normal looking spores in a terminal mass at the end of branches
64.2 I 3.8
PN582” and PN651d
Normal streaming territory size
Normal number of whorls and branches
Normal
Strain
and
shapes and sizes
’ Sorocarp developmental was assessed after depletion of E. coli on LPP agar plates. b Microcyst formation was induced in 120 mM KC1 as described under Materials and Methods. Percentage means i: SD. ’ Mutant strains were obtained after mutagenesis of WS-320 as described under Materials and Methods. d Mutant strains were obtained from D. Francis (University of Delaware, Newark, Del.).
These clones were first maintained as stock cultures on LPP plates with E. coli (B/r). Spores or amoebae (in the case of sporeless mutants) were subcultured from the stock plates into 10 g MSM and when the cell density reached 1 to 3 X lo6 cells/ml, aliquots containing 5 X lo6 cells were inoculated into loo-ml volumes of 15 g MSM (O’Day, 1974). After growing the cells for 7 days, two clones (mic-1 and mic-2) that showed abnormal encystment kinetics were selected for the present study. Induction of encystment. The amoebae of WS-320 and the mutant strains (mic-1, mic-2, PN582, and PN651) were grown axenically in 10 g MSM at 22 f 1°C to between 1 and 3 X lo6 cells/ml. The cells were pelleted by centrifugation at 10009 for 5 min, resuspended at lo7 cells/ml in an encystment inducing solution, and shaken at 22 + 1°C. Encystment was monitored under phasecontrast microscopy. Encystment solutions. Routinely, 120 mM KC1 (Toama and Raper, 1967) was used to induce encystment and served as the control solution. Conditioned medium was obtained from 12 and 25 hr KC1 induced WS-320 cultures by pelleting the cells at 3000 g for 5 min. The osmotic pressure of 120 mM KC1 was determined to be 240 mosm using an osmometer (Model G-62, Fiske Assoc. Inc., Mass.). To study the effects of ammonia on encystment, solutions containing 2.3, 11.5, and 57 mM NH4Cl were made isoosmotic (i.e., 240 mosm) by the addition of KCl. Microcyst germination. Three-day-old microcysts of WS-320 that had been formed in 120 mM KC1 were suspended in 10 mM phosphate buffer at a concentration
microcysts
0
are represented
as
of lo7 cells/ml at 22 & 1°C. The cells were shaken at 200 rpm until germination was complete (O’Day, 1974). Measurement of extracellular ammonia. Extracellular medium was prepared by removing cultures and pelleting the cells at 1OOOgfor 5 min. The ammonia in the extracellular medium was measured using Nessler’s reagent (Sigma Chemical Co., St. Louis, MO.) and quantified from a standard curve prepared from dilutions of NH4Cl (BDH Chemicals). Since the pK of NHJ NH: is 9.26 and the pH of the liquid medium used throughout this study was 6.5, the ratio of NH::NH, was calculated to be 59O:l. Thus in the encystment medium, ammonia existed predominantly as ammonium ion (NH:). RESULTS
Encystment
Pro&les of WS-320 and Mutants
Amoebae of wild-type and the mutants mic-1 and mic2 of P. pallidurn can be induced to form microcysts in 120 m&f KC1 (Figs. lA-D). At 22 & 1 “C, wild-type amoebae began to encyst at about 10 hr (Fig. 2). Thereafter, encystment followed a sigmoidal profile until 93% of the cell population had encysted by 26 hr. The mutant strain mic-1 did not begin encystment until about 16 hr (Fig. 2). About 21% microcysts were produced by mic1 by 26 hr (Fig. 2) with a maximum of about 40% microcysts being formed by 40 hr (data not shown). The mutant strain mic-2 began to encyst at about the same time as the wild type but only about 64% microcysts
CHOI AND O’DAY
Microcysts
359
of P. pallidurn
tures, an increase in the extracellular level of ammonia was observed which resulted in a E-fold increase of ammonia to 2 pmole/107 cells/ml by 22 hr (Fig. 3). Levels of Ammonia Mutant Strains
in the Extracellular
Media of the
The levels of extracellular ammonia in the mutant strains mic-1, mic-2, PN582, and PN651 were measured and compared with those of WS-320. Among these, only mic-I produced levels of ammonia comparable to that of WS-320 (Fig. 4). Considerably lower levels of ammonia were accumulated in the extracellular medium of the mutant strains mic-2 (Fig. 4) and PN582 and PN651 (Fig. 5). When about 66% of microcysts were observed, a level of 1.0 pmole/107 cells/ml was detected in mic-2 cultures as compared to 1.8 pmole in WS-320 (Fig. 4). In 120 mM KCl. PN582 and PN651 did not form
2.0
3
1.6
3 i
0
6
12 HOURS
18
FIG. 3. Accumulation of extracellular ammonia in cultures of P. pallidurn WS-320 cultures. The extracellular media of vegetative (A), germinating (8) and encysting (A) cultures of WS-320 were collected and their ammonia content was determined by Nessler’s reagent as described under Materials and Methods. The vertical bars represent standard deviations.
: h
1.2
0
.-
2 y 0 5 I
.*
+< I
was obtained by 26 hr (Fig. 2). Under the same conditions, PN582 and PN651 did not form microcysts (Fig. 2). Some characteristics of these mutant strains are summarized in Table 1. A more detailed analysis of these mutants will be reported elsewhere. Levels of Extracellular Ammonia in Vegetative, Germinating Microcyst and Encysting Cultures Measurements of extracellular ammonia showed that a constant level of approximately 0.16 pmole/107 cells/ ml was present in the medium of both vegetatively growing cells and germinating microcysts as well as during early encystment (0 to 10 hr) cultures (Fig. 3). When microcysts began to appear in the encysting cul-
\
2 z
.4
0
40 PERCENT MICROCYSTS
80
FIG. 4. Accumulation of extracellular ammonia during encystment of in WS-320, mic-1 and mic-2. Cells of WS-320 (A), mic-1 (m), and mic-2 (v) were induced in 120 mM KCI. Extracellular medium was collected and ammonia concentrations were measured as detailed under Materials and Methods. Ammonia concentrations were plotted as a function of percentage microcysts.
360
DEVELOPMENTAL BIOLOGY
/
2
VOLUME 92, 1982
On the other hand, the mutant strain mic-2 (Fig. 8) began encystment earlier and ultimately produced more microcysts in the conditioned medium than in KCl. For example, at 14,18, and 22 hr; 29,76, and 88% microcysts were observed, respectively, in the 25-hr medium and 24, 64, and 86% were observed in the 12-hr medium while only 16, 55, and 66% were observed in 120 mM KCl. Surprisingly, the mutant strain PN582 which was unable to form any microcysts in KC1 could be induced to form some microcysts by the conditioned medium (Fig. 1E and Table 2). At 72 hr, about 12 and 20% microcysts were observed after shaking the cells of PN582 in 12- and 25-hr conditioned medium respectively.
I 0
20 HdUORS
FIG. 5. Accumulation of extracellular ammonia in the mutant strains PN582 and PN651. Cells of WS-320 (A), PN582 (0) and PN651 (+) were shaken in 120 mM KCl. The ammonia concentrations of the extracellular medium were measured as detailed under Materials and Methods. The vertical bars represent standard deviations.
microcysts and by 20 hr, contained only 0.25 pmole of ammonia/lo7 cells/ml (Fig. 5) which was close to the levels in nonencysting cultures. Effects of Conditioned
Medium
on Encystment
The effects of conditioned medium from 12- and 25hr encystment cultures of WS-320 on the timing of encystment of WS-320 cells are shown in Fig. 6. Cells contained in the 25-hr conditioned medium began encysting much earlier than those in the 12-hr conditioned medium which in turn started encysting earlier than those of the control (120 mMKC1). Cells of the mutant strain mic-1 encysted similarly in both the WS-320 conditioned medium and in 120 mM KC1 (Fig. 7) while PN651 remained cystless in all the solutions (data not shown).
I
10
1
I
I
16
I
I
I
22
HOURS FIG. 6. The effects of conditioned medium on the encystment of WS-320. Conditioned medium at (12 and 25 hr) was collected from encysting (120 mM KCl) WS-320 cultures. The effects of 12-hr (A.... . A) and 25.hr (A - - - A) conditioned medium on encystment of WS-320 were compared with the effects of 120 mM KC1 (A __ A). The vertical bars represent standard deviations.
361
CHOI AND O’DAY
containing NH4C1 and KC1 could induce microcyst formation in the cystless mutant PN582 (Table 3) with this strain producing 30 and 50% microcysts at 17 and 26 hr of encystment, respectively. This mutant eventually reached 91% microcysts by 72 hr in 57 mM NH&l (Table 3). The mutant strain mic-1 did not respond to the isoosmotic solutions in any manner different from the KC1 control (Fig. 11). Furthermore, microcyst differentiation was not induced by any isoosmotic solution in the cystless mutant PN651 (data not shown). DISCUSSION
Ammonia is a natural cellular component pears to play an important role as a regulator
ib”RS
that apof fruit-
I-
FIG. 7. The effects of 12-hr (m. * * . .m) and 25hr (W - - - W) conditioned medium on the encystment of the mutant mic-1 were comn ). The vertical bars reppared with those of 120 mM KC1 (m ~ resent standard deviations.
Effects oj’ Exogenous Ammonia
on Encystment
The effects of isoosmotic solutions containing KC1 and 2.3, 11.5, 57, and 115 mM NH4C1 on the five strains of P. pall&urn are summarized in Figs. 9-11 and Table 3. The response of these strains to the exogenously supplied ammonia was found to be similar to those seen in the experiments with the 12- and 25-hr conditioned medium. At concentrations of 2.3 and 11.5 mM, NHICl in the presence of KC1 had pronounced enhancing effects on the encystment of WS-320 (Fig. 9) and mic-2 (Fig. 10). At 22 hr, about 88% microcysts were observed in these isoosmotic solutions as compared to 60% in 120 mM KC1 (Fig. 10). Although 57 mM NH4C1 accelerated the encystment of WS-320 in the presence of KC1 (Fig. 9), it had an inhibitory effect on the encystment of mic1 (Fig. 10). It was also noted that isoosmotic solutions
A
I.
’
10
I
I
I
16 HOURS
I
I
I
22
FIG. 8. The effects of conditioned medium on the encystment of the mutant mic-2. The effects of 12-hr (v. * . . 0~) and 25-hr (V- - - - 7) conditioned medium and 120 mM KC1 (V ~ V) on the encystment of mic-2 were compared. The vertical bars represent standard deviations.
362
DEVELOPMENTAL BIOLOGY
VOLUME 92, 1982
TABLE 2 THE EFFECTS OF CONDITIONED MEDIUM ON MICROCYST FORMATION IN THE CYSTLESS MUTANT PN582 AND IN WS-320” Percentage 120 mM KC1
microcysts*
12-hr conditioned
medium
25-hr conditioned
medium
Time (hr)
WS-320
PN582
WS-320
PN582
WS-320
PN582
0 17 26 72
0 34.4 f 4.3 90.2 f 5.1 95.6 f 2.4
0 0 0 0
0 41.0 f 4.9 94.5 f 3.2 95.1 * 1.2
0 1.0 f 0.7 6.3 f 2.7 12.3 f 4.1
0 74.5 f 4.5 94.6 ? 3.6 94.8 2 2.3
0 7.3 f 3.0 8.6 * 2.8 20.0 f 2.3
’ Conditioned media were collected at 12 and 25 hr from encysting (120 mM KCl) WS-320 cultures. The amoebae of WS-320 and PN582 were grown in 10 g MSM and were shaken in the conditioned media and 120 mM KCI (control) as detailed under Materials and Methods. b Percentage microcysts are represented as means f SD.
ing body development in cellular slime molds (Feit, 1969; Schindler and Sussman, 1977a,b; Thadani et aE., 1977). Also, the work of Lonski (1976) and Paterno and O’Day (unpublished data) have revealed that ammonia can induce microcyst development in Polysphondylium pallidurn. However, no study has shown that ammonia is normally associated with the development of microcysts. As a result we carried out an in-depth analysis of this problem, the results of which suggest that ammonia acts as an inducer of microcyst differentiation. Measurements of extracellular levels of ammonia in cultures of vegetative amoebae and germinating microcysts of wild-type Polysphondylium pallidurn (WS320) reveal the presence of low ammonia levels. In solutions of KCl, amoebae at first show similar ammonia concentrations but a 12-fold increase occurs during encystment. This elevation may be crucial to encystment because the mutant strains (mic-2, PN582, and PN651) that lack wild-type levels of ammonia are also defective in microcyst formation. Further experiments suggest that the increase in ammonia may be responsible for inducing microcyst formation. Encystment in WS-320 and in mic-2 was initiated earlier when amoebae were placed in conditioned medium as compared to KCl. In addition, increased numbers of microcysts were induced in mic-2 cells treated with the conditioned medium. The mutant strain PN582 did not encyst in KC1 solutions but a significant number of microcysts developed when cells were placed in conditioned medium. It is probable that the encystment I 1111 I I 1 I inducing activity of the conditioned medium is in part 19 23 15 attributed to ammonia because isoosmotic solutions containing NH4C1 and KC1 produced similar results. It HOURS was found that NHICl in the presence of KC1 induced FIG. 9. The effects of isoosmotic solutions on the encystment of WSearlier encystment in WS-320 and mic-2. Furthermore, 320.2.3mM(A---A),ll.5mM(A--A)57mM(A~~~~~A),and mixtures of NH4Cl and KC1 were able to induce higher 115 mM (A -.A) NH,Cl and 120 mM KC1 (A A). The vertical percentage of microcyst in mic-2 than KC1 alone and bars represent standard deviations.
CH~I
AND O’DAY
iwicrocysts
363
qf P. pullidum
was not affected by exogenous ammonia or the ammonia in conditioned medium. Also, the mutant PN651 which produced low levels of ammonia could not be induced to encyst by either conditioned medium or ammonia. Since these mutants cannot be induced to encyst by ammonia, it appears that deficiencies other than those involved in ammonia metabolism are responsible for their encystment defects. The studies of Toama and Raper (1967) and the work presented in the present communication are the only in-depth investigations of factors that influence microcyst formation in P. pallidurn. The work of Toama and Raper (1967) suggested that encystment in P. pallidurn requires high osmotic pressure and showed that a number of exogenously added ions possesss the ability to
3c
I14
HOk
2: ,
FIG. 10. The effects of isoosmotic solutions of NH,Cl on the encystment of the mutant mic-2 were compared with those of 120 mM KCl.2.3mM(~---~),11.5mM(~---~),57mM(~~~~~~~),and 115 mM (V -*V) NHICl and 120 mM KC1 (V ~ v). The vertical bars represent standard deviations.
to induce in KCI.
encystment
in PN582 which
was cystless
It has been shown that factor(s) other than ammonia are also required for differentiation of Dictyostelium amoebae in clumps of submerged cells (Sternfeld and David, 1979). Our results also suggest that other factor(s) may be involved in the differentiation of microcysts. Two lines of evidence support this. First, both WS-320 and mic-2 entered encystment earlier in 12-hr conditioned medium than in KCl. Since the concentration of ammonia in 12-hr conditioned medium was only slightly higher than the basal level, the encystment enhancing activity would not be likely attributed to this low level of ammonia. Second, encystment in the mutant mic-1, which produced wild-type amounts of ammonia,
I
I
I
I
I
I
I
I
22
16 tZ"RS
FIG. 11. The effects of isoosmotic solutions of NH&l and KC1 on the encystment of the mutant mic-1. 2.3 mM (m - - - n ), 11.5 mM (m---m), 57 rnM(W*.* * em), and 115 mM (~-an ) NH,Cl, and 120 mM KC1 (B ~ u). The vertical bars represent standard deviations.
364
DEVELOPMENTAL BIOLOGY
TABLE 3 THE EFFECTS OF AMMONIUM CHLORIDE ON MICROCYST FORMATION IN THE CYSTLESS MUTANT PN582 AND IN WS-320 Percentage 120 mM KC1
microcysts” 57 mM NH4Clb
Time (hr)
WS-320
PN582
WS-320
PN582
0 17 26 72
0 34.4 f 4.3 90.2 f 5.1 95.6 ?z 2.4
0 0 0 0
0 62.1 + 5.1 92.1 + 4.4 93.1 f 3.7
0 30.0 f 5.8 50.4 f 3.9 90.9 f 3.8
a Percentage microcysts are expressed as means f SD. *The osmotic pressure of the 57 mM NH&l was adjusted to 240 mosm by the addition of KCI. See Materials and Methods for details.
induce encystment. Of the ions studied, K+ and Cl- were the most effective. The specific requirement for K+ was exemplified by the inability of NaCl to replace KC1 as an inducer of microcyst formation (Toama and Raper, 1967). In this study, we have shown that 115 mMNH,Cl was not as effectve as 120 mMKC1 in inducing microcyst formation but lower concentrations of NH4C1 (2.3, 11.5, and 57 m&I) in conjunction with KC1 were more effective than KC1 or NH&l alone. Since elevated concentrations of ammonia are produced during microcyst formation and since added ammonia in isoosmotic solutions containing NH&l and KC1 enhances encystment in wildtype cells and induces encystment in certain otherwise cystless mutants, we have uneqivocally shown that NH: can enhance microcyst formation. This study is supported in part by a grant (Grant A6807) from the Natural Sciences and Engineering Research Council of Canada.
REFERENCES BLASKOVICS, J. C., and RAPER, K. B. (1957). Encystment stages of Dictyostelium. Biol BuU 113, 58-88. DIMOND, R. L., BRENNER, M., and LOOMIS, W. F. (1973). Mutations affecting N-acetylglucosaminidase in Dictyostelium dticoideum. Proc. Nat. Acad Sci. USA 70, 3356-3360. ENNIS, H. I., PENNICA, D., and HILL, J. M. (1978). Synthesis of macromolecules during microcyst germination in the cellular slime mold Polysphondylium pallidurn Dev. Biol. 65, 251-259. ERDOS, G. W., and HOHL, H. R. (1980). Freeze-fracture examination of the plasma membrane of the cellular slime mould Polysphodylium pallidurn during microcyst formation and germination. Cytobios29,7-11. FEIT, I. N. (1969). “Evidence for the Regulation of Aggregation Den-
VOLUME 92, 1982
sity by the Production of Ammonia in the Cellular Slime Molds.” Ph. D. thesis, Princeton University. GWYNNE, D., and O’DAY, D. H. (1978). RNA and protein synthetic patterns during germination of Polysphondylium pallidum microcysts. Canad. J. Microbial. 24, 480-486. HOHL, H. R., MIURA-SANTO, L. Y., and COTTER, D. A. (1970). Ultrastructural changes during formation and germination of microcysts in Polysphondylium pallidurn, a cellular slime mould. J. Cell Sci 7, 285-305. LONSKI, J. (1976). The effect of ammonia on fruiting body size and microcyst formation in the cellular slime molds. Dev. Biol. 51,158165. O’DAY, D. H. (1973a). Intracellular and extracellular acetylglucosaminidase activity during microcyst formation in Polysphondylium pallidum. Exp. Cell Res. 79, 186-190. O’DAY, D. H. (1973b). cu-Mannosidase and microcyst differentiation in the cellular slime mould Polysphwndylium pallidum. J. Bacteriol 113, 192-197. O’DAY, D. H. (1974). Intracellular and extracellular enzyme patterns during microcyst germination in the cellular slime mold Polysphondylium pallidurn. Dev. Biol. 36, 400-410. O’DAY, D. H. (1976). Acid protease activities during germination of the cellular slime mold Polysphondylium pallidurn. .I Bacterial. 125, 8-13. O’DAY, D. H. (1977). Microcyst germination in the cellular slime mold Polysphwndylium pallidum: Requirements for macromolecular synthesis and specific enzyme accumulation. In “Eucaryotic Microbes as Model Developmental Systems” (D. H. O’Day and P. H. Horgen, eds.), Vol. 2, pp. 353-371. Dekker, New York. O’DAY, D. H., and FRANCIS, D. (1973). Patterns of alkaline phosphatase activity during alternative developmental pathway in the cellular slime mold, Polysphondyliumpallidum. Canau! J. Zool. 51,301310. O’DAY, D. H., GWYNNE, D. I., and BLAKEY, D. H. (1976). Microcyst germination in the cellular slime mold, Polysphundylium pallidum. Exp. Cell Res. 97, 359-365. O’DAY, D. H., AND PATERNO, G. D. (1979). Intracellular and extracellular CM-cellulase and fl-glucosidase activity during germination of Polysphondylium pallidum microcysts. Arch. Microbial. 121,231234. SCHINDLER, J., and SUSSMAN, M. (1977a). Effect of NH3 on c-AMP associated activities and extracellular c-AMP production in Dietyostelium discoideum. B&hem. Biophys. Res. Cmnmun. 79,611-617. SCHINDLER, J., and SUSSMAN, M. (19’77b). Ammonia determines the choice of morphogenetic pathways in Dictyostelium discoideum. J. Mol Biol. 116, 161-169. STERNFELD, J., and DAVID, C. N. (1979). Ammonia plus another factor are necessary for differentiation in submerged clumps of Dictyostelium. J. Cell Sci. 38, 181-191. SUSSMAN, M. (1963). Growth of the cellular slime mold Polysphurdylium pallidum in a simple nutrient medium. Science 139, 338. THADANI, V., PAN, P., and BONNER, J. T. (1977). Complementary effects of ammonia and CAMP on aggregation territory size in the cellular slime mold Dictyostelium mucoroides. Exp. Cell Res. 108, 75-78. TOAMA, M. A., and RAPER, K. B. (1967). Microcysts of the cellular slime mold Polysphondylium pallidum. I. Factors influencing microcyst formation. J. BacterioL 94, 1143-1149.
BIOLOGY
g&356-364
(1982)
Ammonia and the Induction of Microcyst Differentiation in Wild-Type and Mutant Strains of the Cellular Slime Mold Polysphondylium pallidurn A. H. C. CHOI AND D. H. O’DAY Department
of Zoology, Erindale
Campus, University
of Toronto, Mississauga,
Received December 11, 1981; accepted in revised form
March
Ontario
L5L lC6, Canada
15, 1982
Although low levels (0.16 rmole/107 cells/ml) of ammonia are present in vegetative, germinating, and young encystment cultures of wild-type (WS-320) cells, a 12-fold increase in extracellular ammonia occurs during microcyst differentiation in the cellular slime mold Polysphondylium pallidurn. When WS-320 amoebae were placed in conditioned medium from other wild-type encystment cultures, microcyst formation was initiated earlier than in control (120 mM KCI) cultures. Isoosmotic solutions containing NH&l and KC1 also caused WS-320 cells to encyst earlier. Similar results were obtained with mutant strains. In 120 mM KCl, strains mic-I and mic-2 produce 21 and 64% microcysts, respectively, while strains PN582 and PN651 do not encyst. The mutant mic-1 secretes wild-type levels of ammonia, while mic-2, PN582, and PN651 all secrete much lower amounts. Conditioned medium and solutions containing NH&l and KC1 increased the rate of development and the number of microcysts produced by mic-2 and induced microcyst development in the cystless mutant PN582. Taken together these data indicate that ammonia acts as an inducer of microcyst differentiation. INTRODUCTION
The amoebae of the cellular slime mold Polysphondylium pallidurn can follow either the sorocarp, macrocyst, or microcyst developmental pathway (Blaskovits and Raper, 1957). Of these three alternative pathways, the microcyst cycle has the unique feature that cells encyst to form a homogeneous population of unicellular microcysts, each of which can subsequently germinate to release a single amoeba. Some of the ultrastructural events (Hohl et al., 1970; Erdos and Hohl, 1980) and biochemical changes (O’Day, 1973a,b, 1974, 1976, 1977; O’Day and Francis, 1973; O’Day et al., 1976; Ennis et al., 1978; Gwynne and O’Day, 1978; O’Day and Paterno, 1979) that accompany the encystment-germination cycle have been detailed. Encystment is induced when amoebae are exposed to solutions of high osmotic pressure. After testing the effectiveness of over a dozen osmotically active agents, Toama and Raper (1967) concluded that KCl, glucose, and sucrose were especially effective in inducing encystment; and Lonski (1967) has shown that cells of P. pallidurn switch from sorocarp development of microcyst differentiation when cultures are exposed to high concentrations of NH,Cl. Ammonia is also an important morphogen during sorocarp development in P. pallidurn (Lonski, 1976), P. violaceum (Thadani et al., 1977), Dictyostelium mucoroides (Thadani et al., 1977), and D. discoideum (Schindler and Sussman, 1977a,b). In submerged cultures of P. pallidurn (Paterno and O’Day,
unpublished data) and D. discoideum (Sternfeld and David, 1979), ammonia is secreted and is required for spore and stalk differentiation. In the sorocarp pathway, local accumulations of excreted ammonia can regulate territory size (Feit, 1969; Lonski, 1976) and determine whether a newly formed aggregate immediately constructs a fruiting body or transforms into a migrating slug (Schindler and Sussman, 1977a,b). Apparently, ammonia can also interact with cyclic-AMP, another important morphogen in the development of Dictyostelium species (Schindler and Sussman, 1977a,b; Thadani et al., 1977). Because of the importance of ammonia in cellular slime mold development, we decided to critically examine its role as an inducer of microcyst development. Although Lonski (1976) had measured the levels of ammonia produced by fruiting cultures, data were not provided on the amount of ammonia produced during encystment. We have extended the preliminary work of Lonski (1976) by quantifying the levels of ammonia produced during vegetative growth, encystment, and germination in strain WS-320 of P. pallidurn and during encystment in four mutant strains. These data have enabled us to correlate the release of ammonia with the processes of encystment and germination. In addition, by culturing wild-type and mutant cells in conditioned medium from wild-type encystment cultures and in isoosmotic solutions containing NHICl and KCl, we have been able to show that ammonia induces microcyst formation. 356
0012-1606/82/080356-09$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
Microcysts
357
of P. pallidurn
taining 10 g proteose peptonejliter of distilled water (hereafter called 10 g MSM; O’Day and Francis, 1973). During log phase (1 to 3 X lo6 cells/ml), the amoebae were resuspended in 10 mM phosphate buffer (pH 6.5). Sterile NTG was added to give a final concentration of 1 mg/ml at a cell density of 1 X lo6 cells/ml. After shaking (200 rpm for 30 min at 22 * l”C), the cells were washed three times in phosphate buffer and then resuspended in 10 g MSM for 4 hr. The cell density was readjusted so that each well of the multitest trays (Model IS-MRC-96, Linbro Scientific, Inc., Hamden, Conn.) received, on the average, 0.7 cell/well. When growth was detected after shaking (200 rpm at 22 * l”C), replicates were made into new wells containing 15 g MSM which contains 15 g proteose peptone/liter of distilled water (O’Day, 1974). Under these conditions, strain WS-320 formed 72 to 85% microcysts. Clones that formed few microcysts were isolated for further studies.
FIG. 1. Phase-contrast micrographs of P. pallidurn cells. (A) WS320 amoebae at 0 hr and (B) WS-320 microcysts at 26 hr in KC]. Amoebae and microcysts of the mutant strains (C) mic-1 and (D) mic2 after 26 hr in KCI. (E) Cells of PN582 after 26 hr in 57 mM NH,Cl and 60 mM KCI. x700. MATERIALS
)-
AND METHODS
Stock cultures. Five strains of P. pallidum were used in this study. These included the wild type (WS-320) and two mutant strains (mic-1 and mid?) that were isolated after nitrosoguanidine (NTG) mutagenesis of WS-320 amoebae. In addition, two mutant strains (PN582 and PN651) isolated and kindly supplied by D. Francis (University of Delaware, Newark, Del.) were employed. Stock cultures of all strains were maintained on Escherichia coli (strain B/r) growing on LPP agar plates (0.1% lactose, 0.1% proteose peptone, and 1.5% agar). Mutant isolation. The method for isolating the mutant strains, mic-1 and mic-2, was modified from that of Dimond et al. (1973). Vegetative amoebae were grown axenically in modified Sussman’s (1963) medium con-
IO
22 16 HOURS
28
FIG. 2. Encystment profiles of the WS-320 and mutant strains of P. pallidurn. The WS-320 (A), mic-I, (m), mic-2 (V), PN582 (0) and PN651 (e) cells were cultured in 120 mM KC1 at pH 6.5 and 22 k 1°C as detailed under Materials and Methods. The vertical bars represent standard deviations.
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DEVELOPMENTALBIOLOGY TABLE
VOL~JME 92, 1982 1
CHARACTERISTICSOFTHEP. pallidum
STRAINSUSED
Fruiting
structure”
Percentage microcysts at 26 hr in KC]*
Aggregation
Stalk
Spore
WS-320
Normal streaming in aggregates, normal size of aggregation territories
A main stalk with about 10 whorls and about 5 branches radiating from each whorl
Normal ellipsoid-shaped spores in spore masses at the tips of stalk and branches
93.0 f 1.9
mic-1’
Few, small aggregation centers
Few short stalks with no branches
No spores
20.3 f 2.2
mic-&
Aggregation centers break up into several smaller clumps
Short stalks of irregular thickness and few or no branches
Normal looking spores in a terminal mass at the end of branches
64.2 I 3.8
PN582” and PN651d
Normal streaming territory size
Normal number of whorls and branches
Normal
Strain
and
shapes and sizes
’ Sorocarp developmental was assessed after depletion of E. coli on LPP agar plates. b Microcyst formation was induced in 120 mM KC1 as described under Materials and Methods. Percentage means i: SD. ’ Mutant strains were obtained after mutagenesis of WS-320 as described under Materials and Methods. d Mutant strains were obtained from D. Francis (University of Delaware, Newark, Del.).
These clones were first maintained as stock cultures on LPP plates with E. coli (B/r). Spores or amoebae (in the case of sporeless mutants) were subcultured from the stock plates into 10 g MSM and when the cell density reached 1 to 3 X lo6 cells/ml, aliquots containing 5 X lo6 cells were inoculated into loo-ml volumes of 15 g MSM (O’Day, 1974). After growing the cells for 7 days, two clones (mic-1 and mic-2) that showed abnormal encystment kinetics were selected for the present study. Induction of encystment. The amoebae of WS-320 and the mutant strains (mic-1, mic-2, PN582, and PN651) were grown axenically in 10 g MSM at 22 f 1°C to between 1 and 3 X lo6 cells/ml. The cells were pelleted by centrifugation at 10009 for 5 min, resuspended at lo7 cells/ml in an encystment inducing solution, and shaken at 22 + 1°C. Encystment was monitored under phasecontrast microscopy. Encystment solutions. Routinely, 120 mM KC1 (Toama and Raper, 1967) was used to induce encystment and served as the control solution. Conditioned medium was obtained from 12 and 25 hr KC1 induced WS-320 cultures by pelleting the cells at 3000 g for 5 min. The osmotic pressure of 120 mM KC1 was determined to be 240 mosm using an osmometer (Model G-62, Fiske Assoc. Inc., Mass.). To study the effects of ammonia on encystment, solutions containing 2.3, 11.5, and 57 mM NH4Cl were made isoosmotic (i.e., 240 mosm) by the addition of KCl. Microcyst germination. Three-day-old microcysts of WS-320 that had been formed in 120 mM KC1 were suspended in 10 mM phosphate buffer at a concentration
microcysts
0
are represented
as
of lo7 cells/ml at 22 & 1°C. The cells were shaken at 200 rpm until germination was complete (O’Day, 1974). Measurement of extracellular ammonia. Extracellular medium was prepared by removing cultures and pelleting the cells at 1OOOgfor 5 min. The ammonia in the extracellular medium was measured using Nessler’s reagent (Sigma Chemical Co., St. Louis, MO.) and quantified from a standard curve prepared from dilutions of NH4Cl (BDH Chemicals). Since the pK of NHJ NH: is 9.26 and the pH of the liquid medium used throughout this study was 6.5, the ratio of NH::NH, was calculated to be 59O:l. Thus in the encystment medium, ammonia existed predominantly as ammonium ion (NH:). RESULTS
Encystment
Pro&les of WS-320 and Mutants
Amoebae of wild-type and the mutants mic-1 and mic2 of P. pallidurn can be induced to form microcysts in 120 m&f KC1 (Figs. lA-D). At 22 & 1 “C, wild-type amoebae began to encyst at about 10 hr (Fig. 2). Thereafter, encystment followed a sigmoidal profile until 93% of the cell population had encysted by 26 hr. The mutant strain mic-1 did not begin encystment until about 16 hr (Fig. 2). About 21% microcysts were produced by mic1 by 26 hr (Fig. 2) with a maximum of about 40% microcysts being formed by 40 hr (data not shown). The mutant strain mic-2 began to encyst at about the same time as the wild type but only about 64% microcysts
CHOI AND O’DAY
Microcysts
359
of P. pallidurn
tures, an increase in the extracellular level of ammonia was observed which resulted in a E-fold increase of ammonia to 2 pmole/107 cells/ml by 22 hr (Fig. 3). Levels of Ammonia Mutant Strains
in the Extracellular
Media of the
The levels of extracellular ammonia in the mutant strains mic-1, mic-2, PN582, and PN651 were measured and compared with those of WS-320. Among these, only mic-I produced levels of ammonia comparable to that of WS-320 (Fig. 4). Considerably lower levels of ammonia were accumulated in the extracellular medium of the mutant strains mic-2 (Fig. 4) and PN582 and PN651 (Fig. 5). When about 66% of microcysts were observed, a level of 1.0 pmole/107 cells/ml was detected in mic-2 cultures as compared to 1.8 pmole in WS-320 (Fig. 4). In 120 mM KCl. PN582 and PN651 did not form
2.0
3
1.6
3 i
0
6
12 HOURS
18
FIG. 3. Accumulation of extracellular ammonia in cultures of P. pallidurn WS-320 cultures. The extracellular media of vegetative (A), germinating (8) and encysting (A) cultures of WS-320 were collected and their ammonia content was determined by Nessler’s reagent as described under Materials and Methods. The vertical bars represent standard deviations.
: h
1.2
0
.-
2 y 0 5 I
.*
+< I
was obtained by 26 hr (Fig. 2). Under the same conditions, PN582 and PN651 did not form microcysts (Fig. 2). Some characteristics of these mutant strains are summarized in Table 1. A more detailed analysis of these mutants will be reported elsewhere. Levels of Extracellular Ammonia in Vegetative, Germinating Microcyst and Encysting Cultures Measurements of extracellular ammonia showed that a constant level of approximately 0.16 pmole/107 cells/ ml was present in the medium of both vegetatively growing cells and germinating microcysts as well as during early encystment (0 to 10 hr) cultures (Fig. 3). When microcysts began to appear in the encysting cul-
\
2 z
.4
0
40 PERCENT MICROCYSTS
80
FIG. 4. Accumulation of extracellular ammonia during encystment of in WS-320, mic-1 and mic-2. Cells of WS-320 (A), mic-1 (m), and mic-2 (v) were induced in 120 mM KCI. Extracellular medium was collected and ammonia concentrations were measured as detailed under Materials and Methods. Ammonia concentrations were plotted as a function of percentage microcysts.
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VOLUME 92, 1982
On the other hand, the mutant strain mic-2 (Fig. 8) began encystment earlier and ultimately produced more microcysts in the conditioned medium than in KCl. For example, at 14,18, and 22 hr; 29,76, and 88% microcysts were observed, respectively, in the 25-hr medium and 24, 64, and 86% were observed in the 12-hr medium while only 16, 55, and 66% were observed in 120 mM KCl. Surprisingly, the mutant strain PN582 which was unable to form any microcysts in KC1 could be induced to form some microcysts by the conditioned medium (Fig. 1E and Table 2). At 72 hr, about 12 and 20% microcysts were observed after shaking the cells of PN582 in 12- and 25-hr conditioned medium respectively.
I 0
20 HdUORS
FIG. 5. Accumulation of extracellular ammonia in the mutant strains PN582 and PN651. Cells of WS-320 (A), PN582 (0) and PN651 (+) were shaken in 120 mM KCl. The ammonia concentrations of the extracellular medium were measured as detailed under Materials and Methods. The vertical bars represent standard deviations.
microcysts and by 20 hr, contained only 0.25 pmole of ammonia/lo7 cells/ml (Fig. 5) which was close to the levels in nonencysting cultures. Effects of Conditioned
Medium
on Encystment
The effects of conditioned medium from 12- and 25hr encystment cultures of WS-320 on the timing of encystment of WS-320 cells are shown in Fig. 6. Cells contained in the 25-hr conditioned medium began encysting much earlier than those in the 12-hr conditioned medium which in turn started encysting earlier than those of the control (120 mMKC1). Cells of the mutant strain mic-1 encysted similarly in both the WS-320 conditioned medium and in 120 mM KC1 (Fig. 7) while PN651 remained cystless in all the solutions (data not shown).
I
10
1
I
I
16
I
I
I
22
HOURS FIG. 6. The effects of conditioned medium on the encystment of WS-320. Conditioned medium at (12 and 25 hr) was collected from encysting (120 mM KCl) WS-320 cultures. The effects of 12-hr (A.... . A) and 25.hr (A - - - A) conditioned medium on encystment of WS-320 were compared with the effects of 120 mM KC1 (A __ A). The vertical bars represent standard deviations.
361
CHOI AND O’DAY
containing NH4C1 and KC1 could induce microcyst formation in the cystless mutant PN582 (Table 3) with this strain producing 30 and 50% microcysts at 17 and 26 hr of encystment, respectively. This mutant eventually reached 91% microcysts by 72 hr in 57 mM NH&l (Table 3). The mutant strain mic-1 did not respond to the isoosmotic solutions in any manner different from the KC1 control (Fig. 11). Furthermore, microcyst differentiation was not induced by any isoosmotic solution in the cystless mutant PN651 (data not shown). DISCUSSION
Ammonia is a natural cellular component pears to play an important role as a regulator
ib”RS
that apof fruit-
I-
FIG. 7. The effects of 12-hr (m. * * . .m) and 25hr (W - - - W) conditioned medium on the encystment of the mutant mic-1 were comn ). The vertical bars reppared with those of 120 mM KC1 (m ~ resent standard deviations.
Effects oj’ Exogenous Ammonia
on Encystment
The effects of isoosmotic solutions containing KC1 and 2.3, 11.5, 57, and 115 mM NH4C1 on the five strains of P. pall&urn are summarized in Figs. 9-11 and Table 3. The response of these strains to the exogenously supplied ammonia was found to be similar to those seen in the experiments with the 12- and 25-hr conditioned medium. At concentrations of 2.3 and 11.5 mM, NHICl in the presence of KC1 had pronounced enhancing effects on the encystment of WS-320 (Fig. 9) and mic-2 (Fig. 10). At 22 hr, about 88% microcysts were observed in these isoosmotic solutions as compared to 60% in 120 mM KC1 (Fig. 10). Although 57 mM NH4C1 accelerated the encystment of WS-320 in the presence of KC1 (Fig. 9), it had an inhibitory effect on the encystment of mic1 (Fig. 10). It was also noted that isoosmotic solutions
A
I.
’
10
I
I
I
16 HOURS
I
I
I
22
FIG. 8. The effects of conditioned medium on the encystment of the mutant mic-2. The effects of 12-hr (v. * . . 0~) and 25-hr (V- - - - 7) conditioned medium and 120 mM KC1 (V ~ V) on the encystment of mic-2 were compared. The vertical bars represent standard deviations.
362
DEVELOPMENTAL BIOLOGY
VOLUME 92, 1982
TABLE 2 THE EFFECTS OF CONDITIONED MEDIUM ON MICROCYST FORMATION IN THE CYSTLESS MUTANT PN582 AND IN WS-320” Percentage 120 mM KC1
microcysts*
12-hr conditioned
medium
25-hr conditioned
medium
Time (hr)
WS-320
PN582
WS-320
PN582
WS-320
PN582
0 17 26 72
0 34.4 f 4.3 90.2 f 5.1 95.6 f 2.4
0 0 0 0
0 41.0 f 4.9 94.5 f 3.2 95.1 * 1.2
0 1.0 f 0.7 6.3 f 2.7 12.3 f 4.1
0 74.5 f 4.5 94.6 ? 3.6 94.8 2 2.3
0 7.3 f 3.0 8.6 * 2.8 20.0 f 2.3
’ Conditioned media were collected at 12 and 25 hr from encysting (120 mM KCl) WS-320 cultures. The amoebae of WS-320 and PN582 were grown in 10 g MSM and were shaken in the conditioned media and 120 mM KCI (control) as detailed under Materials and Methods. b Percentage microcysts are represented as means f SD.
ing body development in cellular slime molds (Feit, 1969; Schindler and Sussman, 1977a,b; Thadani et aE., 1977). Also, the work of Lonski (1976) and Paterno and O’Day (unpublished data) have revealed that ammonia can induce microcyst development in Polysphondylium pallidurn. However, no study has shown that ammonia is normally associated with the development of microcysts. As a result we carried out an in-depth analysis of this problem, the results of which suggest that ammonia acts as an inducer of microcyst differentiation. Measurements of extracellular levels of ammonia in cultures of vegetative amoebae and germinating microcysts of wild-type Polysphondylium pallidurn (WS320) reveal the presence of low ammonia levels. In solutions of KCl, amoebae at first show similar ammonia concentrations but a 12-fold increase occurs during encystment. This elevation may be crucial to encystment because the mutant strains (mic-2, PN582, and PN651) that lack wild-type levels of ammonia are also defective in microcyst formation. Further experiments suggest that the increase in ammonia may be responsible for inducing microcyst formation. Encystment in WS-320 and in mic-2 was initiated earlier when amoebae were placed in conditioned medium as compared to KCl. In addition, increased numbers of microcysts were induced in mic-2 cells treated with the conditioned medium. The mutant strain PN582 did not encyst in KC1 solutions but a significant number of microcysts developed when cells were placed in conditioned medium. It is probable that the encystment I 1111 I I 1 I inducing activity of the conditioned medium is in part 19 23 15 attributed to ammonia because isoosmotic solutions containing NH4C1 and KC1 produced similar results. It HOURS was found that NHICl in the presence of KC1 induced FIG. 9. The effects of isoosmotic solutions on the encystment of WSearlier encystment in WS-320 and mic-2. Furthermore, 320.2.3mM(A---A),ll.5mM(A--A)57mM(A~~~~~A),and mixtures of NH4Cl and KC1 were able to induce higher 115 mM (A -.A) NH,Cl and 120 mM KC1 (A A). The vertical percentage of microcyst in mic-2 than KC1 alone and bars represent standard deviations.
CH~I
AND O’DAY
iwicrocysts
363
qf P. pullidum
was not affected by exogenous ammonia or the ammonia in conditioned medium. Also, the mutant PN651 which produced low levels of ammonia could not be induced to encyst by either conditioned medium or ammonia. Since these mutants cannot be induced to encyst by ammonia, it appears that deficiencies other than those involved in ammonia metabolism are responsible for their encystment defects. The studies of Toama and Raper (1967) and the work presented in the present communication are the only in-depth investigations of factors that influence microcyst formation in P. pallidurn. The work of Toama and Raper (1967) suggested that encystment in P. pallidurn requires high osmotic pressure and showed that a number of exogenously added ions possesss the ability to
3c
I14
HOk
2: ,
FIG. 10. The effects of isoosmotic solutions of NH,Cl on the encystment of the mutant mic-2 were compared with those of 120 mM KCl.2.3mM(~---~),11.5mM(~---~),57mM(~~~~~~~),and 115 mM (V -*V) NHICl and 120 mM KC1 (V ~ v). The vertical bars represent standard deviations.
to induce in KCI.
encystment
in PN582 which
was cystless
It has been shown that factor(s) other than ammonia are also required for differentiation of Dictyostelium amoebae in clumps of submerged cells (Sternfeld and David, 1979). Our results also suggest that other factor(s) may be involved in the differentiation of microcysts. Two lines of evidence support this. First, both WS-320 and mic-2 entered encystment earlier in 12-hr conditioned medium than in KCl. Since the concentration of ammonia in 12-hr conditioned medium was only slightly higher than the basal level, the encystment enhancing activity would not be likely attributed to this low level of ammonia. Second, encystment in the mutant mic-1, which produced wild-type amounts of ammonia,
I
I
I
I
I
I
I
I
22
16 tZ"RS
FIG. 11. The effects of isoosmotic solutions of NH&l and KC1 on the encystment of the mutant mic-1. 2.3 mM (m - - - n ), 11.5 mM (m---m), 57 rnM(W*.* * em), and 115 mM (~-an ) NH,Cl, and 120 mM KC1 (B ~ u). The vertical bars represent standard deviations.
364
DEVELOPMENTAL BIOLOGY
TABLE 3 THE EFFECTS OF AMMONIUM CHLORIDE ON MICROCYST FORMATION IN THE CYSTLESS MUTANT PN582 AND IN WS-320 Percentage 120 mM KC1
microcysts” 57 mM NH4Clb
Time (hr)
WS-320
PN582
WS-320
PN582
0 17 26 72
0 34.4 f 4.3 90.2 f 5.1 95.6 ?z 2.4
0 0 0 0
0 62.1 + 5.1 92.1 + 4.4 93.1 f 3.7
0 30.0 f 5.8 50.4 f 3.9 90.9 f 3.8
a Percentage microcysts are expressed as means f SD. *The osmotic pressure of the 57 mM NH&l was adjusted to 240 mosm by the addition of KCI. See Materials and Methods for details.
induce encystment. Of the ions studied, K+ and Cl- were the most effective. The specific requirement for K+ was exemplified by the inability of NaCl to replace KC1 as an inducer of microcyst formation (Toama and Raper, 1967). In this study, we have shown that 115 mMNH,Cl was not as effectve as 120 mMKC1 in inducing microcyst formation but lower concentrations of NH4C1 (2.3, 11.5, and 57 m&I) in conjunction with KC1 were more effective than KC1 or NH&l alone. Since elevated concentrations of ammonia are produced during microcyst formation and since added ammonia in isoosmotic solutions containing NH&l and KC1 enhances encystment in wildtype cells and induces encystment in certain otherwise cystless mutants, we have uneqivocally shown that NH: can enhance microcyst formation. This study is supported in part by a grant (Grant A6807) from the Natural Sciences and Engineering Research Council of Canada.
REFERENCES BLASKOVICS, J. C., and RAPER, K. B. (1957). Encystment stages of Dictyostelium. Biol BuU 113, 58-88. DIMOND, R. L., BRENNER, M., and LOOMIS, W. F. (1973). Mutations affecting N-acetylglucosaminidase in Dictyostelium dticoideum. Proc. Nat. Acad Sci. USA 70, 3356-3360. ENNIS, H. I., PENNICA, D., and HILL, J. M. (1978). Synthesis of macromolecules during microcyst germination in the cellular slime mold Polysphondylium pallidurn Dev. Biol. 65, 251-259. ERDOS, G. W., and HOHL, H. R. (1980). Freeze-fracture examination of the plasma membrane of the cellular slime mould Polysphodylium pallidurn during microcyst formation and germination. Cytobios29,7-11. FEIT, I. N. (1969). “Evidence for the Regulation of Aggregation Den-
VOLUME 92, 1982
sity by the Production of Ammonia in the Cellular Slime Molds.” Ph. D. thesis, Princeton University. GWYNNE, D., and O’DAY, D. H. (1978). RNA and protein synthetic patterns during germination of Polysphondylium pallidum microcysts. Canad. J. Microbial. 24, 480-486. HOHL, H. R., MIURA-SANTO, L. Y., and COTTER, D. A. (1970). Ultrastructural changes during formation and germination of microcysts in Polysphondylium pallidurn, a cellular slime mould. J. Cell Sci 7, 285-305. LONSKI, J. (1976). The effect of ammonia on fruiting body size and microcyst formation in the cellular slime molds. Dev. Biol. 51,158165. O’DAY, D. H. (1973a). Intracellular and extracellular acetylglucosaminidase activity during microcyst formation in Polysphondylium pallidum. Exp. Cell Res. 79, 186-190. O’DAY, D. H. (1973b). cu-Mannosidase and microcyst differentiation in the cellular slime mould Polysphwndylium pallidum. J. Bacteriol 113, 192-197. O’DAY, D. H. (1974). Intracellular and extracellular enzyme patterns during microcyst germination in the cellular slime mold Polysphondylium pallidurn. Dev. Biol. 36, 400-410. O’DAY, D. H. (1976). Acid protease activities during germination of the cellular slime mold Polysphondylium pallidurn. .I Bacterial. 125, 8-13. O’DAY, D. H. (1977). Microcyst germination in the cellular slime mold Polysphwndylium pallidum: Requirements for macromolecular synthesis and specific enzyme accumulation. In “Eucaryotic Microbes as Model Developmental Systems” (D. H. O’Day and P. H. Horgen, eds.), Vol. 2, pp. 353-371. Dekker, New York. O’DAY, D. H., and FRANCIS, D. (1973). Patterns of alkaline phosphatase activity during alternative developmental pathway in the cellular slime mold, Polysphondyliumpallidum. Canau! J. Zool. 51,301310. O’DAY, D. H., GWYNNE, D. I., and BLAKEY, D. H. (1976). Microcyst germination in the cellular slime mold, Polysphundylium pallidum. Exp. Cell Res. 97, 359-365. O’DAY, D. H., AND PATERNO, G. D. (1979). Intracellular and extracellular CM-cellulase and fl-glucosidase activity during germination of Polysphondylium pallidum microcysts. Arch. Microbial. 121,231234. SCHINDLER, J., and SUSSMAN, M. (1977a). Effect of NH3 on c-AMP associated activities and extracellular c-AMP production in Dietyostelium discoideum. B&hem. Biophys. Res. Cmnmun. 79,611-617. SCHINDLER, J., and SUSSMAN, M. (19’77b). Ammonia determines the choice of morphogenetic pathways in Dictyostelium discoideum. J. Mol Biol. 116, 161-169. STERNFELD, J., and DAVID, C. N. (1979). Ammonia plus another factor are necessary for differentiation in submerged clumps of Dictyostelium. J. Cell Sci. 38, 181-191. SUSSMAN, M. (1963). Growth of the cellular slime mold Polysphurdylium pallidum in a simple nutrient medium. Science 139, 338. THADANI, V., PAN, P., and BONNER, J. T. (1977). Complementary effects of ammonia and CAMP on aggregation territory size in the cellular slime mold Dictyostelium mucoroides. Exp. Cell Res. 108, 75-78. TOAMA, M. A., and RAPER, K. B. (1967). Microcysts of the cellular slime mold Polysphondylium pallidum. I. Factors influencing microcyst formation. J. BacterioL 94, 1143-1149.