Protoplasma (2000) 213:176-183 PROTOPLASMA �9 Springer-Verlag 2000 Printed in Austria

Paramecium calkinsi and P. putrinum (Ciliophora, Protista) harboring alpha-subgroup bacteria in the cytoplasm

S. I. Fokin ~'*, E. V. Sabaneyeva 1, Oo N. Borkhsenious 2, M. Schweikert 3, and H.-D. G6rtz 3

Biological Institute, St. Petersburg State University, St. Petersburg, 2 Department of Biological Science, Louisiana State University, Baton Rouge, Louisiana, and 3 Biological Institute, University of Stuttgart, Stuttgart

Received December 20, 1999 Accepted May 8, 2000

Summary. New intracellular bacteria were detected in the cyto- plasm of Paramecium calkinsi and P. putrinurn. Some of the bacte- ria were not evenly distributed in the cytoplasm of the host but were found in the center of the cell, eventually near the nuclei, but not in the cortex area, whereas another species was found in the cortex area. These peculiarities of intracellular bacteria localization in the host suggest that the conditions in various parts of the cytoplasm favor bacterial maintenance to different extent. Due to the results obtained by transmission electron microscopy and in situ hybridiza- tion using appropriate oligonucleotide probes, the bacteria, three or possibly four species, are Gram-negative and belong to the alpha- subgroup of proteobacteria. Bacteria from one stock of P calkinsi were found to be infectious for bacteria-free cells of P. ealkinsi and P.. nephridiaturn.

Keywords: Cytoplasm; Infection; Fluorescence in situ hybridization; Intracellular bacteria; Paramecium calkinsi; Paramecium putrinurn.

Introduction

In t race l lu lar bacter ia have been observed in the cyto-

plasm, nuclei , and pe r inuc lea r space of several Para-

m e c i u m species (for reviews, see Preer et al. 1974,

Preer and Preer 1984, H e c k m a n n and G6r tz 1991,

Fokin and Karpov 1995). While some P a r a m e c i u m

species, e.g., the species of the R aurel ia complex and

P. cauda tum, were f requent ly descr ibed to ha rbor bac-

teria, observa t ions of in t racel lu lar bacter ia f rom other

paramecia are comparab ly rare. More recently, in

some pa ramec ia f rom brackish water cytoplasmic

bacter ia have b e e n found (Fokin 1989b, Fok in and

Sabaneyeva 1993). This a l ready indicated that a search

* Correspondence and reprints: Biological Institute, St. Petersburg State University, Oranienbaumskoye Shosse 2, Old Peterhof, St. Petersburg 198904, Russia. E-mail: fokin@peterlink.ru

for in t racel lu lar bacter ia in pa ramec ia o ther than P

aurelia and P. c a u d a t u m could be successful. In the fol-

lowing, isolated strains of P. calkinsi and P. p u t r i n u m

were found to ha rbor bacter ia in their cytoplasm. The

different features of the bacter ia concern ing their

infectivity and the site of their res idence in the host

cytoplasm are regarded as a fur ther indica t ion for a

great diversity of in t racel lu lar bacter ia in ciliates. Most

of the e ndonuc l e a r bacter ia extensively s tudied in

paramecia have b e e n p roved to be long to the alpha-

subgroup of p ro teobac te r ia (Fokin et al. 1996). The

residents of the cytoplasm have not b e e n s tudied in

this respect as yet. Thus, the a im of the p resen t s tudy

was to shed some light u p o n their diversity and

affiliation.

Material and methods

The following paramecium strains, bearing cytoplasmic bacteria, were used in the study: P. calkinsi strains OK-6-2, isolated at the coastline of Kolguev island, Barents Sea, Russia (1989), and GN5-3, isolated at the North Sea coastline, Carolinensiel, Federal Republic of Germany (1996), and R putrinurn strain UG-1, isolated from a pond in the city of Ulm, Federal Republic of Germany (1996), were used, all of them bearing cytoplasmic bacteria. Several uninfected laboratory stocks of P. calkinsi, P. nephridiaturn, P. duboscqui, and P. putrinurn of different clonal age and geographical origin were used for infection experiments.

Cells were cultivated at 20 ~ on lettuce medium inoculated with Enterobacter aerogenes. Living cells were immobilized for observa- tion with the help of a compression device (Skovorodkin 1990). For the detection of intracellular bacteria the material was fixed by Bouin's fluid and stained by the Feulgen procedure or by lacto- aceto-orcein after ethanol-acetic acid fixation (G6rtz and Dieck- mann 1987). Living and fixed cells were examined by phase-contrast or Nomarski interference-contrast (DIC) microscopy with a Zeiss

S. I. Fokin et al.: Bacteria in paramecium cytoplasm 177

Axioskop microscope. For electron microscopy cells were processed as described elsewhere (Fokin 1989a). Infectivity and killing capa- bilities were determined by feeding uninfected cells with homo- genates of bacteria-bearing cells prepared as described by Preer (1969).

For in situ hybridization, cells were fixed with 4% formaldehyde (w/v, freshly prepared from paraformaldehyde) in phosphate- buffered saline solution (PBS), pH 7.2, for 2 h and washed with phosphate-buffered saline. Cells were incubated with oligo- nucleotide probes in hybridization buffer (Fokin et al. 1996). A eubacteria-specific probe, 5'-GCTGCCTCCCGTAGGAGT-3', labeled with fluorescein isothiocyanate, and a probe specific to the alpha-subgroup of proteobacteria, 5 ' - G CGTTCGCTCTGAGCCAG-5 ', labeled with tetramethylrhodamine isothiocyanate (Amann et al. 1990, 1991) were used.

Specimens were investigated with a Zeiss LSM 410 confocal laser scanning microscope equipped with a plan neofluar xl00 oil immer- sion objective. For detection of fluorescein and tetramethylrho- damine isotl]iocyanate, an argon-ion laser (488 nm wavelength) and a helium-neon laser (543 nm wavelength) with appropriate emission filters (BP, 510-525 nm and 575-640 nm wavelength, respectively)

were used. Color pictures were taken by Polaroid recorder on Zeiss LSM 410.

Results

Bacteria in the cytoplasm of P. calkinsi

In an earl ier s tudy of the cells of P. calkinsi stock OK-

6-2, the presence of bacter ia in the protist cytoplasm

was briefly descr ibed (Fokin and Sabaneyeva 1993).

Bacter ia were visible in squashed p repara t ions of

living cells; in intact cells, bac ter ia could be seen by

D I C and phase-cont ras t microscopy or after Feulgen-

staining. The p resen t inves t iga t ion revealed two mor-

phological ly different forms of bacteria. D u e to their

cell wall s t ructure bo th appeared to be Gram-nega t ive .

O n e form of bac ter ia was rodlike, 1.5-2 g m long

and 0.3-0.4 g m wide. Each bac t e r ium was found en-

closed in an indiv idual host vacuole (Fig. 1). Bacter ia

Figs. 1-3. Bacteria in the cytoplasm of P. calkinsi stock OK-6-2 encircled by host vacuole. Electron micrographs. Arrows indicate host vac- uoles; cisternae of endoplasmic reticulum with and without ribosome are indicated by open (Fig. 3) and solid (Fig. 2) arrowhead, respec- tively. B Bacterium. Bars: 0.5 gm

178 S. 1. Fokin et ai.: Bacteria in paramecium cytoplasm

Figs. 4-7. Bacteria in the cytoplasm of P. calkinsi stock OK-6-2 without host vacuole. Electron micrographs. Arrows in Figs. 4 and 6 indi- cate pili-like structures on the surfsce of bacteria; open arrowheads indicate ER cisternae with ribosomes. B Bacterium; K kinetosome; M mitochondria. Bars: 0.5 gm

were always located within the inner cytoplasmic (endoplasmic) area; sometimes cisternae of endoplas-

mic reticulum (ER) with or without r ibosomes are situated close to some of the bacteria (Figs. 2 and 3).

Sometimes, the cisternae formed additional double- membrane vacuoles around the bacteria. In the latter case r ibosomes were predominant ly located on the

inner surface of the secondary vacuole (Figs. 4-6). Bacteria encircled by such double-membrane vacuoles had no individual vacuoles. Possibly, the membranes of individual vacuoles disappeared when the endobionts

were encircled by cisternae of ER. Some bacteria located within these double-membrane vacuoles showed small pili-like structures on their surfaces

(Fig. 4).

The second form of bacteria was found in the cortex

(ectoplasmic) area of the cells. These bacteria were

quite similar to the form described above in their shape and size, but they never showed any pili-like

structures and were not surrounded by host mem- branes. The bacteria of this type were only found close to the cell surface below the kinetosome layer (Fig. 7).

In P. calkinsi stock GN5-3 (Figs. 8-10 and 14)

numerous bacteria were found to be located in dif- ferent parts of the cytoplasm (Fig. 8), some of them very close to the nuclei (Fig. 9), but never in the cortex (Fig. 14). The bacteria were detected in squash preparat ions and in intact cells after Feulgen-staining. The rod-shaped bacteria measured 1.0-1.5 by 0.25- 0.30 gm. The two-membrane cell wall structure corre-

S. I. Fokin et al.: Bacteria in paramecium cytoplasm 179

Figs. 8-10. Bacteria in the cytoplasm of P. calkinsi stock GN5-3. Electron micrographs. B Bacterium; MI micronucleus; C cilium. Bar: Figs. 8 and 9, 0.5 gm; Fig. 10, 0.3 gm

Figs. U and 12. Bacteria in the cytoplasm of P putrinum. Electron micrographs. M Mitochondrion, MA macronucleus. Bar: 0.5 gm

180 S.I. Fokin et at.: Bacteria in paramecium cytoplasm

sponded to that of Gram-negative bacteria (Fig. 10). No host membrane surrounding the bacteria was observed; instead, some empty space separated the bacteria from the host cytoplasmic structures (Figs. 8-10). Several bacteria were repeatedly found closely packed to each other (Fig. 10).

Incubation of bacteria-free cells of other stocks of P. calkinsi, P. nephridiatum, and P. duboscqui with the homogenate of infected cells did not reveal any killer effect of the latter (data not shown). However, the homogenate proved to be infectious for one stock of P. calkinsi and one stock of P. nephridiatum in two out of four experiments carried out for each species.

Both, originally and experimentally infected stocks maintained stable infection for almost three and two years, respectively. Infection was stable under differ- ent temperature conditions (4-25 ~ However, P. calkinsi is known to have a temperature limitation at about 20 ~ uninfected cell cultures being usually depressed above this temperature.

Bacteria in the cytoplasm of P. putrinum

By means of light microscopy, living cells of the strain UG-1 of P. putrinum were found to harbor bacteria in the cytoplasm. This observation was confirmed by Feulgen and lacto-aceto-orcein staining of fixed cells (data not shown). Electron microscopy revealed that the bacteria were of coccoid shape and measured about 0.5-1.0 by 0.25-0.35 gm. They were not encir- cled by host membranes (Figs. 11 and 12). Most of these bacteria were found in the central part of the host cell (Figs. 12, 15, and 16), but occasionally they could be located close to the pellicle as well (Fig. 11). The electron microscopic aspect of the cell wall mem- branes corresponded to that of Gram-negative bacte- ria (Figs. 11 and 12).

No killer effect of the bacteria was revealed by adding a homogenate of infected cells to bacteria-free P. putrinum. The bacteria did not confer infection to

three other P. putrinum stocks in two experiments for each stock. Cells of strain UG-1 have been keeping their intracellular bacteria for almost three years by now. Bacteria were not lost from the host cells when cultivated in a temperature range from 10 to 25 ~

In situ hybridization and phylogenetic position of bacteria

Using the eubacterial probe for in situ hybridization, bacteria were labeled in P. calkinsi as well in P putrinum. In strain OK-6-2, labeled bacteria were dis- tributed throughout the cytoplasm (Fig. 13), but in strain GN5-3 cells, labeled bacteria were not found in the cortex (Fig. 14). Additional use of the probe spe- cific for the alpha-subgroup of proteobacteria resulted in a double labeling of all intracellular bacteria in both host strains with one exception, whereas prey bacteria in the food vacuoles (phagosomes) were labeled by the eubacterial probe only. The exception was that some of the bacteria in the endoplasmic areas of strain OK- 6-2 cells were marked as eubacteria only (Fig. 13).

In P. putrinum, labeling with the eubacterial probe revealed that most of the bacteria were situated in the central part of the cell. This was in agreement with the conventional staining methods. However, whereas all bacteria were labeled by the eubacterial probe, only a part of them was labeled by the probe for alpha- subgroup proteobacteria (Figs. 15 and 16).

Discussion

The cytoplasm is an initial environment for any types of endocytobionts. Evidently, among the cytoplasmic bacteria phylogenically old and young endocytobiont species can be found. Thus, the investigations on the cytoplasmic bacteria or the cytoplasmic stages of endonucleobionts may have an important theoretical meaning.

Figs, 13-16. In situ hybridization, laser scanning microscopy. FFood vacuole. Bars: 15 ~tm

Fig. 13. Bacteria in the cytoplasm of R calkinsi stock OK~6-2. A double labeling with eubacteria- and alpha-subgroup-specific probes. Part of the bacteria in the endoplasmic area as well as prey bacteria in the food vacuoles are labeled as eubacteria only (green color)

Fig. 14. Bacteria in the cytoplasm of P. calkinsi stock GN5-3. A double labeling with eubacteria- and alpha-subgroup-specific probes. All bacteria except prey bacteria in the food vacuoles are labeled as alpha-subgroup bacteria (orange color)

Fig. 15. Bacteria in the cytoplasm of P. putrinum labeled by alpha-subgroup-specific probe

Fig. 16. Bacteria in the cytoplasm of P. putrinum. A double labeling with eubacteria- and alpha-subgroup-specific probes of the same cell as presented on Fig. 15. Part of the bacteria are labeled as eubacteria only (green color)

S. I. Fokin et al.: Bacteria in paramecium cytoplasm 181

182 S.I. Fokin et al.: Bacteria in paramecium cytoplasm

The finding of new bacteria in the cytoplasm of P calkinsi and P.. putrinum shows again that ciliates are suitable hosts for microorganisms. Many earlier reports already pointed out that intracellular bacteria are site specific, residing in the cytoplasm, in the micro- or macronuclei, or in the perinuclear space. The present study showed that different bacteria prefer dif- ferent areas of the host cytoplasm. One may speculate that nutrients and metabolites are not uniformly avail- able in all areas of the cytoplasm. At the same time, the partial pressure of oxygen may be higher in the cortex area than in the center of the cell. The parame- ters of this kind may finally cause different niches of intracellular bacteria.

Site-specific location according to different condi- tions in the various areas of the host cell cytoplasm may be of special importance for the intracellular bacteria not encircled by a host vacuole. The majority of the bacteria in P calkinsi and P putrinum are not found in a host vacuole. Such "naked" bacteria could actively find their favorite site in the host cells if they were motile. There is, however, no evidence for motil- ity for the bacteria we observed in these ciliates. There- fore, these intracellular bacteria may be supposed to have access to the cytoskeleton of the host cell and to be distributed and located by means of the host.

The pili-bearing bacteria in P calkisi could be dis- tributed in a different way. Pili have been reported to function in attachment or movement in some species of bacteria (Gromov 1985). Sometimes, these struc- tures exist only for limited periods of the bacterial life cycle (Duguid 1968, Clegg and Gerlach 1987), which may also be the case for the bacteria in P calkinsi not found in individual vacuoles. Therefore, it cannot be excluded that pili-bearing bacteria in P calkinsi may actively take part in their distribution in the host cytoplasm.

Although the data obtained are not sufficient for a detailed species description and denomination of the new bacteria, they enable us to distinguish three forms of bacteria in the endoplasmic area of P. calkinsi strain OK-6-2 cells: with individual vacuoles (most of the bacteria); without individual vacuoles but with sec- ondary, double-membrane (ER) vacuole; and, some- times, free bacteria with small pill-like structures on their surface (Figs. I-6). With respect to these mor- phological features and to their distribution and inter- action with host membranes, it seems possible that the different bacterial forms in OK-6-2 cells represent dif- ferent stages of the same bacterium species, but their

belonging to different species cannot be excluded either. Partly, this question could be answered by means of in situ hybridization experiments roughly showing the phylogenetic position of the intracellular bacteria. The observation that only part of the bacte- ria in P. calkinsi (stock OK-6-2) and P putrinum was labeled with the probe specific to the alpha-subgroup indicates that the host cells in these cases harbor dif- ferent bacterial species, in each paramecium species both bacterial species being Gram-negative eubacte- ria, but only one of them belonging to the alpha- subgroup of proteobacteria (for a review, see Moreno 1998). This is the first special in situ study of intracel- lular bacteria in the cytoplasm of Paramecium species (see Brigge et al. 1999).

This is the first description of intracellular bacteria in the cytoplasm of P. putrinum as well. It must be expected that the investigation of other ciliates, and probably other protozoa, may reveal the existence of many new intracellular bacteria. The diversity of intra- cellular bacteria may therefore be much greater than of free-living ones.

Few of the bacteria have been found to be infectious for different host species; most of them seem to be strictly host-specific (for a review, see Heckmann and G6rtz 1991). While many endonuclear bacteria are highly infectious, the many species of intracellular bac- teria (traditionally called symbionts) in the cytoplasm of paramecia were not found to be truly infectious (Preer et al. 1974, Heckmann and G6rtz 1991, Fokin et al. 1996). The bacteria found in P calkinsi stock GN5-3 may therefore be of special experimental interest with respect to studies of the invasion of the cytoplasm; the more so, as they were not found to reside in a host vacuole.

Acknowledgments

We are grateful to Dr. E Br~3mmer for the sampling of P, putrinum and to Dr. T. Brigge for the help with the confocaI laser scanning microscope in the early phase of the work. This work was supported by a grant from the Volkswagen Stiftung (AZ/72 898) to H.-D. G6rtz. The writing of this article partly was supported by a Grant- in-Aid for Scientific Research (B) (hr. 11694211) from the Japan Society for the Promotion of Science to S. I. Fokin.

References

Amann RI, Binder B J, Oslon R J, Chrisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial popu- lation. Appl Environ Microbiol 56:1219-1925

S. I. Fokin et al.: Bacteria in paramecium cytoplasm 183

- Springer N, Ludwig W, G6rtz H-D, Scheifer K-H (1991) Identifi- cation in situ and phylogeny of uncultured bacteria endosym- bionts. Nature 351:161-164

Brigge T, Fokin SI, Brtimmer E G6rtz H-D (1999) Molecular probes for localization of endosymbiotic bacteria in ciliates and toxic dinoflagellales. J Euk Microbiol 46: l l A

Clegg S, Gerlach GT (1987) Enterobacterial fimbriae. J Bacteriol 169:934-938

Duguid JP (1968) The function of fimbriae. Arch Immunol Therap Exp 16:173-188

Fokin SI (1989a) Bacterial endobionts of the ciliate Paramecium woodruffi I: endobionts of the macronucleus. Cytologia (Sankt Petersburg) 31:829-844

- (1989b) Bacterial endobionts of the ciliate Paramecium woodruffi III: endobionts of the cytoplasm. Cytologia (Sankt Petersburg) 31: 964-970

- K a r p o v SA (1995) Bacterial endocytobionts inhabiting the perinuclear space of protista. Endocyt Cell Res 11: 81- 94

- Sabaneyeva EV (1993) Bacterial endocytobionts of the ciliate Paramecium caIkinsi. Eur J ProtistoI 29:390-395

- Brigge T, Brenner L GOrtz H-D (1996) Holospora species infect- ing the nuclei of Paramecium appear to belong into two groups of bacteria. Eur J Protistol 32 Suppl 1:19-24

G6rtz H-D, Dieckmann J (1987) Leptomonas ciliatorum n. sp. in the macronucleus of hypotrich ciliate J Protozool 34:259-265

Gromov BV (1985) Structure of bacteria. LGU, Leningrad Heckmann K, GOrtz H-D (1991) Prokaryotic symbionts of ciliates.

In: Balows A, Truper HG, Dworkin M, Harder W, Scheifer K-H (eds) The prokaryotes, 2nd edn. Springer, Berlin Heidelberg New York Tokyo, pp 3865-3890

Moreno E (1998) Genome evolution within the alpha Proteobacte- ria: why do some bacteria not possess plasmids and others exhibit more than one different chromosome? FEMS Microbiol Rev 22: 255-275

Preer JR, Preer LB (1984) Endosymbionts of protozoa. In: Krieg NR (ed) Bergey's manual of systematic bacteriology, vol 1. Willams and Wilkins, Baltimore, pp 795-811

- Preer LB, Jurand A (1974) Kappa and other endosymbionts in Paramecium aurelia. Bacteriol Rev 38:113-163

Preer LB (1969) Alpha, an infectious macronuclear symbiont of Paramecium aureIiu. J ProtozooI 16:570--578