Effects of color variation and physiological state on ascidian microbiomes.

Ascidians, known for their color variation, host species-specific microbial symbiont communities. Some ascidians can also transition into a nonfiltering (resting) physiological state. Recent studies suggest that the microbial symbiont communities may vary across different physiological states and color morphs of the host. The colonial ascidian, Polyclinum constellatum, which exhibits several color morphs in the Caribbean Sea, periodically ceases its filtering activity. To investigate if color variation in P. constellatum is indicative of sibling speciation, we sequenced fragments of the ribosomal 18S rRNA and the mitochondrial cytochrome oxidase subunit I genes. Additionally, we sequenced a fragment of the 16S rRNA gene to characterize the microbial communities of two common color morphs (red and green) in colonies that were either actively filtering (active) or nonfiltering (resting). Phylogenetic analyses of both ascidian genes resulted in well-supported monophyletic clades encompassing all color variants of P. constellatum. Interestingly, no significant differences were observed among the microbial communities of the green and red morphs, suggesting that color variation in this species is a result of intraspecific variation. However, the host's physiological state significantly influenced the microbial community structure. Nonfiltering (resting) colonies hosted higher relative abundances of Kiloniella (Alphaproteobacteria) and Fangia (Gammaproteobacteria), while filtering colonies hosted more Reugeria (Alphaproteobacteria) and Endozoicomonas (Gammaproteobacteria). This study demonstrates that microbial symbiont communities serve as reliable indicators of the taxonomic state of their host and are strongly influenced by the host's feeding condition.

Prokaryotic symbiont communities in three ascidian species introduced in both Ireland and New Zealand.

Ascidians or sea squirts are among the marine taxa with the most introduced species worldwide. These animals have a suite of biological characteristics that contribute to their successful establishment, including long reproductive seasons, rapid growth rates, and resistance to pollution. Here, we sequenced a fragment of the 16S ribosomal RNA gene to characterize symbiont diversity and host-specificity in the solitary species Syela clava and Ascidiella aspersa, and the colonial species Didemnum vexillum. Samples were collected from introduced populations in several marinas and mussel facilities around Ireland, and a marina in New Zealand. Two additional colonial species Botrylloides violaceus and Didemnum sp. were collected in Ireland, and ambient seawater was sampled from both countries for comparison. Data revealed a strong effect of host species and location on prokaryote symbiont composition, consistent with recent ascidian microbiome literature. However, a location effect did not manifest in alpha diversity metrics (e.g., the same ascidian species at different locations exhibited similar diversity) but was evident in beta diversity metrics (greater intra-specific differences across locations than within locations). Location effects were stronger than species effects only for the solitary species (i.e., A. aspersa from New Zealand was more similar to S. clava from New Zealand than to A. aspersa from Ireland). D. vexillum and A. aspersa hosted a high abundance of prokaryotic symbionts that were previously found in other ascidian species, while S. clava symbiotic community was more closely related to bacteria common in the marine environment. Further studies should aim to unravel host-microbe coevolutionary patterns and the microbial role in facilitating host establishment in different habitats.

Discovery of an Antarctic Ascidian-Associated Uncultivated Verrucomicrobia with Antimelanoma Palmerolide Biosynthetic Potential.

The Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially produced hybrid nonribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74-kbp candidate BGC encoding the multimodular enzymatic machinery (hybrid type I-trans-AT polyketide synthase-nonribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide gene targets and a single 16S rRNA gene variant (R = 0.83 to 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a nearly complete genome (10 contigs) of the BGC-producing organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as "Candidatus Synoicihabitans palmerolidicus." The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium. IMPORTANCE Palmerolide A has potential as a chemotherapeutic agent to target melanoma. We interrogated the microbiome of the Antarctic ascidian, Synoicum adareanum, using a cultivation-independent high-throughput sequencing and bioinformatic strategy. The metagenome-encoded biosynthetic machinery predicted to produce palmerolide A was found to be associated with the genome of a member of the S. adareanum core microbiome. Phylogenomic analysis suggests the organism represents a new deeply branching genus, "Candidatus Synoicihabitans palmerolidicus," in the Opitutaceae family of the Verrucomicrobia phylum. The Ca. Synoicihabitans palmerolidicus 4.29-Mb genome encodes a repertoire of carbohydrate-utilizing and transport pathways, a chemotaxis system, flagellar biosynthetic capacity, and other regulatory elements enabling its ascidian-associated lifestyle. The palmerolide producer's genome also contains five distinct copies of the large palmerolide biosynthetic gene cluster that may provide structural complexity of palmerolide variants.

Paenihalocynthiibacter styelae gen. nov., sp. nov., isolated from stalked sea squirt Styela clava.

A Gram-stain-negative, strictly aerobic, non-motile and rod-shaped bacterial strain, MYP1-1T, was isolated from the intestine of a stalked sea squirt (Styela clava) of the South Sea in the Republic of Korea. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain MYP1-1T clustered with the type strains of Halocynthiibacter species and Pseudohalocynthiibacter aestuariivivens. Strain MYP1-1T exhibited 16S rRNA gene sequence similarity values of 97.0-97.6 % to the type strains of Halocynthiibacter namhaensis, Halocynthiibacter arcticus and P. aestuariivivens. The phylogenetic tree based on genomic sequences showed that strain MYP1-1T formed a distinct branch separating it from the type strains of two Halocynthiibacter species and P. aestuariivivens and other taxa. The DNA G+C content of strain MYP1-1T from its genomic sequence was 55.0 mol%. Strain MYP1-1T contained Q-10 as the predominant ubiquinone and C18 : 1 ω7c as the major fatty acid. The major polar lipids of strain MYP1-1T were phosphatidylcholine, phosphatidylglycerol, one unidentified lipid and one unidentified aminolipid. The differences in fatty acid and polar lipid profiles and other differential phenotypic properties made it reasonable to distinguish strain MYP1-1T from the genera Halocynthiibacter and Pseudohalocynthiibacter. On the basis of the polyphasic taxonomic investigations, we conclude that strain MYP1-1T constitutes a new genus and species within the class Alphaproteobacteria, for which the name Paenihalocynthiibacter styelae gen. nov., sp. nov. is proposed. The type strain is MYP1-1T (=KCTC 82143T=NBRC 114355T).

Marine Natural Products from Tunicates and Their Associated Microbes.

Marine tunicates are identified as a potential source of marine natural products (MNPs), demonstrating a wide range of biological properties, like antimicrobial and anticancer activities. The symbiotic relationship between tunicates and specific microbial groups has revealed the acquisition of microbial compounds by tunicates for defensive purpose. For instance, yellow pigmented compounds, "tambjamines", produced by the tunicate, Sigillina signifera (Sluiter, 1909), primarily originated from their bacterial symbionts, which are involved in their chemical defense function, indicating the ecological role of symbiotic microbial association with tunicates. This review has garnered comprehensive literature on MNPs produced by tunicates and their symbiotic microbionts. Various sections covered in this review include tunicates' ecological functions, biological activities, such as antimicrobial, antitumor, and anticancer activities, metabolic origins, utilization of invasive tunicates, and research gaps. Apart from the literature content, 20 different chemical databases were explored to identify tunicates-derived MNPs. In addition, the management and exploitation of tunicate resources in the global oceans are detailed for their ecological and biotechnological implications.

Existence in cellulose shelters: industrial and pharmaceutical leads of symbiotic actinobacteria from ascidian Phallusia nigra, Andaman Islands.

The diversity of actinobacteria associated with marine ascidian Phallusia nigra from Andaman Islands was investigated. A total of 10 actinobacteria were isolated and based on the biochemical and molecular characterization, the isolates were assigned to 7 different actinobacterial genera. Eight putatively novel species belonging to genera Rhodococcus, Kineococcus, Kocuria, Janibacter, Salinispora and Arthrobacter were identified based on 16S rDNA sequence similarity with the NCBI database. The organic extracts of ten isolates displayed considerable bioactivity against test pathogens, which were Gram-positive and Gram-negative in nature. PCR-based screening for type I and type II polyketide synthases (PKS-I, PKS-II) and nonribosomal peptide synthetases (NRPS) revealed that, 10 actinobacterial isolates encoded at least one type of polyketide synthases biosynthesis gene. Majority of the isolates found to produce industrially important enzymes; amylase, protease, gelatinase, lipase, DNase, cellulase, urease, phosphatase and L-asparaginase. The present study emphasized that, ascidians are a prolific resource for novel bioactive actinobacteria with potential for novel drug discovery. This result expands the scope to functionally characterize the novel ascidian associated marine actinobacteria and their metabolites could be a source for the novel molecules of commercial interest.

Taxonomic Composition and Biological Activity of Bacterial Communities Associated with Marine Ascidians from Andaman Islands, India.

Marine invertebrates, particularly ascidians, constitute an important source of potential active and biofunctional natural products. The microbial diversity associated with ascidians is little recognized, although these microorganisms play a vital role in marine ecosystems. The objective of this study was to investigate bacterial population diversity in four ascidian samples: Phallusia nigra, Phallusia fumigata, Eudistoma viride, and Rhopalaea macrothorax, collected from the North Bay, Andaman and Nicobar Islands. Microbial strains identified up to the species level revealed 236 distinct species/ribotypes out of 298 bacterial strains. Of 298 ascidian-associated bacteria, 72 isolates belong to the class Gammaproteobacteria and the genus Endozoicomonas. The results from this investigation will contribute a broaden knowledge of microbial diversity associated to marine ascidians, and as a promising source for the discovery of new natural products.

Microbiome Variability across the Native and Invasive Ranges of the Ascidian Clavelina oblonga.

Ascidians are prolific colonizers of new environments and possess a range of well-studied features that contribute to their successful spread, but the role of their symbiotic microbial communities in their long-term establishment is mostly unknown. In this study, we utilized next-generation amplicon sequencing to provide a comprehensive description of the microbiome in the colonial ascidian Clavelina oblonga and examined differences in the composition, diversity, and structure of symbiont communities in the host's native and invasive ranges. To identify host haplotypes, we sequenced a fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI). C. oblonga harbored a diverse microbiome spanning 42 bacterial and three archaeal phyla. Colonies in the invasive range hosted significantly less diverse symbiont communities and exhibited lower COI haplotype diversity than colonies in the native range. Differences in microbiome structure were also detected across colonies in the native and invasive range, driven largely by novel bacteria representing symbiont lineages with putative roles in nitrogen cycling. Variability in symbiont composition was also observed among sites within each range. Together, these data suggest that C. oblonga hosts a dynamic microbiome resulting from (i) reductions in symbiont diversity due to founder effects in host populations and (ii) environmental selection of symbiont taxa in response to new habitats within a range. Further investigation is required to document the mechanisms behind these changes and to determine how changes in microbiome structure relate to holobiont function and the successful establishment of C. oblonga worldwide.IMPORTANCE Nonnative species destabilize coastal ecosystems and microbial symbionts may facilitate their spread by enhancing host survival and fitness. However, we know little of the microorganisms that live inside invasive species and whether they change as the host spreads to new areas. In this study, we investigated the microbial communities of an introduced ascidian (Clavelina oblonga) and tracked symbiont changes across locations within the host's native and invasive ranges. Ascidians in the invasive range had less-diverse microbiomes, as well as lower host haplotype diversity, suggesting that specific colonies reach new locations and carry select symbionts from native populations (i.e., founder effects). Further, ascidians in the invasive range hosted a different composition of symbionts, including microbes with the potential to aid in processes related to invasion success (e.g., nutrient cycling). We conclude that the putative functionality and observed flexibility of this introduced ascidian microbiome may represent an underappreciated factor in the successful establishment of nonnative species in new environments.

A marine microbiome antifungal targets urgent-threat drug-resistant fungi.

New antifungal drugs are urgently needed to address the emergence and transcontinental spread of fungal infectious diseases, such as pandrug-resistant Candida auris. Leveraging the microbiomes of marine animals and cutting-edge metabolomics and genomic tools, we identified encouraging lead antifungal molecules with in vivo efficacy. The most promising lead, turbinmicin, displays potent in vitro and mouse-model efficacy toward multiple-drug-resistant fungal pathogens, exhibits a wide safety index, and functions through a fungal-specific mode of action, targeting Sec14 of the vesicular trafficking pathway. The efficacy, safety, and mode of action distinct from other antifungal drugs make turbinmicin a highly promising antifungal drug lead to help address devastating global fungal pathogens such as C. auris.

Shotgun Proteomics of Ascidians Tunic Gives New Insights on Host-Microbe Interactions by Revealing Diverse Antimicrobial Peptides.

Ascidians are marine invertebrates associated with diverse microbial communities, embedded in their tunic, conferring special ecological and biotechnological relevance to these model organisms used in evolutionary and developmental studies. Next-generation sequencing tools have increased the knowledge of ascidians' associated organisms and their products, but proteomic studies are still scarce. Hence, we explored the tunic of three ascidian species using a shotgun proteomics approach. Proteins extracted from the tunic of Ciona sp., Molgula sp., and Microcosmus sp. were processed using a nano LC-MS/MS system (Ultimate 3000 liquid chromatography system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer). Raw data was searched against UniProtKB - the Universal Protein Resource Knowledgebase (Bacteria and Metazoa section) using Proteome Discoverer software. The resulting proteins were merged with a non-redundant Antimicrobial Peptides (AMPs) database and analysed with MaxQuant freeware. Overall, 337 metazoan and 106 bacterial proteins were identified being mainly involved in basal metabolism, cytoskeletal and catalytic functions. 37 AMPs were identified, most of them attributed to eukaryotic origin apart from bacteriocins. These results and the presence of "Biosynthesis of antibiotics" as one of the most highlighted pathways revealed the tunic as a very active tissue in terms of bioactive compounds production, giving insights on the interactions between host and associated organisms. Although the present work constitutes an exploratory study, the approach employed revealed high potential for high-throughput characterization and biodiscovery of the ascidians' tunic and its microbiome.

Antimicrobial Activities of Rhopalaea-Associated Fungus Aspergillus flavus strain MFABU9.

BACKGROUND AND OBJECTIVE: Rhopalaea is a genus of ascidian belonging to the family Diazonidae. Ascidians provide niches for various microorganisms including fungi. This present study describes the potential new source for natural bioactive compounds from Rhopalaea-associated fungi obtained from Bunaken marine park. MATERIALS AND METHODS: As part of an on-going research program to explore the chemical diversity of marine derived fungi, we performed an antimicrobial bioactivity-guided screening of EtOAc extracts of the fungi isolated from ascidian Rhopalaea sp. RESULTS: The study confirms that the ascidian obtained from Bunaken marine park was Rhopalaea sp. The fungus isolated from the ascidian was Aspergillus flavus which showed antimicrobial activity against bacteria Escherichia coli, Staphylococcus aereus, Aeromonas hydrophila and antifungal against the human pathogenic fungus Candida albicans. CONCLUSION: Aspergillus flavus isolated from ascidian Rhopalaea sp. has the potential as antibacterial and antifungal.

Uncovering the Core Microbiome and Distribution of Palmerolide in Synoicum adareanum Across the Anvers Island Archipelago, Antarctica.

Polar marine ecosystems hold the potential for bioactive compound biodiscovery, based on their untapped macro- and microorganism diversity. Characterization of polar benthic marine invertebrate-associated microbiomes is limited to few studies. This study was motivated by our interest in better understanding the microbiome structure and composition of the ascidian, Synoicum adareanum, in which palmerolide A (PalA), a bioactive macrolide with specificity against melanoma, was isolated. PalA bears structural resemblance to a hybrid nonribosomal peptide-polyketide that has similarities to microbially-produced macrolides. We conducted a spatial survey to assess both PalA levels and microbiome composition in S. adareanum in a region of the Antarctic Peninsula near Anvers Island (64° 46'S, 64° 03'W). PalA was ubiquitous and abundant across a collection of 21 ascidians (3 subsamples each) sampled from seven sites across the Anvers Island Archipelago. The microbiome composition (V3-V4 16S rRNA gene sequence variants) of these 63 samples revealed a core suite of 21 bacterial amplicon sequence variants (ASVs)-20 of which were distinct from regional bacterioplankton. ASV co-occurrence analysis across all 63 samples yielded subgroups of taxa that may be interacting biologically (interacting subsystems) and, although the levels of PalA detected were not found to correlate with specific sequence variants, the core members appeared to occur in a preferred optimum and tolerance range of PalA levels. These results, together with an analysis of the biosynthetic potential of related microbiome taxa, describe a conserved, high-latitude core microbiome with unique composition and substantial promise for natural product biosynthesis that likely influences the ecology of the holobiont.

Phylogenetic analysis and screening of antimicrobial and cytotoxic activities of culturable bacteria associated with the ascidian Botryllus schlosseri.

AIMS: Isolating culturable bacteria associated with ascidian (Botryllus schlosseri) and investigating their bioactivities to discover new marine microbial resources with potential to produce novel bioactive natural products. METHODS AND RESULTS: A total of 357 bacteria were isolated from the ascidian B. schlosseri from the coast of Weihai in the north Yellow Sea, China. Of these, 203 isolates were identified by 16S rRNA gene sequencing and they belonged to 52 genera from 30 families in five phyla. The antimicrobial activities and cytotoxic activities of all isolates were determined. Of the 357 isolates, 135 isolates demonstrated antimicrobial activities, and the crude extracts of five isolates showed strong cytotoxicity against human hepatocellular carcinoma Bel 7402 or human cervical carcinoma HeLa cells. CONCLUSIONS: Our study revealed the diversity of bacteria associated with the ascidian B. schlosseri and reported a broad spectrum of antimicrobial and cytotoxic activities displayed by these isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the culturable bacteria associated with the ascidian B. schlosseri may be a potential source for novel bioactive compounds.

Phylogenetic Analysis and Screening of Antimicrobial and Antiproliferative Activities of Culturable Bacteria Associated with the Ascidian Styela clava from the Yellow Sea, China.

Over 1,000 compounds, including ecteinascidin-743 and didemnin B, have been isolated from ascidians, with most having bioactive properties such as antimicrobial, antitumor, and enzyme-inhibiting activities. In recent years, direct and indirect evidence has shown that some bioactive compounds isolated from ascidians are not produced by ascidians themselves but by their symbiotic microorganisms. Isolated culturable bacteria associated with ascidians and investigating their potential bioactivity are an important approach for discovering novel compounds. In this study, a total of 269 bacteria were isolated from the ascidian Styela clava collected from the coast of Weihai in the north of the Yellow Sea, China. Phylogenetic relationships among 183 isolates were determined using their 16S rRNA gene sequences. Isolates were tested for antimicrobial activity against seven indicator strains, and an antiproliferative activity assay was performed to test for inhibition of human hepatocellular carcinoma Bel 7402 and human cervical carcinoma HeLa cell proliferation. Our results showed that the isolates belonged to 26 genera from 18 families in four phyla (Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes). Bacillus and Streptomyces were the most dominant genera; 146 strains had potent antimicrobial activities and inhibited at least one of the indicator strains. Crude extracts from 29 strains showed antiproliferative activity against Bel 7402 cells with IC50 values below 500 µg·mL-1, and 53 strains showed antiproliferative activity against HeLa cells, with IC50 values less than 500 µg·mL-1. Our results suggest that culturable bacteria associated with the ascidian Styela clava may be a promising source of novel bioactive compounds.

Konamycins A and B and Rubromycins CA1 and CA2, Aromatic Polyketides from the Tunicate-Derived Streptomyces hyaluromycini MB-PO13T.

Four new aromatic polyketides, konamycins A (1) and B (2) and rubromycins CA1 (3) and CA2 (4), were isolated from the culture extract of the tunicate-derived Streptomyces hyaluromycini MB-PO13T. Compounds 1 and 2 possess a benzo[ b]fluorene aglycon modified by C-glycosylation with l-amicetose. Compounds 3 and 4 are the new congeners of rubromycin in which a naphthoquinone and carboxylated isocoumarin are joined through a spiroketal carbon. The structures of these compounds were determined by extensive analysis of 1D and 2D NMR spectroscopic data. Compound 1 showed radical scavenging activity in DPPH and superoxide quenching assays, and 3 and 4 displayed antimicrobial activity against Gram-positive bacteria.

Physicochemical Properties and Bacterial Communities of Meongge (Halocynthia roretzi) Jeotgal Prepared with 3 Different Types of Salts.

Three types of meongge (Halocynthia roretzi) jeotgal (MJ) were prepared with 3 different types of salts (12%, w/v): purified salt (PS), solar salt aged for 3 years (SS), and bamboo salt that had been recrystalized 3 times (BS). One set of MJ was fermented with starters, Bacillus subtilis JS2 and Tetragenococcus halophilus BS1-37 (each 6 log CFU/g), and another set without starters for 42 days at 10°C. The LAB count of the SSMJ (non-starter) was highest at day 28 (2.30 log CFU/g). The pH of the PSMJ and SSMJ was 5.72-5.77 at day 0, and 5.40-5.50 at day 42. BSMJ showed higher pH and lower titratable acidities than other samples. Amino-type nitrogen (ANN) increased continuously, and SSMJ showed higher values than other samples from day 14. Bacterial species of non-starter MJ were examined by culture independent method. Clone libraries of 16S rRNA genes were constructed in Escherichia coli from total DNA from nonstarter MJ samples at day 0, 14, and 28. Thirty clones per each sample were randomly selected and DNA sequences were analyzed. Variovorax sp., uncultured bacterium, and Acidovorax sp. were the most dominant group at day 0, 14, and 28, respectively. Lactobacillus sakei and Streptococcus sp. were the next dominant group in SSMJ at day 28. A Streptococcus sp. was detected from PSMJ at day 28. Sensory evaluation for MJ samples at day 28 showed that SSMJ got higher overall acceptability scores. These results showed that solar salt can cause desirable changes in the microbial community of fermented foods, thereby positively affecting their overall quality.

Insights into the Mechanism of the Cyanobactin Heterocyclase Enzyme.

Cyanobactin heterocyclases share the same catalytic domain (YcaO) as heterocyclases/cyclodehydratases from other ribosomal peptide (RiPPs) biosynthetic pathways. These enzymes process multiple residues (Cys/Thr/Ser) within the same substrate. The processing of cysteine residues proceeds with a known order. We show the order of reaction for threonines is different and depends in part on a leader peptide within the substrate. In contrast to other YcaO domains, which have been reported to exclusively break down ATP into ADP and inorganic phosphate, cyanobactin heterocyclases have been observed to produce AMP and inorganic pyrophosphate during catalysis. We dissect the nucleotide profiles associated with heterocyclization and propose a unifying mechanism, where the γ-phosphate of ATP is transferred in a kinase mechanism to the substrate to yield a phosphorylated intermediate common to all YcaO domains. In cyanobactin heterocyclases, this phosphorylated intermediate, in a proportion of turnovers, reacts with ADP to yield AMP and pyrophosphate.

Anti-glioma trichobamide A with an unprecedented tetrahydro-5H-furo[2,3-b]pyrrol-5-one functionality from ascidian-derived fungus Trichobotrys effuse 4729.

Trichobamide A (1), a novel pyrrocidine alkaloid with an unprecedented tetrahydro-5H-furo[2,3-b]pyrrol-5-one moiety, was isolated from the ascidian-derived fungus Trichobotrys effuse 4729. Trichobamide A (1) showed significant inhibition of the proliferation of two glioma cell lines, U251 and SNB19. It induced the up-regulation of P53 expression, which in turn induces the up-regulation of downstream pro-apoptotic gene expression and the down-regulation of anti-apoptotic gene expression.

Characterization of a Fibrinolytic Enzyme Secreted by Bacillus velezensis BS2 Isolated from Sea Squirt Jeotgal.

Bacillus sp. BS2 showing strong fibrinolytic activity was isolated from sea squirt (munggae) jeotgal, a traditional Korean fermented seafood. BS2 was identified as B. velezensis by molecular biological methods.B. velezensis BS2 grows well at 15% NaCl and at 10oC. When B. velezensis BS2 was cultivated in TSB broth for 96 h at 37°C, the culture showed the highest fibrinolytic activity (131.15 mU/µl) at 96 h. Three bands of 27, 35 and 60 kDa were observed from culture supernatant by SDS-PAGE, and fibrin zymography showed that the major fibrinolytic protein was the 27 kDa band. The gene (aprEBS2) encoding the major fibrinolytic protein was cloned, and overexpressed in heterologous hosts, B. subtilis WB600 and E. coli BL21 (DE3). B. subtilis transformant showed 1.5-fold higher fibrinolytic activity than B. velezensis BS2. Overproduced AprEBS2 in E. coli was purified by affinity chromatography. The optimum pH and temperature were pH 8.0 and 37°C, respectively. Km and Vmax were 0.15 mM and 39.68 µM/l/min, respectively, when N-succinyl-Ala-Ala-Pro-Phe-pNA was used as the substrate. AprEBS2 has strong α-fibrinogenase and moderate ß-fibrinogenase activity. Considering its high fibrinolytic activity, significant salt tolerance, and ability to grow at 10°C, B. velezensis BS2 can be used as a starter for jeotgal.

Symbiotic bacteria associated with ascidian vanadium accumulation identified by 16S rRNA amplicon sequencing.

Ascidians belonging to Phlebobranchia accumulate vanadium to an extraordinary degree (≤ 350 mM). Vanadium levels are strictly regulated and vary among ascidian species; thus, they represent well-suited models for studies on vanadium accumulation. No comprehensive study on metal accumulation and reduction in marine organisms in relation to their symbiotic bacterial communities has been published. Therefore, we performed comparative 16S rRNA amplicon sequence analyses on samples from three tissues (branchial sac, intestine, and intestinal lumen) involved in vanadium absorption, isolated from two vanadium-rich (Ascidia ahodori and Ascidia sydneiensis samea) and one vanadium-poor species (Styela plicata). For each sample, the abundance of every bacteria and an abundance value normalized to their abundance in seawater were calculated and compared. Two bacterial genera, Pseudomonas and Ralstonia, were extremely abundant in the branchial sacs of vanadium-rich ascidians. Two bacterial genera, Treponema and Borrelia, were abundant and enriched in the intestinal content of vanadium-rich ascidians. The results suggest that specific selective forces maintain the bacterial population in the three ascidian tissues examined, which contribute to successful vanadium accumulation. This study furthers the understanding of the relationship between bacterial communities and metal accumulation in marine life.