Petroni, Giulio

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.

ABSTRACT In the past 10 years, the number of endosymbionts described within the bacterial order Rickettsiales has constantly grown. Since 2006, 18 novel Rickettsiales genera inhabiting protists, such as ciliates and amoebae, have been described. In this work, we characterize two novel bacterial endosymbionts from Paramecium collected near Bloomington, IN. Both endosymbiotic species inhabit the cytoplasm of the same host. The Gram-negative bacterium “ Candidatus Bealeia paramacronuclearis” occurs in clumps and is frequently associated with the host macronucleus. With its electron-dense cytoplasm and a distinct halo surrounding the cell, it is easily distinguishable from the second smaller symbiont, “ Candidatus Fokinia cryptica,” whose cytoplasm is electron lucid, lacks a halo, and is always surrounded by a symbiontophorous vacuole. For molecular characterization, the small-subunit rRNA genes were sequenced and used for taxonomic assignment as well as the design of species-specific oligonucleotide probes. Phylogenetic analyses revealed that “ Candidatus Bealeia paramacronuclearis” clusters with the so-called “basal” Rickettsiales , and “ Candidatus Fokinia cryptica” belongs to “ Candidatus Midichloriaceae.” We obtained tree topologies showing a separation of Rickettsiales into at least two groups: one represented by the families Rickettsiaceae , Anaplasmataceae , and “ Candidatus Midichloriaceae” (RAM clade), and the other represented by “basal Rickettsiales ,” including “ Candidatus Bealeia paramacronuclearis.” Therefore, and in accordance with recent publications, we propose to limit the order Rickettsiales to the RAM clade and to raise “basal Rickettsiales ” to an independent order, Holosporales ord. nov., inside Alphaproteobacteria , which presently includes four family-level clades. Additionally, we define the family “ Candidatus Hepatincolaceae” and redefine the family Holosporaceae . IMPORTANCE In this paper, we provide the characterization of two novel bacterial symbionts inhabiting the same Paramecium host (Ciliophora, Alveolata). Both symbionts belong to “traditional” Rickettsiales , one representing a new species of the genus “ Candidatus Fokinia” (“ Candidatus Midichloriaceae”), and the other representing a new genus of a “basal” Rickettsiales . According to newly characterized sequences and to a critical revision of recent literature, we propose a taxonomic reorganization of “traditional” Rickettsiales that we split into two orders: Rickettsiales sensu stricto and Holosporales ord. nov. This work represents a critical revision, including new records of a group of symbionts frequently occurring in protists and whose biodiversity is still largely underestimated.