Hans-Dieter Görtz

Prokaryotic Symbionts of Ciliates

Prokaryotes living in ciliates were first noticed over a century ago by J. Muller (1856). Rod-shaped structures were observed in the macro-nuclei and micronuclei of a number of ciliates, and less commonly, in their cytoplasm. In the beginning, it was not clear whether they were parasites or spermatozoa because the micronucleus was considered to be a testis and the macronucleus an ovary, while chromosome filaments and endonuclear symbionts were mistaken for spermatozoa. This view was corrected by Butschli (1876), who also wrote the first review on parasites in ciliates (Butschli, 1889). Accounts of early observations of bacteria in protozoa that followed this initial period were reviewed by Kirby (1941), Wichterman (1953), and Ball (1969).

Killer Particles in the Macronucleus of Paramecium caudatum

One of the earliest known examples of cytoplasmic inheritance is the killer trait in paramecium. For many years it was not known that such killer traits are determined by bacterial endosymbionts; and kappa, the first symbiont discovered in the Parumecium aurelia complex, was initially recognized only by its killing action on sensitive paramecia. Kappa and some other endosymbionts, all members of the genus Caedibacter, have two distinct morphological forms, one of which contains a large inclusion body known as a refractile body, or R body. Bacteria of this form are commonly called “brights” whereas symbionts without R bodies are referred to as “nonbrights. ” R bodies are proteinaceous ribbons, and several investigators have shown that the killing of sensitive paramecia is associated with such R bodies.’ All R body producing bacteria from P. aurelia live in the cytoplasm, whereas those observed in P. caudatum inhabit the macronucleus. Generally, many endosymbionts are endocytoplasmatic forms in P. aurelia host cells and endonuclear ones, living either in the microor macronucleus, if P. caudatum harbors them. We discovered R body producing bacteria, looking similar to those described by Estkve,’ in the macronuclei of two P. caudutum lines which were isolated from a pond in Munster, and after their cultivation in the laboratory we observed that one of the two lines (C220) lost its ability to produce R bodies. Macronuclei of paramecia from this line now contain nonbrights only. They are uniform in size with a diameter of 0.4 pm. The average length is 1.0-1.5pm, but occasionally much longer rods can be found (FIGURES 1 and 2), which resemble a row of nonbrights sticking together end to end. We have been unable to infect symbiont-free P. caudatum cells. For such experiments either isolated nonbrights or homogenates from C 220 cells were used. We have also failed to induce R body production with the UV irradiation method.’ The bacteria appear well adapted to their endonucleobiotic way of life. Rather than being evenly distributed within the macronucleus they are clustered. During cell division they get distributed with the dividing macronuclei of their hosts. Paramecium is obviously not harmed by the nonbright endonucleobionts. Brights are always present in line C 221. Their numbers are low in rapidly growing cultures and increase considerably when such cultures starve. Brights are bigger than nonbrights, about 0.7 pm wide and 1.5-2.5pm long. C 221 host cells suffer and eventually die under certain conditions, e.g., in cultures that have starved for more than a week. Their death is probably caused by their endosymbionts. We established two methods for the isolation and purification of nonbrights, brights, and R bodies. The first method involves ECTEOLA column chromatography of partly purified paramecium homogenates. High yields of pure nonbrights are obtained with this procedure (FIGURE 1). Brights, however, are lost irreversibly. They and their R