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.

FIG 1

Species-specific in situ detection of “Candidatus Bealeia paramacronuclearis.” (A and B) Green fluorescent signal from probe Bealeia_1245 (A) and “Candidatus Fokinia cryptica” red fluorescent signal from probe FokCry_198 (B) in Paramecium biaurelia isolate US_Bl 11III1, both with 0% formamide. (C and D) 4′,6-Diaminidino-2-phenylindole (DAPI) signal (C), also illustrated in a merged image of the Bealeia_1245, FokCry_198, and DAPI signals (D). Relative positioning of both endosymbionts with respect to each other and within the host cell is shown. Scale bar, 10 μm.

FIG 2

Bayesian inference phylogenetic tree of “Candidatus Bealeia paramacronuclearis,” based on prokaryotic SSU rRNA gene sequences, including 18 Rickettsiales, 70 Holosporales, and 7 members of other orders of Alphaproteobacteria as an outgroup. The GTR+I+G model was employed. A total of 1,251 nucleotide columns were used to calculate the tree. Numbers indicate maximum likelihood bootstrap values after 1,000 pseudoreplicates and Bayesian posterior probabilities after 1,500,000 iterations. Values below 50% and 0.5 are not shown. Scale bar, 8 nucleotide substitutions per 100 positions. Ca., Candidatus. *, a sequence which clusters within “Candidatus Paracaedibacteraceae,” including associated sequences in the ML tree (see Fig. 3).

FIG 3

Phylogenetic tree topology of the maximum likelihood analysis employing the GTR+I+G model, showing a different relationship between the families of the order Holosporales with respect to the Bayesian inference tree (Fig. 2). Species selection and nucleotide columns used to calculate the tree are the same as those for the tree in Fig. 2. Numbers indicate maximum likelihood bootstrap values after 1,000 pseudoreplicates and Bayesian posterior probabilities after 1,500,000 iterations (values below 50% and 0.5 are not shown). Scale bar, 10 nucleotide substitutions per 100 positions. Ca., Candidatus.

FIG 4

Bayesian inference phylogenetic tree (GTR+I+G model) of “Candidatus Fokinia cryptica,” based on prokaryotic SSU rRNA gene sequences, including 23 other “Candidatus Midichloriaceae” and 9 Anaplasmataceae as an outgroup. Nucleotide columns used to calculate the tree: 1,311. Maximum likelihood bootstrap values after 1,000 pseudoreplicates and Bayesian posterior probabilities after 1,500,000 iterations are shown down to 50% and 0.5. Scale bar: 7 nucleotide substitutions per 100 positions. Ca., Candidatus.

FIG 5

Transmission electron microscopic images of “Candidatus Bealeia paramacronuclearis,” as observed for Paramecium biaurelia isolates US_Bl 11III1 and US_Bl 15I1. The symbiotic bacteria are oriented in parallel, in groups (A, B, and C), and were often found associated with the macronucleus (C and D), sometimes even lying in its folds (D). White arrowheads indicate the nuclear envelope. Scale bars, 0.5 μm (A, B, and C) or 1.0 μm (D).

FIG 6

Transmission electron microscopic images of Paramecium biaurelia isolate US_Bl 11III1. (A) For this isolate, the secondary symbiont “Candidatus Fokinia cryptica,” indicated by black arrows, was observed in cooccurrence with “Candidatus Bealeia paramacronuclearis.” (B) “Candidatus Bealeia paramacronuclearis” was frequently surrounded by host lysosomes (white asterisks). (C and D) Parts of the host's digestive vacuole (food vacuole [FV]) containing bacteria morphologically similar to “Candidatus Bealeia paramacronuclearis” were seen to bud off, producing vesicles enclosing them. Scale bars, 0.5 μm (A, B, and D) or 1.0 μm (C).