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J. Cell Set. 64, 137-146 (1983) 137 Printed in Great Britain © The Company of Biologists Limited 1983 INFECTION OF MACRONUCLEAR ANLAGEN OF PARAMECIUM CAUDATUM WITH THE MACRONUCLEUS-SPECIFIC SYMBIONT HOLOSPORA OBTUSA MASAHIRO FUJISHIMA Biological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753, Japan AND HANS-DIETER GORTZ Zoologisches Institut der Universitdt Munster, D-4400 Munster, Federal Republic of Germany SUMMARY The gram-negative bacterium Holospora obtusa is an endonuclear symbiont of Paramecium caudatum, which is incorporated into the host cells via the food vacuoles and infects their macronucleus exclusively, but never the micronucleus. Since these two kinds of nuclei originate from a fertilization nucleus, it is assumed that the macronucleus acquires a property necessary for it to be recognized by the bacterium at a certain time during the nuclear differentiation process. We found that this property is acquired by four of the eight postzygotic nuclei as soon as the four nuclei differentiate morphologically into the macronuclear anlagen. INTRODUCTION Ciliated protozoa contain two kinds of nuclei, which differ in ploidy, morphology, function and genetic content. The polyploid macronucleus has a high transcriptional activity (Gorovsky & Woodard, 1969; Ron & Urieli, 1977), whereas the diploid micronucleus has condensed, inactive chromatin compared to that of the macronucleus (Rao & Prescott, 1967; Pasternak, 1967) and functions as germ nucleus. Some micronuclear DNA sequences are not present in the macronucleus (Ammermann, Steinbriick, Berger & Henning, 1974; Lauth, Spear, Heumann & Prescott, 1976; McTavish & Sommerville, 1980; Yao, 1982). Despite the apparently different nature of these nuclei, however, both originate from a common fertilization nucleus. How the macro- and the micronucleus, of common genetic origin, differen- tiate into two morphologically and functionally different nuclei within a cell is un- resolved. In Paramecium caudatum, four species of bacterial endonuclear symbionts are known (Hafkine, 1890; Gortz, 1980): Holospora elegans, H. undulata, H. obtusa and ms-2. The former two inhabit the micronucleus exclusively, but never the macronucleus or cytoplasm. On the other hand, the latter two inhabit the macronucleus only. The Holospora species can easily infect the specific nucleus via the food vacuoles when a homogenate of symbiont-bearing cells is added to symbiont- free cells (Ossipov & Ivakhnyuk, 1972; Ossipov, Skoblo & Rautian, 1975; Gortz &

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J. Cell Set. 64, 137-146 (1983) 137Printed in Great Britain © The Company of Biologists Limited 1983

INFECTION OF MACRONUCLEAR ANLAGEN OFPARAMECIUM CAUDATUM WITH THEMACRONUCLEUS-SPECIFIC SYMBIONT HOLOSPORA

OBTUSA

MASAHIRO FUJISHIMABiological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753, Japan

AND HANS-DIETER GORTZZoologisches Institut der Universitdt Munster, D-4400 Munster, Federal Republic ofGermany

SUMMARY

The gram-negative bacterium Holospora obtusa is an endonuclear symbiont of Parameciumcaudatum, which is incorporated into the host cells via the food vacuoles and infects theirmacronucleus exclusively, but never the micronucleus. Since these two kinds of nuclei originatefrom a fertilization nucleus, it is assumed that the macronucleus acquires a property necessary forit to be recognized by the bacterium at a certain time during the nuclear differentiation process. Wefound that this property is acquired by four of the eight postzygotic nuclei as soon as the four nucleidifferentiate morphologically into the macronuclear anlagen.

INTRODUCTION

Ciliated protozoa contain two kinds of nuclei, which differ in ploidy, morphology,function and genetic content. The polyploid macronucleus has a high transcriptionalactivity (Gorovsky & Woodard, 1969; Ron & Urieli, 1977), whereas the diploidmicronucleus has condensed, inactive chromatin compared to that of themacronucleus (Rao & Prescott, 1967; Pasternak, 1967) and functions as germnucleus. Some micronuclear DNA sequences are not present in the macronucleus(Ammermann, Steinbriick, Berger & Henning, 1974; Lauth, Spear, Heumann &Prescott, 1976; McTavish & Sommerville, 1980; Yao, 1982). Despite the apparentlydifferent nature of these nuclei, however, both originate from a common fertilizationnucleus. How the macro- and the micronucleus, of common genetic origin, differen-tiate into two morphologically and functionally different nuclei within a cell is un-resolved.

In Paramecium caudatum, four species of bacterial endonuclear symbionts areknown (Hafkine, 1890; Gortz, 1980): Holospora elegans, H. undulata, H. obtusa andms-2. The former two inhabit the micronucleus exclusively, but never themacronucleus or cytoplasm. On the other hand, the latter two inhabit themacronucleus only. The Holospora species can easily infect the specific nucleus viathe food vacuoles when a homogenate of symbiont-bearing cells is added to symbiont-free cells (Ossipov & Ivakhnyuk, 1972; Ossipov, Skoblo & Rautian, 1975; Gortz &

138 M. Fujishima and H.-D. Gortz

Dieckmann, 1980; Gortz, 1980). Although the cause of the nuclear specificity of thesebacteria has not been clarified, it is at least clear that they possess the ability to infecta specific nucleus, either the macro- or the micronucleus. This ability of the symbiontsprovides an opportunity to examine the stage at which the postzygotic nuclei differen-tiate into macro- and micronuclei, with respect to the acquisition of a property necess-ary for them to be recognized and infected by the symbionts.

We demonstrate here, by using the macronucleus-specific symbiont//. obtusa, thatthe macronuclear anlagen acquire the property needed by the macronucleus for aninfection with H. obtusa as soon as the postzygotic nuclei differentiate morphologi-cally into the macronuclear anlagen. The result suggests that this property is acquiredwhen macronuclear differentiation begins.

MATERIALS AND METHODS

Strains and culture conditionsThe cells used in this study were Paramecium caudatitm syngen 3, mating type V, strain 27aG3,

and mating type VI, strain 27aG3BC8-l. These strains were kindly supplied by Dr Y. Tsukii, HoseiUniversity. The original Holospora obtusa-bearing strain C101 (syngen unknown) was collected inMunster, FRG. Later, the symbionts infected strain 27aG3 and this newly infected strain was usedfor obtaining the symbionts in this study. The culture medium used was 1 -25 % (w/v) fresh lettucejuice in Dryl's solution (Dryl, 1959) inoculated with a non-pathogenic strain of Klebsiellapneumoniae 1 day before use (Hiwatashi, 1968). In ordinary cultures several hundred cells wereinoculated into 2 ml culture medium and then 4 ml, 10 ml and 10 ml of fresh medium were addedon successive days. Cultures were kept at 25 °C. One or two days after the final feeding, matingreactivity increased to its maximum intensity.

InfectionHolospora species show two morphologically distinct forms in their life cycle (Ossipov &

Ivakhnyuk, 1972; Ossipov et al. 1975; Gortz & Dieckmann, 1980): a reproductive short form andan infectious long form. The former is observed predominantly in nuclei of vegetatively growinghost cells and the latter in that of starved cells. Only the long form is infectious. Infection ofmacronuclei or macronuclear anlagen of P. caudatum with H. obtusa was achieved as follows.Cultures of symbiont-bearing cells in stationary phase were strained through four layers of fine gauzeto remove gross debris. Then the cells were harvested by centrifuging for 3 min at 1000 rev./min,and homogenized by hand in a Teflon homogenizer at 0—4CC. The density of the symbionts in thehomogenate was counted with a blood-counting chamber and adjusted to 4 X lO'symbionts/ml byadding Dryl's solution. Recipient cells and the homogenate containing//, obtusa were mixed indepression slides at 1500 cells/ml and 4 X lO^ymbionts/ml, respectively, at 25 °C.

Cytological methods for observation of nuclei and symbiontsCells infected with//, obtusa were harvested by centrifugation in a hand-operated centrifuge, air-

dried on glass slides, and fixed in a mixture of acetic acid and ethanol (1:3, v/v) for 10 min at roomtemperature. Preparations were stained by the Feulgen reaction and counterstained with 0-25 %(w/v) fast green FCF. Observations were made using a differential interference-contrastmicroscope at a magnification of X1000. Temporal observation of the symbionts in host macronucleiwas made as follows. Cells were pipetted with a few microlitres of culture medium onto glass slidesand fixed in 4 % (w/v) OsO« vapour by inverting the slide over a small vial of the fixative for 3-4 s.The cells were then observed either unstained, or stained with aceto-orcein.

Observation of food vacuolesCell suspensions were mixed with a drop of Indian ink solution for. 15 min at a density of about

Macronuclear differentiation in Paramecium 139

1500 cells/ml at 25 °C. Then the cells were fixed and stained by the Feulgen reaction and with fastgreen FCF as described before. The cells able to form food vacuoles are expected to ingest Indianink and form many black-coloured food vacuoles.

RESULTS

Time-course of infection vnth H. obtusa

In order to determine the time needed for infection of the macronucleus of P.caudatum with H. obtusa, symbiont-free cells were mixed with the homogenate ofsymbiont-bearing cells at 25°C (see Materials and Methods). Then the cells werefixed every 10 min for the first 2 h after mixing, and stained as described. The resultsare shown in Fig. 1. Ten minutes after mixing the cells with the symbiont-containinghomogenate, the macronucleus was infected with//, obtusa in about 20 % of the cells.The symbionts were also observed in the cytoplasm, some were in food vacuoles andsome apparently outside the food vacuoles. The proportion of cells bearing symbiontsin their macronuclei rose to about 60 % at 30 min after mixing and to about 100 % at60 min. Mean numbers of the symbionts observed in the individual macronuclei at 10,30 and 60 min after mixing were about 1, 2 and 3, respectively. The symbionts werenever observed in the micronucleus. The data show that the infection of themacronucleus with H. obtusa begins remarkably quickly after mixing. Since thesymbionts can be observed not only near the macronucleus but also near the

5.8

cell

5a?

100

90

80

70

60

50

40

30

20

10

010 20 30 40 50 60

Time after mixing with H. obtusa (min)

10

120

Fig. 1. Time-course of infection of the macronucleus of P. caudatum with Holosporaobtusa. H. obtusa-iTee cells were mixed with an homogenate of the symbiont-bearing cells(see Materials and Methods). At each time-point, a hundred cells were observed, and theratios of cells that had symbionts in the macronucleus ( • • ) and the mean numbersof the symbionta per macronucleus (O O) were plotted. C.L. is confidence limit.

u

I

"56cc<0

140 M. Fujishima and H.-D. Gortz

micronucleus, it is evident that the symbionts can infect the macronucleus but not themicronucleus. Similar observations were reported by Ossipov and colleagues (seeOssipov, 1981, for a review).

Infection of macronuclear anlagen of exconjugant cells with H. obtusa

As shown in previous studies (Calkins & Cull, 1907; Klitke, 1916; Saito & Sato,1961; Mikami & Hiwatashi, 1975; Mikami, 1980), in P. caudatum the synkaryon(fertilization nucleus) normally divides three times successively; four of the resultantnuclei become macronuclear anlagen, one becomes a micronucleus, and the remain-ing three degenerate. The old macronucleus is transformed into a loosely wound skein(skein formation) at about the time of the third (last) postzygotic division andsubsequently breaks into many fragments (macronuclear fragmentation). Recently,it has been found by Mikami (1980) that the determination of the macro- and themicronuclear differentiation occurs immediately after the third nuclear division andis closely related to a brief localization of the nuclei at the opposite ends of the cell,with the prospective macronuclei in the posterior region and the prospectivemicronuclei in the anterior region. Similar results were also reported in P. tetraurelia(formerly called syngen 4 of P. aurelia; Sonneborn, 1975) (Grandchamp & Beisson,1981). Hereafter, this stage will be tentatively called the 'determination stage fornuclear differentiation'. The earliest morphological change of the nuclei after thedetermination stage is the appearance of heterochromatic aggregates in the fourmacronuclear anlagen, which is followed by increased stainability with fast green.The heterochromatic aggregates then disintegrate and disappear, accompanied by anincrease in the volume of the anlagen (Saito & Sato, 1961).

The high degree of macronuclear specificity of H. obtusa and its rapid infectivityto the macronucleus make it possible to use the symbiont to ask the following ques-tion: when do the division products of the synkaryon differentiate into macronucleiwith respect to the infection with//, obtusa? To examine this problem, 15, 24 and 30 hafter the beginning of the mating reaction at 25 °C, cells were mixed with thehomogenate of H. obtusa-bearing cells at densities of about 1500 cells/ml and4 x 104symbionts/ml, respectively. Conjugating pairs usually separate about 15 hafter the beginning of the mating reaction at 25 °C. The degree of synchrony of thenuclear changes during the conjugation process is relatively high, as the cells enterinto the process almost simultaneously during the mating reaction, but it is notabsolute. Therefore, in cells harvested at 15, 24 and 30 h after the beginning of themating reaction, a series of stages from late conjugation to early postconjugation canbe observed (Saito & Sato, 1961; Mikami & Hiwatashi, 1975), i.e. the third pregamicdivision, pronuclear exchange between the two mates, synkaryon, three postzygoticdivisions, determination stage for nuclear differentiation, eight postzygotic nucleiafter the determination stage (post-determination stage) and development ofmacronuclear anlagen. Two hours after mixing at 25 °C, the cells were fixed andstained as described before.

Uptake of the symbionts into the cells was observed when all eight postzygoticnuclei had migrated from the opposite ends of the cell into a common region, after the

Macronuclear differentiation in Paramecium 141

2A

B

Fig. 2. Photomicrographs of exconjugants that were mixed at 24 h (A) and 30 h (B) , respec-tively, after the beginning of the mating reaction with the homogenate of//, obtusa-bearingcells for 2h, then fixed and stained (see text). Arrowheads indicate four macronuclearanlagen among eight postzygotic nuclei. The anlagen in A are at the beginning of the firstvisible differentiation of the macronuclear anlagen. At this stage heterochromaticaggregates appear in the anlagen. Since the anlagen stain well with fast green, these anlagenare easily distinguished from the other postzygotic nuclei and the fragments of an oldmacronucleus. It is evident that each of four anlagen is infected with H. obtusa.

In B, the macronuclear anlagen have increased in volume and only a few heterochromat-ic aggregates remain at the core of the anlagen. Although four macronuclear anlagen areinfected with one or two//, obtusa, the symbionts in two anlagen (second and fourth onesfrom the left of the photograph) are out of focus.

In A and B four macronuclear anlagen can easily be found, but the other four postzygoticnuclei cannot be distinguished from the many fragments of an old macronucleus that arepresent. X1300.

142 M. Fujishima and H.-D. Gb'rtz

determination stage for nuclear differentiation (post-determination stage), which isthe stage just before the first visible differentiation of the macronuclear anlagen. Someof the symbionts were observed in food vacuoles and some were apparently outsidethe food vacuoles, in the host cytoplasm. However, symbionts were not observed inparamecia at earlier stages, i.e. the stages of synkaryon, two or four postzygotic nucleiand the determination stage for nuclear differentiation. After the appearance of thefirst symbionts within the cells, it was confirmed that none of the postzygotic nucleiimmediately before their first visible differentiation were infected with the symbionts.Then, almost simultaneously with the first visible differentiation of the macronuclearanlagen all of them were infected with the symbionts (Fig. 2A, B). Some of the oldmacronuclear fragments were also infected, but the new micronucleus, which derivedfrom the synkaryon, was not infected. Notwithstanding that the diameter of the firststage of the macronuclear anlagen was shorter than the length of H. obtusa, thesymbionts could infect each of the four anlagen. Because of their length, the sym-bionts appeared to protrude out of the anlagen (Fig. 2A) . It should be emphasized thatthe postzygotic nuclei immediately before the first visible differentiation of themacronuclear anlagen were not infected with the symbionts, despite the presence ofsymbionts in the cytoplasm.

These results indicate that the macronuclear anlagen acquire the property of themacronucleus that is needed for recognition by and infection with//, obtusa at almostthe same time as the first recognizable change in the macronuclear anlagen.Therefore, this strongly suggests that this property is acquired in close associationwith the initiation of structural or functional differentiation in the macronucleus.

Capacity for food vacuole formation in exconjugant cells

Uptake of the symbionts was not observed in early exconjugant cells, i.e. at thestages of synkaryon, two or four postzygotic nuclei and determination of nucleardifferentiation. In order to examine why the uptake of the symbionts did not occurin these cells, their capacity for food vacuole formation was examined. The cells at IS,24 and 30 h after mixing of complementary mating types at 25 °C were suspended inIndian ink solution for 15 min at a density of about 1500 cells/ml at 25 °C, then fixedand stained (see Materials and Methods). Fifteen minutes of incubation with Indianink solution is sufficient for ingestion of ink and formation of black food vacuoles (seealso Takagi et al. 1981). Therefore, the capacity for food vacuole formation at dif-ferent stages in exconjugant cells can be determined. As mentioned before, by har-vesting the cells at three different times (15, 24 and 30 h) after the mixing of com-plementary mating types, cells at various stages from the third prezygotic division tothe development of macronuclear anlagen were obtained. Furthermore, since theincubation time is only 15 min, the nuclear stages of the cells do not change muchduring the incubation. Therefore, the ability to form food vacuoles at each of thestages from the late conjugation process to the early post-conjugation process can bedetermined. As a control experiment, cells in stationary phase were incubated inIndian ink solution in the same way. The results are summarized in Table 1. At thesynkaryon stage, the cells are still paired at the paroral regions. The pairs usually

Macronuclear differentiation in Paramecium 143

Table 1. Comparison of food vacuole formation in the late conjugants and early post-conjugants of ¥. caudatum*

Nuclear stage of the cells

Vegetative GifSynkaryonTwo postzygotic nucleiFour postzygotic nucleiEight postzygotic nuclei

Determination stagePost-determination stage

Four macronuclear anlagen

-n-2§-31

Ability of food

rPresence (A)

181000

036

2618723

vacuole formationA

Absence (B)

01752

378

7214

000

A/A+B (xlOO)(%)

100000

072

100100100

• Fifteen, 24 and 30 h after mixing of complementary mating types, the cells were incubated inIndian ink solution for ISmin at about 1500cells/ml, at 25°C (see text).

f As a control, cells of 27aG3 (mating type V) in stationary phase were used. Cells in this phasehave a G\ macro- and a G\ micronucleus (Fujishima, 1983).

X Heterochromatic aggregates appear in these macronuclear anlagen.§ Disintegration of the heterochromatic aggregates occurs in these anlagen.1j The heterochromatic aggregates disappear, accompanied by an increase in volume of the an-

lagen. These stages of the macronuclear anlagen were classified after Saito & Sato (1961).

separate at the stage of two postzygotic nuclei. However, as shown in the table, theexconjugant cells could not form food vacuoles up to the determination stage fornuclear differentiation. Initiation of food vacuole formation was observed after thedetermination stage. These results agree well with the finding that the first symbiontappeared in exconjugant cells at the post-determination stage, suggesting that theinability of the symbionts to infect early exconjugant stages is due to the inability ofthe cells to form food vacuoles.

DISCUSSION

In Paramecium to date, bveHolospora species are known as endonuclear symbionts(Hafkine, 1890; Preer, 1969; Ossipov, Borchsenius & Podlipaev, 1980; Preer &Preer, 1982): H. obtusa, H. elegans and H. undulata in P. caudatum, H. caryophila(formerly called alpha; Preer, 1969) in P. biaurelia (formerly called syngen 2 of P.aurelia; Sonneborn, 1975), and//, acuminata in P. bursaria. All of these symbiontsshow a nuclear specificity in their infectivity. In the present study, the uptake of//.obtusa into the cell started at the stage when the cell could form food vacuoles (Table1). The result strongly suggests that the uptake is mainly by way of the food vacuoles,which agrees with other cytological observations (see Gortz, 1983, for a review).

An electron-microscopic observation of the infection process of P. caudatum with

144 M. Fujishima and H.-D. Gortz

H. obtusa has been reported by Ossipov & Podlipaev (1977). However, many detailsof this process (i.e. protection against the digestive enzymes in the food vacuoles,migration from the food vacuoles to the macronucleus and nuclear specificity of theinfection) are not yet understood. It has been reported thatHolospora species infect thehost nucleus within 1-2 h of the addition of the symbionts to the external medium ofsymbiont-free cells (Preer, 1969; Ossipov & Podlipaev, 1977; Gortz & Dieckmann,1980). However, we found that the actual time needed for the appearance of the firstsymbiont in the macronucleus is less than 10 min (Fig. 1). This rapid infectivity of//.obtusa indicates that it can escape from food vacuoles even earlier. This may protect thesymbionts from the digestive enzymes in the food vacuoles, because lysosomal fusionto the newly formed food vacuoles begins 8 min after vacuole formation (Fok, Lee &Allen, 1982). As mentioned before, all Holospora species exhibit nuclear specificity intheir habitat. Although the mechanism by which the nuclear specificity is controlledremains unknown, it can be assumed that these symbionts are able to distinguish be-tween the macro- and the micronucleus in some way. Even when the macronucleus iswell-infected by//, obtusa, the symbionts are never observed inside the micronucleus,despite the presence of symbionts nearby. Since, as far as we have observed, H. obtusais unable to pass through the nuclear membrane of the micronucleus, it is suggestedthat the symbiont may have the ability to distinguish between the nuclear membranesof the two kinds of nuclei, or something associated with them.

Besides the differences in morphology, DNA content and transcriptional activity,several differences have been found between the macro- and micronuclei of ciliates:ribosomal RNA gene amplification (Gall, 1974; Yao, Blackburn & Gall, 1979; Yao,1981) and lack of some micronuclear DNA sequences in the macronucleus (Ammer-mann et at. 1974; Lauth et al. 1976; McTavish & Sommerville, 1980; Yao, 1982),and the presence of a micronuclear-specific histone (Allis, Glover & Gorovsky, 1979).However, the stage at which these differences develop during the differentiationprocesses of the two nuclei is not known. In the present study, we determined thestage at which the nuclei acquired the quality needed for infection with H. obtusa; //.obtusa could infect the macronuclear anlagen as soon as the anlagen become morpho-logically distinguishable from other postzygotic nuclei. The symbionts could notinfect any of the postzygotic nuclei before the occurrence of the first morphologicalchange, or the new micronucleus. The results clearly indicate that the propertyneeded for infection with H. obtusa is acquired at the first stage in the visible dif-ferentiation of macronuclear anlagen. As far as we know, the appearance of thisproperty is the earliest of the known characteristics of the macronucleus. Thisproperty is assumed to be intimately correlated with a fundamental characteristic ofthe macronucleus, because it is present from the beginning of macronuclear dif-ferentiation to macronuclear degradation (macronuclear fragmentation).

By using micronuclear-specific symbionts, H. elegans or H. undulata, it should bepossible to discover when one of the postzygotic nuclei differentiates into themicronucleus, as recognizable by the symbionts. These methods will provide us witha new approach to analysing the timing and the mechanism of macro- andmicronuclear differentiation in genetically identical nuclei.

Macronuclear differentiation in Paramecium 145

The earlier part of this study was supported by a fellowship from the Alexander von Humboldt-Foundation to M. F., and was carried out while the author was a guest in the laboratory of Dr KlausHeckmann, Zoologisches Institut der Universitat, Miinster, FRG.

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{Received 14 February 1983-Accepted 25 May 1983)