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.

Recently, the family Midichloriaceae has been described within the bacterial order Rickettsiales. It includes a variety of bacterial endosymbionts detected in different metazoan host species belonging to Placozoa, Cnidaria, Arthropoda and Vertebrata. Representatives of Midichloriaceae are also considered possible etiological agents of certain animal diseases. Midichloriaceae have been found also in protists like ciliates and amoebae. The present work describes a new bacterial endosymbiont, “Candidatus Fokinia solitaria”, retrieved from three different strains of a novel Paramecium species isolated from a wastewater treatment plant in Rio de Janeiro (Brazil). Symbionts were characterized through the full-cycle rRNA approach: SSU rRNA gene sequencing and fluorescence in situ hybridization (FISH) with three species-specific oligonucleotide probes. In electron micrographs, the tiny rod-shaped endosymbionts (1.2 x 0.25–0.35 μm in size) were not surrounded by a symbiontophorous vacuole and were located in the peripheral host cytoplasm, stratified in the host cortex in between the trichocysts or just below them. Frequently, they occurred inside autolysosomes. Phylogenetic analyses of Midichloriaceae apparently show different evolutionary pathways within the family. Some genera, such as “Ca. Midichloria” and “Ca. Lariskella”, have been retrieved frequently and independently in different hosts and environmental surveys. On the contrary, others, such as Lyticum, “Ca. Anadelfobacter”, “Ca. Defluviella” and the presently described “Ca. Fokinia solitaria”, have been found only occasionally and associated to specific host species. These last are the only representatives in their own branches thus far. Present data do not allow to infer whether these genera, which we named “stand-alone lineages”, are an indication of poorly sampled organisms, thus underrepresented in GenBank, or represent fast evolving, highly adapted evolutionary lineages.

Members of the order Rickettsiales are often found in association with ciliated protists. An interesting case is the bacterial endosymbiont “Candidatus Megaira”, which is phylogenetically closely related to the pathogen Rickettsia. “Candidatus Megaira” was first described as an intracellular bacterium in several ciliate species. Since then it has been found in association with diverse evolutionary distantly-related hosts, among them other unicellular eukaryotes, and also algae, and metazoa, such as cnidarians. We provide the characterization of several new strains of the type species “Candidatus Megaira polyxenophila”, and the multidisciplinary description of a novel species, “Candidatus Megaira venefica”, presenting peculiar features, which highlight the diversity and variability of these widespread bacterial endosymbionts. Screening of the 16S rRNA gene short amplicon database and phylogenetic analysis of 16S rRNA gene hypervariable regions revealed the presence of further hidden lineages, and provided hints on the possibility that these bacteria may be horizontally transmitted among aquatic protists and metazoa. The phylogenetic reconstruction supports the existence of at least five different separate species-level clades of “Candidatus Megaira”, and we designed a set of specific probes allowing easy recognition of the four major clades of the genus.

Bacteria of the family Rickettsiaceae have always been largely studied not only for their importance in the medical field, but also as model systems in evolutionary biology. In fact, they share a recent common ancestor with mitochondria. The most studied species, belonging to genera Rickettsia and Orientia, are hosted by terrestrial arthropods and include many human pathogens. Nevertheless, recent findings show that a large part of Rickettsiaceae biodiversity actually resides outside the group of well-known pathogenic bacteria. Collecting data on these recently described non-conventional members of the family is crucial in order to gain information on ancestral features of the whole group. Although bacteria of the family Rickettsiaceae, and of the whole order Rickettsiales, are formally described as non-flagellated prokaryotes, some recent findings renewed the debate about this feature. In this paper we report the first finding of members of the family displaying numerous flagella and active movement inside their host cells. These two new taxa are hosted in aquatic environments by protist ciliates and are described here by means of ultrastructural and molecular characterization. Data here reported suggest that the ancestor of Rickettsiales displayed flagellar movement and re-evaluate the hypothesis that motility played a key-role in the origin of mitochondria. Moreover, our study highlights that the aquatic environment represents a well exploited habitat for bacteria of the family Rickettsiaceae. Our results encourage a deep re-consideration of ecological and morphological traits of the family and of the whole order.

The order Rickettsiales (Alphaproteobacteria) encompasses multiple diverse lineages of host-associated bacteria, including pathogens, reproductive manipulators, and mutualists. Here, in order to understand how intracellularity and host association originated in this order, and whether they are ancestral or convergently evolved characteristics, we built a large and phylogenetically-balanced dataset that includes de novo sequenced genomes and a selection of published genomic and metagenomic assemblies. We perform detailed functional reconstructions that clearly indicates “late” and parallel evolution of obligate host-association in different Rickettsiales lineages. According to the depicted scenario, multiple independent horizontal acquisitions of transporters led to the progressive loss of biosynthesis of nucleotides, amino acids and other metabolites, producing distinct conditions of host-dependence. Each clade experienced a different pattern of evolution of the ancestral arsenal of interaction apparatuses, including development of specialised effectors involved in the lineage-specific mechanisms of host cell adhesion and/or invasion.