**1. Introduction**

*Paramecium* 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* [1–4]. 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 μ m 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 [1,6]. 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 [1,7,8]. 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 [6,8].

With the recent advance of sequencing techniques, the phylogeny of genus *Holospora* has been revised [9–13]. 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*. "*Ca.* 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 [11,15]. 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 [10,11].

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 sugges<sup>t</sup> this bacterium to be classified as a new species "*Ca.* Gortzia yakutica" sp. n.

#### **2. Materials and Methods**

#### *2.1. Sampling and Identification of Paramecium*

The ciliate *P. putrinum* YA111-52 was originally isolated from a freshwater pond in Yakutia (62◦02 N 129◦44 E), Sakha Republic, Russia in the summer of 2013. Monoclonal cultures of this species were maintained under standard conditions at the room temperature in lettuce medium inoculated with bacterium *Enterobacter aerogenes* as the food source [16]. The host was identified by cell morphology, the structure of the micronucleus, and a contractile vacuole [17,18]. Live observations and images were made at the St. Petersburg State University Center for Culturing Collection of Microorganisms with a Leica DM2500 microscope equipped with differential interference contrast (DIC).

The syngen of *P. putrinum* YA111-52 was determined by series of crossing with *P. putrinum* test-clones from two syngens (syngen 1: clones ABT1-3, ALT27-6, syngen 2: clones BBR51-12, YA1-8). All cultures were fed the day before the experiment. Approximately 100 cells of the testing clones were mixed with an equal number of test-clones' cells [19,20].

#### *2.2. Phenotypic Characterization of the Symbionts*

The infectious capability of the new HLB was proved by adding IFs of the bacteria to a non-infected *P. putrinum* culture. Cross-infection experiments were performed with four *P. putrinum* clones from both syngens listed above. *Paramecium* cells containing IFs of the new HLB were concentrated at 4500 g for 10 min and homogenized using 1% solution of detergent Nonidet P-40 (Sigma-Aldrich Cat No. 21-3277 SAJ). A small amount of the homogenate was checked at 200× magnification to verify that all ciliate cells had been broken. Equal amounts of homogenate were mixed with recipient *Paramecium* cultures and incubated at the room temperature. Cells were observed at 24 and 48 h post-infection. Additional checks of the mixed cultures were performed every two weeks during the following two months [20].

#### *2.3. Purification and Sequencing of Symbionts*

The cell culture of *P. putrinum* containing IFs of the new HLB was concentrated and homogenized as stated above. The infectious forms of the endosymbiont were isolated from the homogenate by centrifugation in Percoll density gradient (Sigma-Aldrich, St. Louis, MO, USA, Cat No. P1644) as described previously [13]. DNA from the purified IFs was isolated with the DNeasy Blood and Tissue kit (QIAGEN Cat No. 69504) using a modified protocol as described previously [21].

Bacterial universal primers 27F1 (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-GGTTA CCTTGTTACGACTT-3) were used for the amplification of 16S rDNA [22]. PCR products were gel purified and cloned with the TIAN Quick Midi Purification Kit (Tiangen, Beijing, China) following the manufacturer's recommendations. Purified rDNA inserted in the PTZ57 RT plasmid vector (InsTAclone PCR Clone Kit, Fermentas), the recombinant plasmids were transformed to competent cells Trans5 α (TransGen Biotech, Beijing, China). The positive clones were digested with HhaI (Fermentas, Thermo Scientific, Waltham, MA, USA). Clones determined to be unique by the RFLP analysis were sequenced by an automated ABI DNA sequencer (model 373, PE Applied Biosystems) with primers M13. In this study, 50 positive clones were randomly selected and analyzed using RFLP with enzyme HhaI, 26 unique clones were sequenced.

#### *2.4. Fluorescence In Situ Hybridization (FISH)*

Fluorescence in situ hybridization (FISH) with rRNA-targeted probes was performed to visualize the localization of the endosymbiont. The probe was designed specifically for the new HLB—Gyak567 (5-AGGTAGCCACCTACACA-3). The probe was tested against the SILVA r138 database using TestProbe 3.0 [23] allowing 0 mismatches. There was one match found for the sequence GYAK567 in the REFNR sequence collection belonging to uncultured bacterium clone lp146, environmental sample from apple orchard, China (GenBank KC331364). The efficiency of the probe was tested in silico using mathFISH tool (mathfish.cee.wisc.edu), resulting in Gooverall of −12.2 kcal/mol, and 0.9954 hybridization efficiency. The designed probe was found to have at least three mismatches with other *Holospora*-like bacteria shown in the supplementary Figure S1.

The probe was labeled with the cyanine 5 (Cy5) fluorescent dye at the 5 end. We also used the Eub338 probe for *Bacteria* labeled with Fluorescein as a positive control [24]. *P. putrinum* cell culture containing the new HLB was concentrated using centrifugation at 3000 g for 10 min. Cells were fixed in 4% paraformaldehyde in the 1X PBS buffer at 4 ◦C for 3 h shaken every 30 min, the cells were pelleted by centrifugation, and washed twice with the PBS solution to remove the residual fixative. The hybridization buffer (0.9 M NaCl, 20 mM Tris-HCI pH 7.2, 0.01% SDS) and the probe stock to the final concentration of 5 ng/ μL were added. The hybridization was followed by three 20 min post-hybridization washes at 48◦C in the washing buffer (0.9 M NaCl, 20 mM Tris-HCI pH 7.2, 0.01% SDS). Cells were embedded on slides in Mowiol 4-88 mounting medium (Sigma, St. Louis, MO, USA), prepared as described in Cold Spring Harbor protocols [25]. All experiments included a negative control without probes to test for autofluorescence. The slides were imaged with Leica TCS SP5 confocal laser scanning microscope in The Chromas Research Facility at Saint Petersburg State University.

## *2.5. Phylogenetic Analysis*

Seventy-nine individual sequences of 16S rRNA genes were used for the phylogenetic analysis of 36 *Rickettsiales*, *Holosporales*, and other related bacteria (see Table S1 for the accession numbers). Seventy-two sequences were obtained from GenBank [26] and seven more were extracted from assembled genomes [27] as follows: all GenBank sequences were used as *BLASTN* queries against seven genome assemblies, then the intervals overlapping high-scoring hits (alignment length > 1300) were extracted as an interval BED file, merged using *bedtools* v2.29.0 [28] (*bedtools merge*), and the corresponding sequence together with 500 bp flanking on each side was extracted using *bedtools getfasta*. Only Genbank sequences longer than 1200 bp were used.

The initial multiple alignment was constructed using *ssu-align* v0.1.1 [29] with the default settings and then filtered using *ssu-mask* v0.1.1, yielding a multiple sequence alignment (MSA) of 1397 bp (see Figure S2 for the structural analysis of the retained sites). The MSA was further analyzed using *BMGE* v1.12 [30], and the alignment was additionally trimmed to account for the shortest sequences; to this end, 154 bp at the 5' end and 164 at the 3' end were removed, resulting in the final multiple alignment of 1079 bp.

Sequence similarity was calculated from the trimmed MSA using a custom Perl script, and visualized using *ggplot2* [31] and *R*.

To select the model that best fits our data, *modeltest-ng* [32] was run on the trimmed MSA with the default parameters. All three criteria used by *modeltest-ng* (BIC, AIC, AICc) have indicated similar models: GTRGAMMAI was found to be the best model using BIC, and GTRGAMMAIX was selected using AIC/AICc. Therefore, the latter model was selected for the following analysis. RAxML v8.2.12 [33] was run on the MSA using "-m GTRGAMMAIX -f a -x 123 -N 1000 -p 456" options, generating 1000 bootstraps. The resulting phylogenetic tree was visualized using Interactive Tree of Life v4 [34]. The 16S rRNA of the 21 sequenced clones of "*Ca.* Gortzia yakutica" strain YA111-52 were deposited in the GenBank database under the accession numbers MT421875.1–MT421895.1. The resulting phylogenetic tree has clonal and outgroup sequences collapsed, while the complete tree is presented as Figure S3. Additionally, we have run the Bayesian inference of phylogeny using *MrBayes* v3.2.7 [35]. The tree topology comparison ("tanglegram") was generated using *Dendroscope* [36], and is available as Figure S4.

Exact commands used in the analysis, the code to reproduce the visualization, and the analysis scripts are available at https://github.com/apredeus/yakutica.
