1. IntroductionSponges (phylum Porifera) are ancient, multicellular metazoans [1]. These organisms are sessile, filter feeding invertebrates living in marine and freshwater ecosystems and contain symbiotic microorganisms such as bacteria, viruses, archaea, cyanobacteria, microalgae, fungi, and protozoa [2,3,4,5,6,7,8,9]. Freshwater sponges (Demospongiae, Spongillida, and Lubomirskiidae) dominate the fauna of the littoral zone of Lake Baikal [10,11]. Endemic Baikal sponges live in symbiosis with various species of eukaryote and prokaryote microorganisms, including chlorophyll-producing algae [12,13,14]. In recent years, several reports were published on diseases and the mortality of endemic sponges in Lake Baikal [15,16,17]. The number of L. baikalensis sponges that are most susceptible to disease has decreased significantly [16]. Annually, up to 10–20% of diseased sponges are observed to die during the winter period [16]. Currently, diseased and dying sponges have been observed in many Lake Baikal areas [15,16]. However, the etiology and ecology of the disease and death of sponges remains unknown.Earlier, an analysis of 16S rRNA gene amplicons revealed a significant increase in the number of opportunistic microorganisms, including Betaproteobacteria of the Oxalobacteraceae family, in diseased freshwater sponges and in the cell culture of primmorphs [17,18]. Moreover, we isolated, sequenced, and analyzed the genome of the strain Janthinobacterium sp. SLB01 from diseased sponges collected from Lake Baikal [19,20,21]. It is known that the bacteria of the Janthinobacterium sp. strain SLB01 can grow, form biofilms, and produce the violet pigment violacein and has proteolytic, lipolytic, and saccharolytic activities. In addition, bacteria contains genes encoding violacein, alpha-amylases, phospho-lipases, a type VI secretion system, and proteins for the synthesis and secretion of polysaccharides [19,21]. The strain Janthinobacterium sp. SLB01 is a psychrotolerant, violacein–producing, rod-shaped, Gram-negative, and aerobic bacterium that contains violacein pigment and can form a floc [19]. We identified five genes encoding the vioA, vioB, vioC, vioD and vioE of violacein biosynthesis, which may be a pathogenic factor in the bacteria, similar to the identified genes in the published J. lividum MTR strain [21,22]. It is known that the bacteria produce the pigment violacein, which is a compound with antibiotic and antiviral properties [23,24]. In addition, it is known that the genus Janthinobacterium has a wide occurrence, including in soil, aquatic sites, marine habitats, and high-altitude environments, with a unique ability to survive and colonize new environments [25,26,27] In the strain Janthinobacterium sp. SLB01, we found key genes of the type VI secretion system (T6SS) [19], which is considered a virulence factor in many Proteobacteria [28]. Moreover, these genes for floc formation, including an operon for the biosynthesis of exopolysaccharides and an operon containing genes of PEP-CTERM/XrtA system glycosyltransferase, the PEP-CTERM system histidine kinase PrsK, and PEP-CTERM-box response regulator transcription factor prsR are homologous to the described genes for floc formation in Zoogloea resiniphila [29].It was shown that the genome of the strain Janthinobacterium sp. SLB01 contains virulence factors [21]. Therefore, we conducted a test of the effect of this strain on the cell culture of primmorphs from the healthy sponge L. baikalensis including reisolation, sequencing, and analysis of the genome of Janthinobacterium sp. The pathogenicity of the strain Janthinobacterium sp. SLB01 for Baikal sponges and primmorphs is not known. The purpose of this study is to show that the strain Janthinobacterium sp. SLB01 isolated from the diseased sponge L. baikalensis is a pathogen for the cell culture of primmorphs. This research will help clarify the etiological factors of the diseases and mortality among Baikal sponges. 2. Materials and Methods 2.1. Sampling of Sponges and Cell Culture of PrimmorphsSpecimens of the healthy sponge L. baikalensis Pallas, 1776 (Demospongiae, Haplosclerida, Spongillida, Lubomirskiidae) were collected by scuba divers in individual containers from Lake Baikal in the Olkhon Gate Strait, Central Siberia, Russia (53°02′21″ N; 106°57′37″ E), at a depth of 10 m (water temperature 3–4 °C). The cell cultures of primmorphs were obtained via the mechanical dissociation of cells according to the previously described technique [30]. A clean sponge sample was crushed, and the obtained cell suspension was subsequently filtered through sterile 200-, 100-, and 29 μm nylon meshes. The gel-like suspension was diluted 10-fold with Baikal water, placed in a refrigerator, and stored for 3 min at 3–6 °C until a dense precipitate formed. Healthy primmorphs were placed into 200–500 mL cultural bottles (Nalge Nunc International, Rochester, NY, USA). Cell cultures of primmorphs were cultivated in natural Baikal water (NBW) at 3–4 °C under illumination with a light intensity of 47 lx or 0.069 W with a 12 h cycle of day and night changes for a month. The cell culture of primmorphs was then used for experimental infection. 2.2. Bacteria IsolationIn this study, we used the strain Janthinobacterium sp. SLB01 isolated from a sample of the diseased sponge L. baikalensis, collected in Lake Baikal, Central Siberia, Russia [19] for subsequent experimental infection. Healthy primmorphs (diameters of 2–4 mm) were transferred to 24-well plates (Nalge Nunc International, Rochester, NY, USA), with one piece per well in 2 mL of NBW, and infected with the Janthinobacterium sp. strain SLB01 with an initial dose of bacteria of 2.5 × 104 CFU/mL in 50 μL. The infection was repeated at least three times. The infected primmorphs were cultivated at 3–6 °C with a 12-h day and night cycle for 14 days. During the infection of primmorphs, observations were carried out with daily descriptions and sampling for DNA isolation and sequencing. The cell suspension from the infected primmorphs was then homogenized and filtered using an MF-Millipore membrane filter of 0.45 µm pore size (Merck, Zug, Switzerland), and 10 µL was transferred to the nutrient medium. The bacteria were cultured on a nutrient media with R2A (0.05% yeast extract, 0.05% tryptone, 0.05% casamino acids, 0.05% dextrose, 0.05% soluble starch, 0.03% sodium pyruvate, 1.7 mM K2HPO4, 0.2 mM MgSO4, final pH 7.2 adjusted with crystalline K2HPO4 or KH2PO4) agar plates (Merck KGaA, Darmstadt, Germany) at pH 7.2. The dishes were inoculated with three repetitions and cultivated at a temperature of 22 °C for 5 days, with the growth of the strain observed daily. 2.3. Microscopy

We observed daily changes in infected cell cultures of primmorphs with Janthinobacterium sp. strain SLB01 over 14 days. The samples were stained with a NucBlue Live ReadyProbes reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA). The cell morphology was determined via light microscopy on an Axio Imager Z2 microscope (Zeiss, Oberkochen, Germany) equipped with fluorescence optics (self-regulating, blue HBO 100 filter, 358/493 nm excitation, 463/520 nm emission).

The samples were prepared for Scanning Electron Microscopy (SEM) analysis. Fixation was performed according to the following procedure: pre-fixation in 1% OsO₄ (10 min), washing in a cacodylate buffer (30 mM, pH 7.9), (10 min), fixation in 1.5% glutaraldehyde solution on a cacodylate buffer (30 mM, pH 7.9), (1 h), washing in a cacodylate buffer (30 mM, pH 7.9), (30 min), postfixation in 1% OsO₄ solution on a cacodylate buffer (30 mM, pH 7.9) (2 h), and washing in filtered Baikal water for 15 min at room temperature followed by dehydration in a graded ethanol series. The specimens were placed into SEM stubs, dried to a critical point, and coated with liquid carbon dioxide (BalTec CPD 030) using a Cressington 308 UHR sputter coater before examination under a Sigma series scanning electron microscope (Zeiss, Oberkochen, Germany) operating at 5.0 190 kV.

The samples were prepared for Transmission Electron Microscope (TEM) analysis. We took both healthy primmorphs and infected primmorphs for 24 h, 3 and 7 days with the strain Jantinobacterium sp. SLB01. The samples were fixed with 2.5% glutaraldehyde in a 0.1 M cacodyllate buffer (pH 7.2) for 24 h at 4 °C. The material was then washed in a 0.1 M cacodyllate buffer (pH 7.2) 3 times for 1 h, and 1% OsO4 diluted in 0.1 M cacodyllate buffer (pH 7.2) was fixed for 30 min. After washing from the fixative in distilled water (3 times for 30 min), the material was dehydrated in a series of increasing concentrations of ethanol and acetone. Next, the material was embedded in a mixture of Epon and Araldite (Sigma, Missouri, MO, USA). Semi-thin and ultra-thin sections were made using a Leica UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany). Ultrathin sections were contrasted with a 0.5% aqueous solution of uranyl acetate (20 min) and Reynolds lead citrate (10 min). Ultrathin sections were analyzed using a Libra 200 FE transmission electron microscope (Carl Zeiss, Oberkochen, Germany) and a Libra 120 (Carl Zeiss, Oberkochen, Germany).

2.4. Genome SequencingGenomic DNA for full-genome sequencing was isolated following a standard bacterial DNA isolation cetyltrimethylammonium bromide (CTAB) protocol (https://jgi.doe.gov/wp-content/uploads/2014/02/JGI-Bacterial-DNA-isolation-CTAB-Protocol-2012.pdf, accessed on 10 September 2020). The sequence library was prepared with a Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA). Genomes were sequenced on the Illumina MiSeq platform using v2 paired-end chemistry (2 × 250 bp, 12,099,942 reads total). 2.5. Genome Assembly, Annotation and Phylogenetic RelationshipRaw read error correction and filtering with FastP tool were performed with default settings [31]. Genomes were assembled with SPAdes version 3.11.0 [32] using the default settings. Contigs from draft assembly with a length of more than 10 Kbp were scaffolded with Ragout version 2.2 with default settings [33] (https://github.com/fenderglass/Ragout, accessed on 9 March 2021) using Janthinobacterium sp. LM6 chromosome (GenBank accession no. CP019510) as the reference. We used the same software set for genome assembly and annotation to prevent genome variations depending on reference and assembly software versions. Although the prokaryotic genome annotation pipeline (PGAP) version for annotating the genome Janthinobacterium sp. strain SLB01 was 4.13 (used when the genome was released in NCBI RefSeq), to annotate Janthinobacterium sp. PLB02, we used the newest available NCBI PGAP version (5.1), which can annotate more genes because its database is richer. Gene annotations were performed using PGAP (https://github.com/ncbi/pgap, accessed on 9 March 2021). Core genome construction was accomplished with Roary version 3.13.0 using default settings [34]. Genome completeness analysis was performed with benchmarking universal single-copy orthologs (BUSCO) version 5.0.0 using the dataset “burkholderiales_odb10” [35]. Strains of Janthinobacterium spp. identification was carried out via phylogenetic analysis with PhyloPhlAn 3.0 [36] based on a comparison of 400 universal marker genes (a maximum-likelihood method) [37] using the “supermatrix_aa” and “low diversity” modes with the “phylophlan” database. We acquired 10 closely related strains of Janthinobacterium by 16S rRNA from the Basic Local Alignment Search Tool (BLAST/-NCBI) to build a phylogenetic tree. 2.6. Statistical Analysis

All of the infection experiments were performed at least three times. The data were reported as the means ± standard deviation (SD). A statistical analysis was then carried out (single-factor (ANOVA) followed by Tukey’s multiple range test) using the SPSS.16 software. Differences in mean values were considered significant at p < 0.05.

4. Discussion

In this study, we showed that the strain Janthinobacterium sp. SLB01 isolated from a diseased sponge L. baikalensis and infected cell culture of primmorphs is the same and that the genomes of the strains are identical. The strains Janthinobacterium sp. SLB01 and Janthinobacterium sp. PLB02 are pathogens for cell cultures of primmorphs and the sponge L. baikalensis. After experimental infection of the cell culture of primmorphs, we found that short rod-shaped bacteria of the strain Janthinobacterium sp. SLB01 grew quickly and parasitized sponge cells and their symbiotic microalgae. We detected the death of the symbiotic microalgae (Chlorophyta) and the sponge cells in the infected primmorphs, as well as increased bacteria counts.

The bacteria Janthinobacterium sp. was found in the mesohyl of cell cultures of primmorphs 24 h after infection and was able to lyse the primmorph cells. The characteristic features of the structure of Janthinobacterium sp. during the development of the infectious process included the presence of a folding outer membrane, an increase in the periplasm, and an electron-transparent zone of lysis around the bacterial cells. It is known that the outer cell membrane and periplasm of Gram-negative bacteria serve as the compartments responsible for the production of secondary metabolites, including proteolytic enzymes and other factors of bacterial cell virulence [39,40]. An increase in the surface area of the outer membrane and the volume of the periplasm in Janthinobacterium sp. over the course of infection indicates the activation of processes aimed at realizing their pathogenic potential. We observed that the Janthinobacterium sp. penetrated the cytoplasm of microalgae and lysed their contents, using nutrients for growth, division, and the formation of colonies of the bacteria. The infection process progressed in the sponge cells of the primmorphs and the microalgae and reached the terminal stage on the day 7 of infection, thus indicating a rapid course of the pathogenic process. We observed the destruction of the photosynthetic apparatus, the loss of chlorophyll autofluorescence, and the death of symbiotic microalgae in all the infected primmorphs. Earlier, we showed that the cell culture of the primmorphs of healthy sponge L. baikalensis is identical to that of sponges, and can be used as a model system for studying the diseases of Baikal sponges [18]. Here, we showed that during the experimental infection of the cell culture of primmorphs with the strain Janthinobacterium sp. SLB01, the bacteria attacked eukaryotic cells of the microalgae and then acquired the released nutrients after cell lysis (Figure 3F,G).A comparison of the two genomes from Janthinobacterium sp. SLB01 and Janthinobacterium sp. PLB02 isolated from the diseased sponge and infected cell cultures of the primmorphs showed that genomes of these bacteria have identical genomic content. The genome sizes, gene counts, and G+C content were very close. The genome size of Janthinobacterium strains slightly differed due to the number of Ns (unknown nucleotides) after the scaffolding procedure. Moreover, we found that these species are rod-shaped Gram-negative bacteria that produce violacein, a compound with antimicrobial and antiviral properties that is toxic to eukaryotic cells [41]. The isolated bacteria Janthinobacterium sp. PLB02 can colonize the space and possibly suppress the grown microalgae with the pigment violacein. This pigment production was observed in the infected primmorphs, and all the genes (operon vioABCDE) were present in its genome. We identified five genes encoding VioA, VioB, VioC, VioD, and VioE proteins related to violacein biosynthesis similar to those identified in published Janthinobacterium sp. SLB01. Earlier, we observed that one essential strategy of the Jantinobacterium sp. strain SLB01 is the secretion of virulence factors through the cell membranes of the victim to achieve a potential target [21,22]. In addition, an identical T6SS secretion system of the strain Jantinobacterium sp. SLB01 was found in the isolated Janthinobacterium sp. PLB02. Both strains’ genomes contained all three categories of genes required for the function of type T6SS [42,43]. Bacterial strains Janthinobacterium spp. SLB01 and PLB02, based on a comparison of complete genomes, showed similarity with the strain J. lividum MTR. Interestingly, J. lividum either caused necrosis on mushroom tissue blocks or colonized the skin of some amphibians, conferring protection against fungal pathogens [27,44]. In addition, isolated bacteria also produced floc formation and strong biofilm in the stationary phase. When cultivating the strains Janthinobacterium sp. SLB01 and Janthinobacterium sp. PLB02, we observed biofilm and floc formation in the diseased sponges and the infected cell cultures of primmorphs of L. baikalensis. A genomic analysis of the two strains found RpoN, PepA, XrtA, PrsK, and PrsR gene clusters present in the formation of floc and 100% similarity between the strains (Table 2). Using an ultrastructural analysis, we found that the symbiotic microalgae were completely enclosed in a thick microbial biofilm during the infection of primmorphs with the strain Jantinobacterium sp. SLB01 (Figure 2B). Moreover, on day 7 after infection, it was discovered that the formation of bacterial colonies was accompanied by utilization of the components of the microalgal cytoplasm; there remained only a polysaccharide shell with bacteria enclosed in it (Figure 3G). Thus, floc formation and biofilm can negatively affect the physiology of the life of the host (sponge L. baikalensis) due to clogging of the pores. These negative effects of biofouling on the functioning of the filter-feeding marine sponge Halisarca caerulea were previously reported [45]. Exopolysaccharides (EPS) are known to be the main component of the biofilm produced by the species of Oxalobacteraceae [46]. It is known that the family Oxalobacteraceae is characterized by the presence of extremely ecologically diverse species of microorganisms and contains environmental saprophytic organisms, phytopathogens, and opportunistic pathogens, including those common in freshwater ecosystems [47]. The genomes of many environmental isolates of Janthinobacterium from ice, water, sediments, and soils were sequenced [25,26,27], but strains of Janthinobacterium sp. strain SLB01 and the new Janthinobacterium sp. strain PLB02 from the Baikal sponge and cell culture of primmorphs were isolated in this study for the first time.The disease and mass mortality of sponges and corals have been observed worldwide in the marine environment in recent years [48,49,50,51,52], and corresponding die-off events threaten overall sponge-associated biodiversity [53,54,55,56]. These changes in sponge–microbe interactions appear to be associated with climate change and the occurrence of opportunistic infections resulting from changes in water temperature caused by global warming, light intensity, and salinity [57,58,59,60,61,62]. Previously, Webster et al., presented a description of the pathogenic bacterial strain NW4327 isolated from an infected marine sponge Rhopaloeides odorabile in the Great Barrier Reef [63]. Choudhury et al. reported the isolation of the pathogenic bacterial strain of Pseudoalteromonas agarivorans found in diseased sea sponges with pathogenicity genes [64].

Thus, in this study, we sought to reproduce Koch’s postulates with a cell culture of primmorphs. The present study is the first of its kind. We were able to isolate the new strain of Janthinobacterium sp. PLB02 after infecting a cell culture of primmorphs using the strain Janthinobacterium sp. SLB01 isolated from a diseased sponge L. baikalensis. We found that the strains are the same and have virulence factors in their genomes. We showed interactions of the Janthinobacterium sp., marking this species as a potential pathogen for cell cultures of primmorphs of the Baikal sponge L. baikalensis. The results of this study will help expand our understanding of microbial interactions in the development of disease and the death of Baikal sponges.