THE use of Koch's technique to isolate bacteria in pure cultures has enabled thousands of bacterial species to be characterized. But for the many microorganisms that have never been cultivated, DNA amplification in vitro using the polymerase chain reaction is now making their genes accessible1–3. Here we use this technique to study bacteria of the genus Holospora, which live in ciliates4 and whose phylogenetic relationship has remained unknown because they are impossible to cultivate. Species of Holospora are highly infectious5–7 and live in the nuclei of their specific host cells: H. elegans and H. undulata infect micronuclei of Paramecium caudatum8, whereas H. obtusa infects the macronucleus in other strains of the same host species9; Holospora species have a common developmental cycle10–13. We have amplified, cloned and sequenced gene fragments encoding ribosomal RNA of H. obtusa. The phylogenetic position of H. obtusa in the α group of Proteobacteria was determined by 16S rRNA sequence analysis. The sequences were then used to design species- as well as genus-specific rRNA hybridization probes, which enabled us to detect and differentiate individual cells of the endosymbionts in situ. The large amount of rRNA in the cells indicates a high physiological activity of the endosymbionts in the host nuclei.

Identification in situ and phylogeny of uncultured bacterial endosymbionts

Class I. Alphaproteobacteria class. nov.

Ciliates may be hosts for numerous bacteria, which can occupy almost all cellular compartments of the protists. About 200 ciliate species are recorded as hosts of different intracellular bacteria, being a small part of the diversity for such types of endocytobiosis in nature. In the Paramecium genus alone close to 60 types of bacteria adapted for intracellular life are known. In this review extensive material concerning the variety of endocytobionts, their categories, and their interaction with host cells is presented. Special attention is paid to endocytobiosis in Paramecium with highly infectious bacteria Holospora, bacteria of the Caedibacter and Polynucleobacter genera, methanogenic bacteria, and "xenosomes" as well as to life cycles and strategies of bacterial endonucleobionts. The above model bacteria and their interactions with hosts have not been exhaustively studied. A number of unsolved problems concerning their interactions within an endocytobiotic system and their ecological implications remain to be studied.

Bacterial Endocytobionts of Ciliophora and Their Interactions with the Host Cell

Paramecium species are extremely valuable organisms to enable experiments for the reestablishment of endosymbiosis. This is investigated in two different systems, the first with Paramecium caudatum and the endonuclear symbiotic bacterium Holospora species. Although most endosymbiotic bacteria cannot grow outside the host cell as a result of their reduced genome size, Holospora species can maintain their infectivity for a limited time. We found that an 89-kDa periplasmic protein has an important function for Holospora's invasion into the target nucleus, and that Holospora alters the host gene expression; the host thereby acquires resistance against various stresses. The second system is the symbiosis between P. bursaria and symbiotic Chlorella. Alga-free P. bursaria and the algae retain the ability to grow without a partner. Consequently, endosymbiosis between the aposymbiotic host cells and the symbiotic algae can be reestablished easily by mixing them. We now found four checkpoints for the reestablishment of the endosymbiosis between P. bursaria and the algae. The findings in the two systems provide excellent opportunities for us to elucidate not only infection processes but also to assess the associations leading to eukaryotic cell evolution. This paper summarizes recent progresses on reestablishment of the primary and the secondary endosymbiosis in Paramecium.

Endosymbionts in Paramecium

osen ring Genus Caedibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441 Genus Pseudocaedibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442 Genus Lyticum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443 Genus Pseudolyticum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443 Genus Tectibacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443 Genus Nonospora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444 Genus Holospora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444 Genus Paraholospora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446

18 Symbiotic Associations Between Ciliates and Prokaryotes

Changes in Morphology,Buoyant Density and Protein Composition in Differentiation from the Reproductive Short Form to the Infectious Long Form of Holospora obtusa,a Macronucleus-Specific Symbiont of the Ciliate Paramecium caudatum

Sensitivity of the infectious long form of the macronuclear endosymbiont Holospora obtusa of the ciliate Paramecium caudatum against chemical and physical factors.

Kazumi Nagahara

Papers

Changes in Morphology,Buoyant Density and Protein Composition in Differentiation from the Reproductive Short Form to the Infectious Long Form of Holospora obtusa,a Macronucleus-Specific Symbiont of the Ciliate Paramecium caudatum

Sensitivity of the infectious long form of the macronuclear endosymbiont Holospora obtusa of the ciliate Paramecium caudatum against chemical and physical factors.