Amoebae play an important ecological role as predators in microbial communities. They also serve as niche for bacterial replication, harbor endosymbiotic bacteria and have contributed to the evolution of major human pathogens. Despite their high diversity, marine amoebae and their association with bacteria are poorly understood. Here we describe the isolation and characterization of two novel marine amoebae together with their bacterial endosymbionts, tentatively named 'Candidatus Occultobacter vannellae' and 'Candidatus Nucleophilum amoebae'. While one amoeba strain is related to Vannella, a genus common in marine habitats, the other represents a novel lineage in the Amoebozoa. The endosymbionts showed only low similarity to known bacteria (85-88% 16S rRNA sequence similarity) but together with other uncultured marine bacteria form a sister clade to the Coxiellaceae. Using fluorescence in situ hybridization and transmission electron microscopy, identity and intracellular location of both symbionts were confirmed; one was replicating in host-derived vacuoles, whereas the other was located in the perinuclear space of its amoeba host. This study sheds for the first time light on a so far neglected group of protists and their bacterial symbionts. The newly isolated strains represent easily maintainable model systems and pave the way for further studies on marine associations between amoebae and bacterial symbionts.

Department of Botany and Zoology Faculty of Science Masaryk University 61137 Brno Czech Republic

Department of Microbiology and Ecosystem Sience University of Vienna Althanstraße 14 A 1090 Vienna Austria

Faculty of Science University of South Bohemia Branišovská 31 370 05 České Budějovice Czech Republic

Institute of Parasitology Biology Centre CAS Branišovská 31 370 05 České Budějovice Czech Republic

Water Research Institute National Research Council of Italy Via Salaria Km 29 300 00015 Monterotondo Roma Italy

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