Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus

Acanthamoeba sp. UWE39. Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently unc haracterized. In this study, the phylogeny of three Gram-negative, rod-shap ed endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebi c cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN -3, Acanthamoeba sp. UWCS and Acanthamoeba sp. UWE39 are related to the Par amecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans a nd Holospora obtuse. With overall 16S rRNA sequence similarities to their c losest relative, C. caryophilus, of between 87% and 93%, these endosymbiont s represent three distinct new species. in situ hybridization with fluoresc ently labelled endosymbiont-specific 16S rRNA-targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisi onally the endosymbiont of Acanthamoeba polyphaga HN-3 as 'Candidatus Caedi bacter acanthamoebae', the endosymbiont of Acanthamoeba sp. strain UWCS as 'Candidatus Paracaedibacter acanthamoebae' and the endosymbiont of Acantham oeba sp. strain UWE39 as 'Candidatus Paracaedibacter symbiosus'. The phylog eny of the Acanthamoeba host cells was analysed by comparative sequence ana lyses of their 18S rRNA. Although Acanthamoeba polyphaga HN-3 clearly group s together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit < 92% 18S rRNA sequence si milarity to each other and to other Acanthamoeba isolates. Therefore, we pr opose two new sequence types (T13 and T14) within the genus Acanthamoeba co ntaining, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.