Isolation and Properties of the Bacterial Strain Janthinobacterium sp. SLB01.

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.

16S rRNA Gene Amplicon Sequencing Data of Bacterial Community of Freshwater Sponge Lubomirskia baicalensis.

Our study was devoted to investigating the mass disease and mortality of freshwater sponges (Lubomirskiidae) in Lake Baikal. The first sights of the disease were discovered in 2011 and were associated with a shift in sponge microbial diversity. To study the microbiome, we performed sequencing of the 16S rRNA amplicon DNA extracted from the freshwater sponges.

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.

Genome Analysis of the Janthinobacterium sp. Strain SLB01 from the Diseased Sponge of the Lubomirskia baicalensis.

The strain Janthinobacterium sp. SLB01 was isolated from the diseased freshwater sponge Lubomirskia baicalensis (Pallas, 1776) and the draft genome was published previously. The aim of this work is to analyze the genome of the Janthinobacterium sp. SLB01 to search for pathogenicity factors for Baikal sponges. We performed genomic analysis to determine virulence factors, comparing the genome of the strain SLB01 with genomes of other related J. lividum strains from the environment. The strain Janthinobacterium sp. SLB01 contained genes encoding violacein, alpha-amylases, phospholipases, chitinases, collagenases, hemolysin, and a type VI secretion system. In addition, the presence of conservative clusters of genes for the biosynthesis of secondary metabolites of tropodithietic acid and marinocine was found. We present genes for antibiotic resistance, including five genes encoding various lactamases and eight genes for penicillin-binding proteins, which are conserved in all analyzed strains. Major differences were found between the Janthinobacterium sp. SLB01 and J. lividum strains in the spectra of genes for glycosyltransferases and glycoside hydrolases, serine hydrolases, and trypsin-like peptidase, as well as some TonB-dependent siderophore receptors. Thus, the study of the analysis of the genome of the strain SLB01 allows us to conclude that the strain may be one of the pathogens of freshwater sponges.

Cooperative Interaction of Janthinobacterium sp. SLB01 and Flavobacterium sp. SLB02 in the Diseased Sponge Lubomirskia baicalensis.

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.

Draft Genome Sequence of the Green Microalga Chlorella sp. Strain BAC9706, Isolated from Lake Baikal, Russia.

Green algae of the phylum Chlorophyta are the most widespread autotrophic picoplankton in Lake Baika (Russia). To expand our molecular biological knowledge of these microalgae and compare them in the future with an endosymbiotic strain, we present here the draft genome sequence of Chlorella sp. strain BAC9706.

Microbiome analysis of healthy and diseased sponges Lubomirskia baicalensis by using cell cultures of primmorphs.

Endemic sponges (Demosponges, Lubomirskiidae) dominate the fauna of the littoral zone of Lake Baikal. These freshwater sponges live in symbiosis with diverse eukaryotes and prokaryotes, including chlorophyll-containing microalgae. Within the last 5 years, the incidence of sponge disease and mortality events in Lake Baikal has increased. The etiology and ecology of these events remain unknown, in part because of the lack of models to study sponge-microbe interactions. In this work, we tested the use of primmorph cell cultures of Lubomirskia baicalensis as a tool for investigating the microbiomes of sponges. We infected primmorphs, cultured in vitro, with samples from diseased sponges and observed, by microscopy, disease symptoms, including loss of green symbionts, associated with mass die-off events. Subsequent sequencing of 16S rRNA gene fragments revealed that the microbiome community of healthy sponge and primmorphs formed a group separate from the community of diseased sponges and infected primmorphs. This confirms the suitability of the primmorph cell culture as a model sponge system. We also discovered mass mortality of green symbionts (Chlorophyta) was associated with a shift in the microbial communities of sponges/primmorphs. Microbes in diseased sponges, and infected primmorphs, belonged mainly to the phyla Bacteroidetes and Proteobacteria and these families Flavobacteriaceae, Burkholderiaceae, and Moraxellaceae. Primmorphs cell culture may provide a model to study interactions between these bacteria and their host and elucidate the cause of mass mortality events.

Draft Genome Sequence of Flavobacterium sp. Strain SLB02, Isolated from the Diseased Sponge Lubomirskia baicalensis.

There are significant changes in the consortium of microorganisms of freshwater Baikal sponges during their mass death, which began in 2011. The alleged cause of disease is a significant increase in the number of opportunistic microorganisms. Here, we report the draft genome sequence of Flavobacterium sp. strain SLB02.

Draft Genome Sequence of Janthinobacterium sp. Strain SLB01, Isolated from the Diseased Sponge Lubomirskia baicalensis.

The draft genome sequence of Janthinobacterium sp. strain SLB01, a violacein-producing psychrotolerant bacterium isolated from the diseased sponge Lubomirskia baicalensis, was determined. We identified five genes encoding VioA, VioB, VioC, VioD, and VioE proteins related to violacein biosynthesis that were like those identified in published Janthinobacterium lividum strains MTR and RIT308.

Diversity and shifts of the bacterial community associated with Baikal sponge mass mortalities.

The disease of freshwater sponges was first discovered in 2011, when pink samples were found in the Central Basin of Lake Baikal. Subsequently, the visible signs of the disease have changed, and now sponges appear with various symptoms of damage to the body, such as discoloration, tissue necrosis, the formation of brown patches and dirty-purple biofilms on some branches. These signs of the disease are accompanied by the mass death of sponges. We identified differences in microbiomes by sequencing 16S rRNA genes and found changes in the consortium of microorganisms of freshwater Baikal sponges. We found that the observed imbalance in the studied microbial communities of diseased sponges is caused by several different conditionally pathogenic microorganisms that increase their negative effect by acting together and in concert, which leads to the death of photosynthetic microalgae and sponges. Sponges are an important component of coastal communities, and the massive loss of sponges can obviously affect the structure of benthic communities and the purity of water.

Evaluating the use of diversity indices to distinguish between microbial communities with different traits.

Several measures of biodiversity are commonly used to describe microbial communities, analyzed using 16S gene sequencing. A wide range of available experiments on 16S gene sequencing allows us to present a framework for a comparison of various diversity indices. The criterion for the comparison is the statistical significance of the difference in index values for microbial communities with different traits, within the same experiment. The results of the evaluation indicate that Shannon diversity is the most effective measure among the commonly used diversity indices. The results also indicate that, within the present framework, the Gini coefficient as a diversity index is comparable to Shannon diversity, despite the fact that the Gini coefficient, as a diversity estimator, is far less popular in microbiology than several other measures.

The signs of adaptive mutations identified in the chloroplast genome of the algae endosymbiont of Baikal sponge.

Background: The study of ecosystems of the great lakes is important as observations can be extended to ecosystems of larger scale. The ecological crisis of Lake Baikal needs investigations to discover the molecular mechanisms involved in the crisis. The disease of Baikal sponges is one of the processes resulting in the degradation of the littoral zone of the lake. Methods: The chloroplast genome fragment for the algae endosymbiont of Baikal sponge was assembled from metagenomic sequencing data. The distributions of polymorphic sites were obtained for the genome fragment, separately for samples from healthy sponge, diseased sponge and dead sponge tissues. Results: The comparative analysis of chloroplast genome sequences suggests that the symbiotic algae from Baikal sponge is close to Choricystis genus of unicellular algae. Also, the distributions of polymorphic sites allowed detection of the signs of extensive mutations in the chloroplasts isolated from the diseased sponge tissues. Conclusions: The study demonstrate the particular case of evolution at the molecular level due to the conditions of a severe crisis of a whole ecosystem in Lake Baikal. The detection of adaptive mutations in the chloroplast genome is an important feature which could represent the behavior of an ecosystem in the event of a severe crisis.

The rearrangement of motif F in the flavivirus RNA-directed RNA polymerase.

In the flavivirus genus, the non-structural protein NS5 plays a central role in RNA viral replication and constitutes a major target for drug discovery. One of the prime challenges in the study of NS5 protein is to investigate the interplay between the two protein domains, namely, the RNA-dependent RNA polymerase (RdRp) domain and the methyltransferase (MTase) domain. These investigations could clarify the multiple roles of NS5 protein in the virus life cycle. Here we present the results of sequence analyses and structural bioinformatics studies of NS5 protein, which suggest that the conserved motif F in the NS5 protein could act as a lock which controls the rearrangement of the domains and as a switch in the protein enzymatic activity.

Characteristics of far eastern strains of tick-borne encephalitis virus.

A comparative study of biological, molecular and genetic characteristics of a collection of ten strains of tick-borne encephalitis virus (TBEV) isolated in Primorsky Krai before 1960 and stored in a lyophilized state for a prolonged period (over 65 years) is presented. The collection includes the Sofjin strain isolated from the brain of a fatal case in Primorsky Krai in 1937 and transferred to the Scientific Research Institute of Epidemiology and Microbiology (Vladivostok) in 1953. All lyophilized viral strains demonstrated great preservation and high infectious activity in the model of 2-day-old non-inbred mice. Whole-genome sequencing showed that all strains belong to the Far East TBEV subtype, comprising three clusters of Sofjin-, Oshima- and Senzhang-like strains. We show that SofjinPYB, Sofjin (Vector) and Sofjin-HO strains form a separate branch of the phylogenetic tree and are closely related to Khabarovsk-Obor-4, but not to the original Sofjin strain. The Sofjin-1953, Sofijin-Chumakov, SofjinKSY and SofjinCDC strains are genetically close to each other and can be used as reference strains for comparative analysis of the tick-borne encephalitis virus population.

The complete mitochondrial genome of Baikalospongia intermedia (Lubomirskiidae): description and phylogenetic analysis.

The complete mitochondrial genome of the Lake Baikal sponge Baikalospongia intermedia was sequenced. The circular mitochondrial genome is 28,327 bp in length and includes 14 protein-coding genes, 2 ribosomal RNA genes and 25 transfer RNA genes. Bayesian comparative analysis of molecular evolution rates was found no acceleration of the mtDNA evolution of B. intermedia. This species clustered with other species of the genus Baikalospongia on the Bayesian tree.

Louping ill virus (LIV) in the Far East.

This study focused on finding, culturing, and identifying the biological and genetic characteristics of three louping ill virus (LIV) strains in the south of the Russian Far East. The Primorye-155-77 and Primorye-20-79 virus strains were isolated from Ixodes persulcatus ticks, and the Primorye-185-91 strain was isolated from the blood of a person after a tick bite. According to the hemagglutination and neutralization tests, Primorye-155-77, Primorye-20-79 and Primorye-185-91 had weak reactivity with antibodies in an antiserum against tick-borne encephalitis virus. In Primorye-155-77 and Primorye-20-79, the sequences of the 5' ends of the 2456-nucleotide-long viral RNA including the 5' untranslated region (UTR) and genes of the capsid protein, prM protein and envelope E protein were determined. The complete genome sequence of Primorye-185-91 was determined. The E protein gene of the Negishi strain differed from those of three analyzed strains, as there were mutations resulting in the replacement of three amino acids: Ala163Thr, Asp193Asn and Ala313Thr. The homology of Primorye-185-91 to LIV 369/T2 was 97.57 %, and to the Penrith strain, it was 98.36 %. Phylogenetic analysis showed that Primorye-155-77, Primorye-20-79 and Primorye-185-91 are related to LI/A and LI/K strains isolated in England and Scotland and to the Negishi strain; these strains have a common progenitor. Negishi-like strains were represented by one subtype of louping ill virus, i.e. the British subtype (LIV-Brit). The possibility is discussed of a single introduction of the virus to the Far Eastern region (Japan and Primorsky Krai) from a single natural locus more than 50 years ago.

The nature of replication of tick-borne encephalitis virus strains isolated from residents of the Russian Far East with inapparent and clinical forms of infection.

We describe the biological properties and molecular characteristics of complete genomes of 33 tick-borne encephalitis virus (TBEV) strains that induced different forms of infection, from inapparent to severe focal ones resulting in fatal outcome. Hemagglutinating activity of Oshima-like strains was higher at pH 5.8, while activity of Sofjin- and Senhzang-like strains were higher at pH 6.2 and 6.8, respectively. We determined susceptibility of porcine kidney (PK) cell cultures to these TBEV strains by cytopathic effect (CPE), plaque formation, and size of plaques. The clinical TBEV strains had higher virus titers both in tissue culture infectious dose 50(TCID50) and in plaque-forming unit (PFU) titers and larger plaques than the inapparent strains. A comparison of virus multiplication kinetics by PFU in culture fluid with kinetics of ELISA antigen and hemagglutinin accumulation suggested a different mechanism of interaction between these virus strains and PK cells at the initial stage of cell infection.

The relationship between the structure of the tick-borne encephalitis virus strains and their pathogenic properties.

Tick-borne encephalitis virus (TBEV) is transmitted to vertebrates by taiga or forest ticks through bites, inducing disease of variable severity. The reasons underlying these differences in the severity of the disease are unknown. In order to identify genetic factors affecting the pathogenicity of virus strains, we have sequenced and compared the complete genomes of 34 Far-Eastern subtype (FE) TBEV strains isolated from patients with different disease severity (Primorye, the Russian Far East). We analyzed the complete genomes of 11 human pathogenic strains isolated from the brains of dead patients with the encephalitic form of the disease (Efd), 4 strains from the blood of patients with the febrile form of TBE (Ffd), and 19 strains from patients with the subclinical form of TBE (Sfd). On the phylogenetic tree, pathogenic Efd strains formed two clusters containing the prototype strains, Senzhang and Sofjin, respectively. Sfd strains formed a third separate cluster, including the Oshima strain. The strains that caused the febrile form of the disease did not form a separate cluster. In the viral proteins, we found 198 positions with at least one amino acid residue substitution, of which only 17 amino acid residue substitutions were correlated with the variable pathogenicity of these strains in humans and they authentically differed between the groups. We considered the role of each amino acid substitution and assumed that the deletion of 111 amino acids in the capsid protein in combination with the amino acid substitutions R16K and S45F in the NS3 protease may affect the budding process of viral particles. These changes may be the major reason for the diminished pathogenicity of TBEV strains. We recommend Sfd strains for testing as attenuation vaccine candidates.

Molecular identification and phylogeny of Dermacentor nuttalli (Acari: Ixodidae).

Dermacentor nuttalli is an epidemiologically important tick in Palearctic Asia which transmits several infectious diseases including tularemia, North Asian tick-borne rickettsiosis, Lyme disease and tick-borne encephalitis. The genetic specificity and phylogeny of D. nuttalli from four geographic localities in Eastern Siberia were characterized using the mitochondrial (mt) 16S ribosomal RNA (rRNA) gene and internal transcribed spacer 2 (ITS2). Low genetic diversity was observed in the populations of ticks distributed from South Siberia to North China. From 11 detected mt 16S haplotypes, one was found in all populations, whereas the others were restricted to specific localities. These results suggested that the genetic structure of D. nuttalli represents integrated populations with no geographic isolation across the distribution area. The phylogenetic reconstructions inferred from the mt 16S rRNA gene and ITS2 were in agreement and showed a distinct D. nuttalli clade within a monophyletic Eurasian lineage of Dermacentor sp.

Identification and first insights into the structure and biosynthesis of chitin from the freshwater sponge Spongilla lacustris.

This work demonstrates that chitin is an important structural component within the skeletal fibers of the freshwater sponge Spongilla lacustris. Using a variety of analytical techniques ((13)C solid state NMR, FT-IR, Raman, NEXAFS, ESI-MS, Morgan-Elson assay and Calcofluor White Staining); we show that this sponge chitin is much closer to α-chitin, known to be present in other animals, than to ß-chitin. Genetic analysis confirmed the presence of chitin synthases, which are described for the first time in a sponge. The presence of chitin in both marine (demosponges and hexactinellids) and freshwater sponges indicates that this important structural biopolymer was already present in their common ancestor.