Disentangling the Taxonomy of Rickettsiales and Description of Two Novel Symbionts (“Candidatus Bealeia paramacronuclearis” and “Candidatus Fokinia cryptica”) Sharing the Cytoplasm of the Ciliate Protist Paramecium biaurelia

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.

Rare Freshwater Ciliate Paramecium chlorelligerum Kahl, 1935 and Its Macronuclear Symbiotic Bacterium “Candidatus Holospora parva”

“…However, our phylogenetic reconstruction of Holosporaceae and Holosporales is in accordance with previous studies [23, 63, 64, 98] and its inner relations are confirmed. Indeed, all H .…”

“…As shown in some recent literature based on 16S rDNA and genome phylogenies [23, 63, 64], the family Holosporaceae is placed as basal to the rest of Rickettsiales and within this order. Other studies utilizing different and less widespread molecular markers, such as 23S rDNA and protein-coding genes, placed Holosporaceae outside of the order Rickettsiales and suggested to rank it as a separate order [95–97]; a recent study formally elevated Holosporaceae to the order Holosporales providing an overall revision of families belonging to this new order [98]. …”

Identifying the causal agent of necrotizing hepatopancreatitis in shrimp: Multilocus sequence analysis approach

“…Finally, sequencing and assembling of whole NHP‐B genome allowed to obtain the complete nucleotide sequences of biomarker genes, very usefully for taxonomic classification. Phylogenetic analysis confirmed that the bacterium formerly included in the order Rickettsiales actually belongs to the new Holosporales order, and particularly to the Holosporaceae family (Szokoli et al., ). Interestingly, a common feature of bacteria belonging to this family is the occupation of specific compartments in their host cells, which are generally rich in metabolites and nutrients; being the case of NHP‐B that thrives exclusively in the hepatopancreatic tissue of crustaceans.…”

Outwitting planarian’s antibacterial defence mechanisms:Rickettsialesbacterial trans-infection fromParamecium multimicronucleatumto planarians

“…For molecular characterization of both ciliate host and bacterial endosymbiont, total DNA was extracted from approximately 50 Paramecium cells as described in [30, 31]. All PCR reactions were carried out with the Takara ExTaq (Takara, Kusatsu, Japan) reaction kit.…”