ciliates (Oligohymenophorea, Ciliophora, Alveolata) host diverse intracellular symbionts, among which the best studied are Holospora
-like bacteria (HLB), obligate intranuclear bacteria of family Holosporaceae
, order Holosporales
, class Alphaproteobacteria
]. HLB have a set of interesting features, such as a complex life cycle involving two morphological stages, infectious and reproductive, and infectious forms (IFs) which are unusually large for bacterial cells (up to 20 µm long). IFs have hypertrophied periplasm forming about half of the cell, and a recognition tip on the periplasm end [5
]; they can survive in ambient conditions for several hours and infect new host cells. The reproductive forms (RFs) are small and able to reproduce by binary fission, and can transform into IFs [1
]. HLB species can distinguish between two types of host nuclei, macronucleus (Ma) and micronucleus (Mi) [2
These features were traditionally used to assign bacteria to genus Holospora
before any molecular information was available. Thus, until the emergence of sequencing methods, all bacteria with the described morphological and physiological features were considered Holospora
species and classified by their host specificity, localization in the host cell, size and shape of IFs and RFs, and the ability to trigger formation of the connecting piece during division of the infected nucleus [1
]. Infectious forms of H. obtusa, H. undulata, H. elegans
, “H. curviuscula
”, and “H. acuminata
” gather near the center of the spindle apparatus of the dividing host nucleus forming the so-called connecting piece, while the reproductive forms mainly appear in the apical parts of the nucleus. Following formation of the connecting piece, IFs escape into the cytoplasm and then into the ambient environment. The second group of the Holospora
species (“H. caryophila”, “H. bacillata
”, and “H. curvata
”) consists of HLB, which do not form the connecting piece [6
With the recent advance of sequencing techniques, the phylogeny of genus Holospora
has been revised [9
]. One of the Holospora
species, H. caryophila
, was recently redescribed as Preeria caryophila
based on low similarity of the 16S rRNA gene with other species from genus Holospora
and the ability to infect several host species [10
]. Boscaro et al. recently reported a new genus of HLB, “Ca.
Gortzia”, currently comprised of two species, “Ca.
Gortzia infectiva” [11
], and “Ca.
Gortzia shahrazadis” [12
], macronuclear symbionts of Paramecium jenningsi
and Paramecium multimicronucleatum
, respectively. These endosymbionts do not induce the formation of the connecting piece in the nucleus during division of Paramecium
Hafkinia simulans” was recently described by Fokin et al. within Holosporaceae
as a macronuclear symbiont of a ciliate Frontonia salmastra
showing typical HLB features [14
]. The 16S rRNA gene sequences obtained from “Ca
. Gortzia” and “Ca
. Hafkinia” differ by approximately 7–10% from the 16S rRNA of Holospora
species, which is beyond the threshold for the genus level [11
]. Together with other discriminating features like inducing formation of the connecting piece (now assigned only to genus Holospora
), and reduced host specificity as shown for Preeria caryophila
, it supports the separation of HLB group into four genera [10
Here we report a new Holospora-like intranuclear bacterium in the macronucleus of ciliate Paramecium putrinum originating from Yakutia (Sakha Republic), Russia. Our microscopical observations, phylogenetic analysis based on the 16S rRNA genes, and fluorescence in situ hybridization assays allow suggestion of its inclusion as a novel member of genus “Ca. Gortzia”. We suggest this bacterium to be classified as a new species “Ca. Gortzia yakutica” sp. n.
Here, we report a new Holospora-like bacterium from the macronucleus of P. putrinum. All features of this bacterium, such as morphology, intracellular localization, complex life cycle, host and nuclear specificity, and infectivity, indicate a close relation of this endosymbiont to other HLB. The phylogenetic analysis based on the 16S rRNA gene sequence also shows that the endosymbiont is close to other HLB and belongs to Holosporaceae family. Recently described macronuclear symbionts “Ca. G. shahrazadis” and “Ca. G. infectiva” are the closest relatives of the new endosymbiont, and together they form a well-supported clade sister to Holospora genus. Consequently, we assign this symbiotic bacterium to the genus “Ca. Gortzia” and name it “Ca. Gortzia yakutica” sp. nov.
As with “Ca.
G. infectiva” and “Ca.
G. shahrazadis”, the new endosymbiont does not induce formation of the connecting piece during the host division, and can escape into host’s cytoplasm; these two features together can be considered to be distinctive for the genus. Another feature to discuss is the manner of transforming IFs to RFs. It is described that IFs of Holospora
species do not undergo a binary division, but constrict at several points forming a specific chain of cells and divide into several cells simultaneously [6
]. On the contrary Serra et al. describe binary fission of IFs for the “Ca
. Gortzia shahrazadis” [12
]. We observed a classical IFs chain formation as well as the behavior similar to what is described for “Ca
. Gortzia” (Figure 2
, shown with the white arrowhead). Our knowledge about the transformation of IFs into RFs for the new HLB is limited and based on observations of live Paramecium
cultures with DIC microscopy, and henceforth is far from comprehensive. At the same time, this phenomenon certainly deserves to be carefully investigated in further studies.
The ability to form the connecting piece also places the new symbiont close to “H. bacillata”
, “H. curvata”
and “H. sp.
from the macronucleus of P. putrinum
]. It is possible that this species was described previously by Fokin et al. as “Holospora
sp. from macronucleus of P. putrinum
” from Germany [1
]. The original description has the information about localization, the shape and sizes of IFs and RFs; the ability to induce the formation of the connecting piece. The new HLB reported here and the endosymbiont from P. putrinum
reported by Fokin et al. have a similar phenotype (same host and localization, same shape of the cell, both do not form the connecting piece), but the described sizes are different, with the new HLB being notably smaller (e.g., the length of IFs is 12 µm vs. 17 µm). As the culture of “Holospora
sp. from macronucleus of P. putrinum
” had been lost precluding a more detailed characterization, it is impossible to establish whether these two endosymbionts belong to the same species.
All HLB have very distinctive morphological and physiological features and form a monophyletic clade within Holosporaceae
includes four HLB genera—Holospora
, and several other genera, which do not share HLB phenotype. Takeshita et al. recently described an endosymbiont from an anaerobic Scuticociliate—“Ca.
Hydrogenosomobacter endosymbioticus” [39
]. This endosymbiont has an uncertain position on the phylogenetic trees based on 16S rRNA genes: according to Takeshita et al. it forms a sister taxon to HLB, but with the low branch support (less than 70%) [39
]. In our analysis this species appears within the HLB clade (Figure 6
), but the branch support is quite low as well. “Ca
. H. endosymbioticus” does not have HLB characteristic features discussed above, and its phylogenetic placement would have to be revised when some additional molecular data become available. Another issue arises with “Ca.
Hafkinia”, which was described as a separate genus within HLB, based on the 93.9–94.5% similarity with Holospora
], whereas our analysis shows 96–96.5% similarity, which places it within the genus Holospora
While phylogenetic analysis based on 16S rRNA gene sequences is undoubtedly useful and widely used to make decisions on bacterial taxonomy, the examples given above show the limitations of such approach. Different thresholds proposed by various authors [15
], different approaches to multiple alignments and substitution models can affect similarity values and topology of phylogenetic trees. It has been recently demonstrated that complete genome sequences could be used to better define bacterial species [41
]. Thus, we can conclude that we would be in a much better position to infer the phylogenetic relationships of the HLB clade when complete genomes of Gortzia
spp. become available.
5. Description of “Candidatus Gortzia yakutica” sp. nov.
Gortzia yakutica (Gor’tzi.a ya.ku’ti.ca; N.L. fem. n. Gortzia, in honour of Professor emeritus Hans-Dieter Görtz; N.L. fem. adj. yakutica, of or belonging to Republic of Yakutia, the name of the region where the bacterium was first collected).
Obligate macronuclear endosymbionts of the free-living ciliate P. putrinum, occasionally can be found in the cytoplasm. Sampled from the freshwater pond in Republic of Yakutia, Russia. Has two life stages: small reproductive forms (1–2 by 2–4 µm) and long infectious forms (1–2 by 7–12 µm, rod-shaped with tapered ends, sometimes slightly curved). No formation of the connecting piece was observed. Basis of assignment: SSU rRNA gene sequence (GenBank accession numbers: MT421875.1–MT421895.1) and positive match with the species-specific FISH oligonucleotide probe Gyak567 (5’-AGGTAGCCACCTACACA-3’).
Type strain is YA111-52 carried by Paramecium putrinum YA111-52 (Culture Collection of Ciliates and their Symbionts, CCCS 1024, St. Petersburg State University). Unculturable outside of host cells so far.