AbstractRickettsialesare a lineage of obligatorily intracellularAlphaproteobacteria, encompassing important human pathogens, manipulators of host reproduction, and mutualists. Here we report the discovery of a novelRickettsialesbacterium associated withParamecium, displaying a unique extracellular lifestyle, including the ability to replicate outside host cells. Genomic analyses show that the bacterium possesses a higher capability to synthesize amino acids, compared to all investigatedRickettsiales. Considering these observations, phylogenetic and phylogenomic reconstructions, and re-evaluating the different means of interaction ofRickettsialesbacteria with eukaryotic cells, we propose an alternative scenario for the evolution of intracellularity inRickettsiales. According to our reconstruction, theRickettsialesancestor would have been an extracellular and metabolically versatile bacterium, while obligate intracellularity and genome reduction would have evolved later in parallel and independently in different sub-lineages. The proposed new scenario could impact on the open debate on the lifestyle of the last common ancestor of mitochondria withinAlphaproteobacteria.
ABSTRACTThe orderRickettsiales(Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae,Anaplasmataceae, andHolosporaceae) and a fourth, family-level cluster, which includes only one candidate species, “CandidatusMidichloria mitochondrii,” as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the “CandidatusMidichloria” clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity ofRickettsiales. In this paper, two new bacteria belonging to the “CandidatusMidichloria” clade, hosted by two different strains of the ciliate protistEuplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the familyRickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the “CandidatusMidichloria” clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the “CandidatusMidichloria” clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, “CandidatusAnadelfobacter veles” and “CandidatusCyrtobacter comes.”