title: Evaluation of Enrichment Protocols for Bacterial Endosymbionts of Ciliates by Real-Time PCR.
abstract: Large-scale studies on obligate bacterial endosymbionts may frequently require preliminary purification and enrichment protocols, which are often elaborate to set up and to evaluate, especially if the host organism is a protist. The purpose of this study was to develop a real-time PCR-based strategy and employ it for assessing two of such enrichment protocols for Holospora caryophila, hosted by the ciliate Paramecium. Four SSU rRNA gene-targeted real-time PCR assays were designed, which allowed to compare the amount of H. caryophila to other organisms, namely the host, its food bacterium (Raoultella planticola), and free-living bacteria present in the culture medium. By the use of the real-time PCR assays in combination, it was possible to conclude that the "cell fractionation" protocol was quite successful in the enrichment of the symbiont, while the "Percoll gradient" protocol will need further refinements to be fully repeatable. The proposed approach has the potential to facilitate and encourage future studies on the yet underexplored field of bacterial endosymbionts of ciliates and other protists. It can also find valuable applications for experimental questions other than those tested, such as fast and precise assessment of symbiont abundance in natural populations and comparison among multiple coexisting symbionts.
authors.affiliation: Department of Biology, University of Pisa, 56126, Pisa, Italy. miccast@tiscali.it.
authors.forename: Michele
authors.initials: M
authors.lastname: Castelli
authors.affiliation: Department of Biology, University of Pisa, 56126, Pisa, Italy.
authors.forename: Olivia
authors.initials: O
authors.lastname: Lanzoni
authors.affiliation: Department of Clinical and Experimental Medicine, University of Pisa, 56126, Pisa, Italy.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Rossi
authors.affiliation: Department of Microbiology, Faculty of Biology, St. Petersburg State University, Saint Petersburg, Russia, 199034.
authors.forename: Alexey
authors.initials: A
authors.lastname: Potekhin
authors.affiliation: Microbiology, Institute of Biology II, University of Freiburg, 79104, Freiburg, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Schrallhammer
authors.affiliation: Department of Biology, University of Pisa, 56126, Pisa, Italy. giulio.petroni@unipi.it.
authors.forename: Giulio
authors.initials: G
authors.lastname: Petroni
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D018407:Bacterial Physiological Phenomena; D015373:Bacterial Typing Techniques / Q000379:methods*; D044822:Biodiversity; D016798:Ciliophora / Q000382:microbiology* / Q000502:physiology; D004269:DNA, Bacterial / Q000235:genetics; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D013559:Symbiosis
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S