Identification in situ and phylogeny of uncultured bacterial endosymbionts

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 accessible^1-3. 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 infectious^5-7 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 cycle^10-13. 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.