Lawrence Berkeley National Laboratory

Bacterial strains affiliated to the phylogenetically shallow subcluster C (PnecC) of the 28 Polynucleobacter cluster, which is characterized by a minimal 16S rRNA gene sequence similarity of approx. 98.5 percent, have been reported to occur as obligate endosymbionts of 30 ciliates (Euplotes spp.), as well as to occur as free-living cells in the pelagic zone of freshwater habitats. We investigated if these two groups of closely related bacteria represent 32 strains fundamentally differing in lifestyle, or if they simply represent different stages of a facultative endosymbiotic lifestyle. The phylogenetic analysis of 16S rRNA gene and 16S34 23S ITS sequences of five endosymbiont strains from two different Euplotes species and 40 pure culture strains demonstrated host-species-specific clustering of the endosymbiont 36 sequences within the PnecC subcluster. The sequences of the endosymbionts showed characteristics indicating an obligate endosymbiotic lifestyle. Cultivation experiments 38 revealed fundamental differences in physiological adaptations, and determination of the genome sizes indicated a slight size reduction in endosymbiotic strains. We conclude that the 40 two groups of PnecC bacteria represent obligately free-living and obligately endosymbiotic strains, respectively, and do not represent different stages of the same complex lifecycle. 42 These closely related strains occupy completely separated ecological niches. To our best knowledge, this is the closest phylogenetic relationship between obligate endosymbionts and 44 obligately free-living bacteria ever revealed.