• genome isolation
• ciliates
• endosymbiosis
• intracellular bacteria

Intracellular bacteria, traditionally called endosymbionts, are very common in all living organisms, and ciliated protists are considered one of the most suitable model organisms for the study of endosymbionts because of their small size and simple cultivation techniques.
The aim of this Thesis was to perform a “complete study” approach on bacterial endosymbionts of ciliates. The study was split into two distinct but related parts, due to time constraints.
The first part consisted in the characterization of symbionts from newly isolated hosts present in environmental samples. The latter were initially collected from different places, thus making more probable to find new symbiont species. Then it was essential to isolate ciliates from their natural environment and establish cell cultures. The subsequent step consisted in performing the screening of ciliates cultures to detect the presence of possible bacterial endosymbionts, with fluorescence in situ hybridization (FISH) using a universal 16S rRNA gene-directed probe. Subsequently the endosymbiont retrieved in a selected culture was molecularly characterized.
The second part consisted in setting up the conditions to perform massive total DNA isolation on the selected bacterial endosymbiont Holospora caryophila, preliminary to whole genome sequencing. A particular strain of Paramecium octaurelia called GFg infected with H. caryophila was selected for our purpose. This intracellular bacterium is uncultivable outside host cells, furthermore in literature it has been reported that rapid growing lines of Paramecium could lose the endosymbiont. Therefore, to obtain the highest amounts of bacterial cells, a growth experiment was designed establishing three different host feeding conditions. For each of them the host cells were enumerated and FISH experiments were performed with a H. caryophila-specific probe to check the infection level. After having found the best feeding condition, massive culture was established in order to isolate the symbiont cells from lysed hosts and subsequently extract their genomic DNA. Different kinds of approaches, previously used in literature for the isolation of other ciliate intracellular bacteria, were adapted and performed. Finally, to evaluate how the isolation procedures worked out, two distinct approaches were set up and utilized: FISH and real-time PCR.