Janthinobacterium sp. Strain SLB01 as Pathogenic Bacteria for Sponge Lubomirskia baikalensis

Sponges (phylum Porifera) are ancient, marine and inland water, filter feeding metazoans. In recent years, diseased sponges have been increasingly occurring in marine and freshwater environments. Endemic freshwater sponges of the Lubomirskiidae family are widely distributed in the coastal zone of Lake Baikal. The strain Janthinobacterium sp. SLB01 was isolated previously from the diseased sponge Lubomirskia baikalensis (Pallas, 1776), although its pathogenicity is still unknown. The aim of this study was to confirm whether the Janthinobacterium sp. strain SLB01 is the pathogen found in Baikal sponge. To address this aim, we infected the cell culture of primmorphs of the sponge L. baikalensis with strain SLB01 and subsequently reisolated and sequenced the strain Janthinobacterium sp. PLB02. The results showed that the isolated strain has more than 99% homology with strain SLB01. The genomes of both strains contain genes vioABCDE of violacein biosynthesis and floc formation, for strong biofilm, in addition to the type VI secretion system (T6SS) as the main virulence factor. Based on a comparison of complete genomes, we showed the similarity of the studied bacterial strains of Janthinobacterium spp. with the described strain of Janthinobacterium lividum MTR. This study will help expand our understanding of microbial interactions and determine one of the causes in the development of diseases and death in Baikal sponges.

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.

Sampling of Sponges and Cell Culture of Primmorphs
Specimens 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.

Bacteria Isolation
In 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 × 10 4 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 K 2 HPO 4 , 0.2 mM MgSO 4 , final pH 7.2 adjusted with crystalline K 2 HPO 4 or KH 2 PO 4 ) 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.

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% OsO 4 (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% OsO 4 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% OsO 4 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).

Genome Assembly, Annotation and Phylogenetic Relationship
Raw 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  [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.

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.

Bacterial Isolation and Microscopy
We used a model cell culture of healthy primmorphs of sponge L. baikalensis for experimental infection with the Janthinobacterium sp. strain SLB01 isolated from a diseased sponge, L. baikalensis, to determine the bacterial pathogenicity. The healthy primmorphs were bright green in color and with bright red autofluorescence of chlorophyll in the cells due to the presence of green symbiotic microalgae belonging to the taxon Chlorophyta in their composition. We observed that the cells of the sponges contained a strict arrangement of symbiotic microalgae in the amoebocytes of the uninfected primmorphs ( Figure 1A,B).
A completely different picture was observed in primmorphs infected with the Janthinobacterium sp. strain SLB01. Dirty scurf, a fetid odor, and biofilm formation were observed in the infected cultures, which were likely associated with the growth of bacteria. The primmorphs lost their green color after infection with the strain Janthinobacterium sp. SLB01 ( Figure 1C). We observed the destroyed cells of amoebocytes and a chaotic arrangement and adhesion of microalgae. On the third day of cultivation of primmorphs infected with Janthinobacterium sp. strain SLB01, we observed the suppression of autofluorescence ( Figure 1D). After 7 days of cultivation of the infected culture of primmorphs, we observed dead cells of sponges and a chaotic arrangement of microalgae with an increase in the number of short rod-shaped bacteria ( Figure 1E,F). The loss of chlorophyll autofluorescence and the death of microalgae were observed in all experimental samples. A completely different picture was observed in primmorphs infected with thinobacterium sp. strain SLB01. Dirty scurf, a fetid odor, and biofilm formation w served in the infected cultures, which were likely associated with the growth of The primmorphs lost their green color after infection with the strain Janthinobacte SLB01 ( Figure 1C). We observed the destroyed cells of amoebocytes and a chaotic ment and adhesion of microalgae. On the third day of cultivation of primmorphs with Janthinobacterium sp. strain SLB01, we observed the suppression of autofluo ( Figure 1D). After 7 days of cultivation of the infected culture of primmorphs, we o dead cells of sponges and a chaotic arrangement of microalgae with an increas The autofluorescence of chlorophyll-containing intracellular microalgae is shown in red (C,D) chaotic arrangements of green microalgae and increases in bacteria in primmorphs infected with Janthinobacterium sp. strain SLB01 on day 3 of cultivation. Bacteria are shown with a blue color (indicated by the arrows); (E,F) the primmorphs infected with Janthinobacterium sp. strain SLB01 on day 7 of bacteria cultivation (blue color; shown by arrows). The samples of primmorphs were stained with the NucBlue Live ReadyProbes reagent for fluorescence microscopy. Scale bars: 10 µm.
Using SEM, we found that the microalgae contained spheroidal cells 2.5-3.0 µm in diameter with a clean cell wall in the healthy primmorphs ( Figure 2A). However, we experimentally observed the interaction of bacteria with host cells in the primmorphs infected with Janthinobacterium sp. strain SLB01 ( Figure 2B). The squa mous epithelium was destroyed, and the symbiotic microalgae were packed entirely in thick microbial biofilm with short rod-shaped bacteria ( Figure 2B).
We observed an interaction of bacteria with the host cells in the primmorphs and symbiotic microalgae infected with the Janthinobacterium sp. strain SLB01 through the us of ultrastructural analysis ( Figure 3).  However, we experimentally observed the interaction of bacteria with host cells in the primmorphs infected with Janthinobacterium sp. strain SLB01 ( Figure 2B). The squamous epithelium was destroyed, and the symbiotic microalgae were packed entirely in a thick microbial biofilm with short rod-shaped bacteria ( Figure 2B).
We observed an interaction of bacteria with the host cells in the primmorphs and symbiotic microalgae infected with the Janthinobacterium sp. strain SLB01 through the use of ultrastructural analysis ( Figure 3).
We found that amoebocyte cells were filled with green symbiotic microalgae in the healthy primmorphs ( Figure 3A). The amoebocyte cells were up to 20 µm in diameter, containing a nucleus with a prominent nucleolus. The cytoplasm of amoebocytes contains dictyosomes of the Golgi apparatus and cisterns of the endoplasmic reticulum. A distinctive feature of amoebocytes is the presence in the cytoplasm of specialized vacuolessymbiosomes with symbiotic microalgae representatives of the Chlorophyceae family enclosed in them. Microalgal cells (2.5-3.0 µm in diameter) have a thin electron-dense polysaccharide envelope separated from the cell's outer membrane by a narrow supramembrane space ( Figure 3B). There is also a chloroplast, which can contain electron-transparent inclusions that are, most notably, starch grains. Granules are often present between the thylakoid membranes and directly in the cytoplasm of the microalgae ( Figure 3B). In addition, it was noted that no bacteria were found in the mesohyl of healthy primmorphs. In the mesohyl of infected cell cultures, rod-shaped bacteria were found in the primmorphs 24 h after infection ( Figure 3D). The structure of the bacteria had an enlarged folded outer membrane and there was an electron-transparent halo observed around the bacterial cells which indicated their ability to lyse the surrounding components of the extracellular matrix. In addition to mesohyl, bacteria were present in amoebocytes, which penetrated via phagocytosis. The system of intracellular membranes was destroyed, and vacuolization was enhanced. The symbiosomes with microalgae enclosed in them were preserved in the cytoplasm of amoebocytes. Some symbiotic microalgae left the host cells and were located within the extracellular matrix. The destruction processes of cells of primmorphs reached the terminal stage on day 7 after infection ( Figure 3E). The microalgae were located directly in the mesoglea, where they became available for the action of bacteria on day 7 after the start of the infection. The cytoplasm in the cells of primmorphs was fragmented, resulting in the absence of whole functionally active cells. We observed that the extracellular matrix also contained symbiotic microalgae infected with bacteria, which, due to division, subsequently formed colonies of bacteria united by contact processes ( Figure 3F). The formation of bacterial colonies was accompanied by the lysis of the components of the microalgal cytoplasm, with the presence of a polysaccharide shell enclosing bacteria ( Figure 3G). Thus, as the infection progressed, the cells of primmorphs became lysed, and microalgae were served on the 14th day of cultivation on the surfaces of symbiotic microalgae. Scale bars: 2 However, we experimentally observed the interaction of bacteria with host the primmorphs infected with Janthinobacterium sp. strain SLB01 ( Figure 2B). Th mous epithelium was destroyed, and the symbiotic microalgae were packed enti thick microbial biofilm with short rod-shaped bacteria ( Figure 2B).
We observed an interaction of bacteria with the host cells in the primmor symbiotic microalgae infected with the Janthinobacterium sp. strain SLB01 through of ultrastructural analysis (Figure 3).  The strain Janthinobacterium sp. PLB02 was isolated from a cell culture of primmorphs infected with the Janthinobacterium sp. strain SLB01. The bacteria were rod-shaped, motile, and aerobic; in addition, the purple pigment violacein appeared on the second day. A morphological analysis showed that the bacteria have short rods up to 2.0 µm long and 0.3 µm in diameter, with a two-layer outer membrane typical of Gram-negative bacteria ( Figure 3C). The cytoplasm, in most cases, was granular, flagellated and electron-dense, with a well-defined nucleoid zone.

Comparison of Genomes of Janthinobacterium spp. Strains
In the present study, we compared the genomic contents of two strains: Janthinobacterium sp. SLB01 and reisolated Janthinobacterium sp. PLB02. We assembled the genome of the Janthinobacterium sp. strain PLB02 from sequence data the same way as was done for the Janthinobacterium sp. SLB01 strain. The final genome assembly statistics of the raw read count, genome size, number of genes, pseudogenes, protein-coding sequences, tRNA noncoding RNA, and references to genome reports are presented in Table 1. A genome completeness analysis with benchmarking universal single-copy orthologs (BUSCO) [35] showed results for the strains Janthinobacterium spp. SLB01 and PLB02, with 99.1% complete (not fragmented) and 0.9% missing BUSCOs. The fully assembled genomes included 6,467,981 bp for strain Janthinobacterium sp. SLB01 and 6,417,505 bp for strain Janthinobacterium sp. PLB02, and exhibited similar G+C contents (62.63% and 62.65%, respectively). Genome annotation with PGAP revealed 5643 genes (5502 proteincoding) for strain SLB01 and 5651 (5510 protein-coding) for strain PLB02, as shown in Table 1. We compared the genomic contents of the Janthinobacterium sp. strain SLB01 and Janthinobacterium sp. strain PLB02 with the data of Roary [34] and found that most of the genes were the same (with a homology of more than 99%).

Phylogenetic Relationship
We built a phylogenetic tree for the Janthinobacterium species to compare the genomic features of both strains-Janthinobacterium spp. SLB01 and PLB02-with closer species [38]. Strain Janthinobacterium sp. PLB02 showed the highest phylogenetic affiliation to strain Janthinobacterium sp. SLB01 of phylum Proteobacterium from the family Oxalobacteraceae ( Figure 4). The result of the phylogenetic tree based on 400 universal marker genes using loPhlAn (a maximum-likelihood method) [35] showed that the genomes of Janthinob rium sp. SLB01 and Janthinobacterium sp. PLB02 are homologous to each other and closely related to the psychrotolerant strain J. lividum MTR (Figure 4).

Analysis of the Virulence Genes
We compared the obtained genomes of strains of the Janthinobacterium sp. PLB02 Janthinobacterium sp. SLB01 with each other and analyzed the coding virulence pro and key genes such as the genes of violacein, floc formation, and the type VI secre system [21]. We found that the strains were 100% homologous to each other in term virulence factors. Earlier, we showed that the strain Janthinobacterium sp. SLB01 was to produce violacein and contained the violacein synthesis operon vioABCDE [21,22] lated from primmorphs, the strain Janthinobacterium sp. PLB02 also produced the pigm violacein, and the genome contained the violacein synthesis operon vioABCDE. The fl ing regions for gene coordinates and locus names are presented in Figure 5 and Tabl  The result of the phylogenetic tree based on 400 universal marker genes using Phy-loPhlAn (a maximum-likelihood method) [35] showed that the genomes of Janthinobacterium sp. SLB01 and Janthinobacterium sp. PLB02 are homologous to each other and very closely related to the psychrotolerant strain J. lividum MTR (Figure 4).

Analysis of the Virulence Genes
We compared the obtained genomes of strains of the Janthinobacterium sp. PLB02 and Janthinobacterium sp. SLB01 with each other and analyzed the coding virulence proteins and key genes such as the genes of violacein, floc formation, and the type VI secretion system [21]. We found that the strains were 100% homologous to each other in terms of virulence factors. Earlier, we showed that the strain Janthinobacterium sp. SLB01 was able to produce violacein and contained the violacein synthesis operon vioABCDE [21,22]. Isolated from primmorphs, the strain Janthinobacterium sp. PLB02 also produced the pigment violacein, and the genome contained the violacein synthesis operon vioABCDE. The flanking regions for gene coordinates and locus names are presented in Figure 5 and Table 2.
and key genes such as the genes of violacein, floc formation, and the type VI secretion system [21]. We found that the strains were 100% homologous to each other in terms of virulence factors. Earlier, we showed that the strain Janthinobacterium sp. SLB01 was able to produce violacein and contained the violacein synthesis operon vioABCDE [21,22]. Isolated from primmorphs, the strain Janthinobacterium sp. PLB02 also produced the pigment violacein, and the genome contained the violacein synthesis operon vioABCDE. The flanking regions for gene coordinates and locus names are presented in Figure 5 and Table 2.    1353472  3909260  1354779  3910567  100  1308  1308  vioB  F3B38_RS17240 J3P46_17340  1354776  3910564  1357796  3913584  100  3021  3021  vioC  F3B38_RS17245 J3P46_17345  1357798  3913586  1359087  3914875  100  1290  1290  vioD  F3B38_RS17250 J3P46_17350  1359087  3914929  1360205  3915993  100  1119  1065  vioE  F3B38_RS17255 J3P46_17355  1360216  3916004  1360216  3916585  100  582  582 We found 100% homology of the strains Janthinobacterium spp. SLB01 and PLB02. We used the generally accepted names of the genes of the violacein synthesis operon vioABCDE instead of the annotated names when comparing their genomes (Table 2).
Our previous study discovered gene clusters involved in floc formation [21,22]. We found that the clusters of genes of the Janthinobacterium sp. strain PLB02 have 100% structural similarity to the genome of the strain Janthinobacterium sp. SLB01 ( Figure 6).
Previously, we showed that the Janthinobacterium sp. strain SLB01 formed a strong biofilm rich in exopolysaccharides (EPS) in the stationary phase [21,22]. Interestingly, the isolated strain Janthinobacterium sp. PLB02 also formed a strong biofilm. The strain produced floc and biofilm via exopolysaccharide biosynthesis and PEP-CTERM/XrtA protein expression. A genome analysis showed that the Janthinobacterium sp. strain PLB02 has all the necessary gene cassettes for flocculation, similar to strain Janthinobacterium sp. SLB01. Both genomes contain the system glycosyltransferase putative exosortase XrtA (previously called EpsH), the PEP-CTERM system histidine kinase PrsK, the PEP-CTERM system associated sugar transferase, the sensor histidine kinase of a two-component system, and the PEP-CTERM-box response regulator transcription factor PrsR. Localization, annotation, and identity percentages of these genes are presented in Table 3.  vioD  F3B38_RS17250  J3P46_17350 1359087 3914929 1360205 3915993  100  1119 1065  vioE  F3B38_RS17255  J3P46_17355 1360216 3916004 1360216 3916585  100  582  582 Our previous study discovered gene clusters involved in floc formation [21,22]. We found that the clusters of genes of the Janthinobacterium sp. strain PLB02 have 100% structural similarity to the genome of the strain Janthinobacterium sp. SLB01 (Figure 6).   Sensor histidine kinase of a two-component system Moreover, we analyzed the type VI secretion system (T6SS) as the primary virulence factor in the genome of the strain Janthinobacterium sp. PLB02 and found that the genes of both strains were 100% identical to each other. As in the strain Janthinobacterium sp. SLB01, the genome of the isolated strain Janthinobacterium sp. PLB02 contained all three categories of genes required for the type VI secretion system to function. The isolated strain Janthinobacterium sp. PLB02 was identical to strain Janthinobacterium sp. SLB01.

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 Gramnegative 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.