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.