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Bacteria of the genus Janthinobacterium are widespread in soils and freshwater ecosystems and belong to the phylum Proteobacteria. The Janthinobacterium sp. SLB01 strain was isolated from diseased freshwater Lubomirskia baicalensis (Pallas, 1776) sponge, and the draft genome was published previously. However, the properties of the SLB01 strain are not known. The aim of the study is to describe some properties of the Janthinobacterium sp. SLB01 strain, isolated from L. baicalensis sponge. The identification of the SLB01 strain was established as Gram-negative, motile, rod-shaped, and psychrotolerant, with growth at 3 and 22 °C. We found that the SLB01 strain has proteolytic, lipolytic, and saccharolytic activity and can use citrates and reduce nitrates. The bacteria Janthinobacterium sp. SLB01 strain can grow, form biofilms, and produce the violet pigment violacein. We identified the pigments violacein and deoxyviolacein by chromatography and mass spectrometry. These metabolites may be of interest to biotechnology in the future. The studied characteristics of the Janthinobacterium sp. SLB01 strain are an important addition to previous studies of the genome of this strain. This study will help us to understand the relationship between the microbial communities of Lake Baikal and sponges. [ABSTRACT FROM AUTHOR]

Bacteria of the genus Janthinobacterium are widespread in soils and freshwater ecosystems and belong to the phylum Proteobacteria. The Janthinobacterium sp. SLB01 strain was isolated from diseased freshwater Lubomirskia baicalensis (Pallas, 1776) sponge, and the draft genome was published previously. However, the properties of the SLB01 strain are not known. The aim of the study is to describe some properties of the Janthinobacterium sp. SLB01 strain, isolated from L. baicalensis sponge. The identification of the SLB01 strain was established as Gram-negative, motile, rod-shaped, and psychrotolerant, with growth at 3 and 22 °C. We found that the SLB01 strain has proteolytic, lipolytic, and saccharolytic activity and can use citrates and reduce nitrates. The bacteria Janthinobacterium sp. SLB01 strain can grow, form biofilms, and produce the violet pigment violacein. We identified the pigments violacein and deoxyviolacein by chromatography and mass spectrometry. These metabolites may be of interest to biotechnology in the future. The studied characteristics of the Janthinobacterium sp. SLB01 strain are an important addition to previous studies of the genome of this strain. This study will help us to understand the relationship between the microbial communities of Lake Baikal and sponges.

The morphological description of morphogenesis accompanying primmorph formation from cell aggregates in the endemic Baikal sponge Lubomirskia baicalensis as well as the description of cell composition of these primmorphs was investigated. Development of small sponges from dissociated cells in L. baicalensis is a stepwise process that can be separated into three stages: 1-primary aggregates; 2-early-stage primmorphs; and 3-primmorphs or young sponges. Primary aggregates are irregular in shape and consist of loosely packed cells. Early-stage primmorph development is characterized by cell sorting, aggregate compactization, surface epithelization and cell differentiation. Primmorphs or young sponges have developed elements of the aquiferous system. The main morphogenesis occurring during primmorph development in L. baicalensis is the epithelization of the cell aggregate surface accompanied by the flattening of poorly connected spherical cells. The main sources of new exopinacoderm development during L. baicalensis primmorph formation are choanocytes and amoebocytes that transdifferentiate into exopinacocytes. The cellular composition of L. baicalensis primmorphs does not differ significantly when compared to intact sponges. The most abundant cells in both primmorphs and in intact sponges are amoebocytes and choanocytes. Our results provide a morphological basis for future investigations using primmorphs of this species as models in biotechnology, evo-devo, cytology and molecular biology approaches. [ABSTRACT FROM AUTHOR]

The diversity of 16S rRNA genes in the microbial community of endemic sponge Lubomirskia baicalensis with bleached patches of tissue was studied. Eight bacterial phyla were identified in the sponge microbiome: Cyanobacteria (27.3%; n = 36; 2 OTU, operational taxonomic unit), Proteobacteria (22.7%; n = 30; 5 OTU), Actinobacteria (16.7%; n = 22; 7 OTU), Verrucomicrobia (15.2%; n = 20; 4 OTU), Planctomycetes (9%; n = 12; 3 OTU), Bacteroidetes (4.5%; n = 6; 3 OTU), WS5 (3%; n = 4; 1 OTU), and TM7 (1.5%; n = 2; 1 OTU). The basic phyla typical for freshwater sponge microbiomes are present in the community. However, in contrast to previously studied L. baicalensis bacterial associations in the bleached sponge community, a dominance of Cyanobacteria and a low number of representatives of the Bacteroidetes and Betaproteobacteria were observed. Phylotypes exhibiting a high percentage of similarity with the microorganisms inhabiting substrates rich in organic matter were also found. Clearly, the bleaching processes of Baikal sponges affect the composition and the ratio of the major taxonomic groups of sponge-associated bacteria. [ABSTRACT FROM AUTHOR]

Bacterial diversity of two Lake Baikal endemic sponges characterized by different life forms, branching Lubomirskia baicalensis and encrusting Baikalospongia sp., was studied using 454 pyrosequencing of the 16S rRNA gene fragments. In the communities associated with L. baicalensis and Baikalospongia sp., 426 and 428 OTUs, respectively, were identified. In microbial associations of these sponges, 24 bacterial phyla with predominance of Bacteroidetes, Proteobacteria, and Actinobacteria were identified. Analysis of the taxonomic composition of bacterial communities of the sponges was carried out by searching the dominant phylotypes within the clusters of phylum level. Comparison of bacterial associations of the sponges with Lake Baikal bacterioplankton revealed both the shared OTUs and the unique ones characteristic of the studied species. [ABSTRACT FROM AUTHOR]

The first reports on the presence of zoochlorellae within the organelles of mesenchymal cells from freshwater sponges were published in the 19th century. Today, it is well-known that freshwater sponges can be found in association with different endosymbiotic algae. However, until now there has been no detailed information about the endosymbiotic chlorophyll-containing algae in the remarkable endemic green sponges from Lake Baikal. In our study we were able for the first time to isolate and identify endosymbionts from primmorphs cultivatedin vitro, and to compare them with those from naturally occurringLubomirskia baicalensissponges. Structural as well as molecular biological investigations show that the endosymbiotic alga is aMychonastesspecies closely related toM. huancayensis.Another novel aspect of our work was to show that it is possible to use primmorphs of endemic sponges for isolation and subsequent cultivation of their endosymbiotic algae. We employed a simple cold-water (3–4°C) approach for cultivatingMychonastessp., both within sponge primmorphs and in culture. [ABSTRACT FROM PUBLISHER]

The screening of metagenomic DNA of the microbial community associated with the Baikalian sponge Lubomirskia baicalensis was performed in order to investigate the presence of polyketide synthase (PKS) genes. PKS enzyme systems take part in the synthesis of a great number of biologically active substances. The cloning and sequencing of amplified products of the ketosynthase domain section of the PKS gene cluster revealed 15 fragments of PKS genes with amino acid sequences differing from each other by 35-65%. A BLASTX analysis showed that all of these sequences belong to KS domains identified in various groups of microorganisms, i.e., Alpha-, Beta-, and Deltaproteobacteria; Verrucomicrobia; Cyanobacteria; and Chlorophyta. Some sequences were related to genes that participate in the biosynthesis of curacin A ( CurI, CurJ), stigmatellin ( StiC, StiG), nostophycin ( NpnB), and cryptophycin ( CrpB). The homology of the found sequences with those of the EMBL database lies in the range of 50-82%, which indicates that the freshwater sponge community contains genes that encode new, not yet studied polyketide substances of potential biotechnological significance. [ABSTRACT FROM AUTHOR]

In this study, for the first time the diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis collected from the Sousern Basin of Lake Baikal was investigated employing cultivation-independent approaches. In total, 102 bacterial 16S rRNA clones were screened using restriction fragment length polymorphism (RFLP) and 30 were selected for sequencing. BLASTN and phylogenetic analysis based on near full length 16S rDNA sequences showed that 22 operational taxonomic units (OTUs) were clustered in six known phyla: Actinobacteria (8 OTUs), alpha-Proteobacteria (4 OTUs), beta-Proteobacteria (4 OTUs), Verrucomicrobia (4 OTUs), Nitrospiracea (1 OTU) and Bacteroidetes (1 OTU). Remarkably all phylotypes were affiliated to uncultured microorganisms, however, all alpha-Proteobacteria sequences were closely related to bacteria derived from the freshwater sponge Spongilla lacustris. Our results reveal a high diversity in the L. baicalensis bacterial community and provide an insight into microbial ecology and diversity within freshwater sponges inhabiting the ancient Lake Baikal ecosystem. [ABSTRACT FROM AUTHOR]

Sponges (Porifera) are among the simplest living and the earliest branching metazoans. They hold a pivotal role for studying genome evolution of the entire metazoan branch, both as an outgroup to Eumetazoa and as the closest branching phylum to the common ancestor of all multicellular animals (Urmetazoa). In order to assess the transcription inventory of sponges, we sequenced expressed sequence tag libraries of two demosponge species, Suberites domuncula and Lubomirskia baicalensis, and systematically analyzed the assembled sponge transcripts against their homologs from complete proteomes of six well-characterized metazoans—Nematostella vectensis, Caenorhabditis elegans, Drosophila melanogaster, Strongylocentrotus purpuratus, Ciona intestinalis, and Homo sapiens. We show that even the earliest metazoan species already have strikingly complex genomes in terms of gene content and functional repertoire and that the rich gene repertoire existed even before the emergence of true tissues, therefore further emphasizing the importance of gene loss and spatio-temporal changes in regulation of gene expression in shaping the metazoan genomes. Our findings further indicate that sponge and human genes generally show similarity levels higher than expected from their respective positions in metazoan phylogeny, providing direct evidence for slow rate of evolution in both “basal” and “apical” metazoan genome lineages. We propose that the ancestor of all metazoans had already had an unusually complex genome, thereby shifting the origins of genome complexity from Urbilateria to Urmetazoa. [ABSTRACT FROM PUBLISHER]

Abstract: Some sponges [phylum Porifera], e.g. the demosponges Lubomirskia baicalensis or Axinella polypoides, show an arborescent growth form. In the freshwater sponge L. baicalensis this morphotype is seen mostly in depths below 4m while in more shallow regions it grows as a crust. The different growth forms are determined in nature very likely by water current and/or light. The branches of this species are composed of modules, arranged along the apical–basal axis. The modules are delimited by a precise architecture of the spicule bundles; longitudinal bundles originate from the apex of the earlier module, while at the basis of each module these bundles are cross-linked by traverse bundles under formation of annuli. Genes encoding putative morphogenetic factors, myotrophin and epidermal growth factor (EGF)-like molecules, and one gene of an antagonist for the Wnt signaling pathway, the soluble frizzled molecule, have been identified and characterized. Their expression levels as well as those of silicatein, one major spicule-forming molecule, have been studied in the crusts and the modules. The data revealed that at the apices of each module higher level of expression of myotrophin and EGF can be detected, while the base of each module is characterized by a high steady-state expression level of soluble frizzled molecule. These results suggest that module formation in L. baicalensis is controlled by a tuned interaction of agonistic (e.g., myotrophin and EGF) as well as antagonistic morphogenetic factors (e.g., soluble frizzled molecule). [Copyright &y& Elsevier]

Abstract: Aquatic animals, especially filter feeders such as sponges [phylum Porifera], are exposed to a higher viral load than terrestrial species. Until now, the antiviral defense system in the evolutionary oldest multicellular organisms, sponges, is not understood. One powerful protection of vertebrates against virus infection is mediated by the interferon (IFN)-inducible 2′-5′-oligoadenylate synthetase [(2-5)A synthetase] system. In the present study we cloned from the freshwater sponge Lubomirskia baicalensis a cDNA encoding a 314 aa long ORF with a calculated size of 35748Da, a putative (2-5)A synthetase, and raised antibodies against the recombinant protein. The native enzyme was identified in a crude extract from L. baicalensis by application of a novel separation procedure based on polymer coated ferromagnetic nanoparticles. The particles were derivatized with a synthetic double-stranded RNA [dsRNA], synthetic poly(I:C), a known allosteric activator of the latent (2-5)A synthetase. These particles were used to separate a single 35kDa protein from a crude extract of L. baicalensis, which cross-reacted with antibodies raised against the sponge enzyme. In situ hybridization studies revealed that highest expression of the gene is seen in cells surrounding the aquiferous canals. Finally primmorphs, an in vitro cell culture system, from L. baicalensis were exposed to poly(I:C); they responded to this dsRNA with an increased expression of the (2-5)A synthetase gene already after a 1-day incubation period. We conclude that sponges contain the (2-5)A synthetase antiviral protection system. [Copyright &y& Elsevier]

Siliceous spicules of Baikal freshwater sponge Lubomirskia baicalensis contain several proteins, including silicateins. Analysis of a L. baicalensis cDNA library revealed four different mRNAs coding for proteins related to marine sponge silicatein α (α1, α2, α3, and α4). The intron-exon structure was determined forthe genomic α1 silicatein gene. The gene is 1988 bp from the initiation to the termination codon and consists of six intron (total size 1007 bp) and seven exons (total size 981 bp). Mass spectrometry of a tryptic digest of spicule proteins revealed peptides of two silicateins α. [ABSTRACT FROM AUTHOR]

Abstract: Silicateins are enzymes, which are restricted to sponges (phylum Porifera), that mediate the catalytic formation of biosilica from monomeric silicon compounds. The silicatein protein is compartmented in the sponges in the axial filaments which reside in the axial canals of the siliceous spicules. In the present study silicatein has been isolated from the freshwater sponge Lubomirskia baicalensis where it occurs in isoforms with sizes of 23 kDa, 24 kDa and 26 kDa. Since the larger protein is glycosylated we posit that it is a processed form of one of the smaller size forms. The silicatein isoforms are post-translationally modified by phosphorylation; at least four isoforms exist with pI''s of 5.4, of 5.2, of 4.9 and of 4.7. Surprisingly silicatein not only mediates polymerization of silicate, but also displays proteolytic activity which is specific for cathepsin L enzymes, thus underscoring the high relationship of the silicateins to cathepsin L. The cDNAs from L. baicalensis for silicatein and cathepsin L, as well as the respective genes, were cloned. It was found that the five introns present in the sponge genes are highly conserved up to human cathepsin L. This analysis has been completed by sequencing of two silicatein genes (both for silicatein-α and -β) and of cathepsin L from another demosponge, Suberites domuncula. A comprehensive phylogenetic analysis with these new sequences shed new light upon the evolution of cathepsin L and silicatein families which occurred at the base of the metazoan phyla. It is concluded, that in parallel with the emergence of these enzymes at first the number of introns increased, especially in the coding region of the mature enzyme. Later in evolution the number of introns decreased again. We postulate that modification of the catalytic triad, especially of its first amino acid, is a suitable target for a chemical modulation of enzyme function of the silicateins/cathepsin L. [Copyright &y& Elsevier]

The endemic freshwater sponge Lubomirskia baicalensis lives in Lake Baikal in winter (samples from March have been studied) under complete ice cover at near 0°C, and in summer in open water at 17 °C (September). In March, specimens show high metabolic activity as reflected by the production of gametes. L. baicalensis lives in symbiosis with green dinoflagellates, which are related to Gymnodinium sanguineum. Here we show that these dinoflagellates produce the toxin okadaic acid (OA), which is present as a free molecule as well as in a protein-bound state. In metazoans OA inhibits both protein phosphatase-2A and protein phosphatase-1 (PP1). Only cDNA corresponding to PP1 could be identified in L. baicalensis and subsequently isolated from a L. baicalensis cDNA library. The deduced polypeptide has a molecular mass of 36 802 Da and shares the characteristic domains known from other protein phosphatases. As determined by western blot analysis, the relative amount of PP1 is almost the same in March (under ice) and September (summer). PP1 is not inhibited by low OA concentrations (100 nm); concentrations above 300 nm are required for inhibition. A sponge cell culture system (primmorphs) was used to show that at low temperatures (4 °C) expression of hsp70 is strongly induced and hsp70 synthesis is augmented after incubation with 100 nm OA to levels measured at 17 °C. In the enriched extract, PP1 activity at 4 °C is close to that measured at 17 °C. Immunoabsorption experiments revealed that hsp70 contributes to the high protein phosphatase activity at 4 °C. From these data we conclude that the toxin OA is required for the expression of hsp70 at low temperature, and therefore contributes to the cold resistance of the sponge. [ABSTRACT FROM AUTHOR]

The freshwater sponge Lubomirskia baicalensis (from Lake Baikal) is characterized by a body plan composed of serial modules which are arranged along an apical–basal axis. In shallow water, the sponge occurs only encrusting, while in deeper environment (>3 m), this species forms branches and grows in an arborescent manner. Each module is stabilized by bundles of spined oxeas (amphioxeae spicules). The spicules are surrounded by an organic matrix. cDNAs for structural proteins (silicatein and mannose-binding lectin (MBL)) as well as for one regulatory protein (mago nashi) were isolated from L. baicalensis. Surprisingly the silicatein α molecule exists in several, at least four, isoforms (a1 to a4). Expression studies revealed that the steady-state levels of transcripts for the silicateins, the mannose-binding lectin, and mago nashi are highest at the top of the branches, while only very low levels are found in cells at the base. Based on in situ hybridization studies, evidence is presented that the spicule formation (1) starts and is completed inside of the bundles, and (2) occurs together with the mannose-binding lectin from the surfaces of the bundles. The data suggest that the modules are sequentially formed. It is speculated that the expression of the silicateins and the mannose-binding lectin might be (partially) controlled by mago nashi. [ABSTRACT FROM AUTHOR]

Abstract: The skeletal elements (spicules) of the demosponge Lubomirskia baicalensis were analyzed; they are composed of amorphous, non-crystalline silica, and contain in a central axial canal the axial filament which consists of the enzyme silicatein. The axial filament, that orients the spicule in its longitudinal axis exists also in the center of the spines which decorate the spicule. During growth of the sponge, new serially arranged modules which are formed from longitudinally arranged spicule bundles are added at the tip of the branches. X-ray analysis revealed that these serial modules are separated from each other by septate zones (annuli). We describe that the longitudinal bundles of spicules of a new module originate from the apex of the earlier module from where they protrude. A cross section through the oscular/apical–basal axis shows that the bundle rays are organized in a concentric and radiate pattern. High resolution magnetic resonance microimaging studies showed that the silica spheres of the spicules in the cone region contain high amounts of ‘mobile’ water. We conclude that the radiate accretive growth pattern of sponges is initiated in the apical region (cones) by newly growing spicules which are characterized by high amounts of ‘mobile’ water; subsequently spicule bundles are formed laterally around the cones. [Copyright &y& Elsevier]

Endemic freshwater sponges (demosponges, Lubomirskiidae) dominate in Lake Baikal, Central Siberia, Russia. These sponges are multicellular filter-feeding animals that represent a complex consortium of many species of eukaryotes and prokaryotes. In recent years, mass disease and death of Lubomirskia baicalensis has been a significant problem in Lake Baikal. The etiology and ecology of these events remain unknown. Bacteria from the families Flavobacteriaceae and Oxalobacteraceae dominate the microbiomes of diseased sponges. Both species are opportunistic pathogens common in freshwater ecosystems. The aim of our study was to analyze the genomes of strains Janthinobacterium sp. SLB01 and Flavobacterium sp. SLB02, isolated from diseased sponges to identify the reasons for their joint dominance. Janthinobacterium sp. SLB01 attacks other cells using a type VI secretion system and suppresses gram-positive bacteria with violacein, and regulates its own activity via quorum sensing. It produces floc and strong biofilm by exopolysaccharide biosynthesis and PEP-CTERM/XrtA protein expression. Flavobacterium sp. SLB02 utilizes the fragments of cell walls produced by polysaccharides. These two strains have a marked difference in carbohydrate acquisition. We described a possible means of joint occupation of the ecological niche in the freshwater sponge microbial community. This study expands the understanding of the symbiotic relationship of microorganisms with freshwater Baikal sponges. [ABSTRACT FROM AUTHOR]