Ruegeria spongiae sp. nov., isolated from Callyspongia elongata.

A Gram-stain-negative, rod-shaped, polar flagellated, aerobic, light-yellow bacterium, designated as 2012CJ41-6T, was isolated from a sponge sample of Callyspongia elongata from Chuja-myeon, Jeju-si, Jeju-do, Republic of Korea. On the basis of 16S rRNA gene sequencing, strain 2012CJ41-6T clustered with species of the genus Ruegeria and appeared closely related to R. halocynthiae DSM 27839T (96.46 % similarity), R. denitrificans CECT 4357T (96.32 %), R. profundi ZGT108T (96.32 %), R. litorea CECT 7639T (96.32 %) and R. atlantica CECT 4292T (96.16 %). The average nucleotide identity and digital DNA-DNA hybridization between strain 2012CJ41-6T and the most closely related strain was 75.3 % and 19.6 %, indicating that 2012CJ41-6T represents a novel species of the genus Ruegeria. Growth occurred at 15-37 °C on marine medium in the presence of 0.5-10 % (w/v) NaCl and at pH 5.5-8.5. The DNA G+C content of the genomic DNA was 60.80 mol%, and ubiquinone-10 (Q-10) was the major respiratory quinone. The major cellular fatty acids (>5 %) were C18 : 1 ω7c and/or C18:1 ω6c (summed feature 8). The polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, one unidentified phospholipid, one unidentified aminolipid, one unidentified aminophospholipid and five unidentified lipids. Physiological and biochemical characteristics indicated that strain 2012CJ41-6T represents a novel species of the genus Ruegeria, for which the name Ruegeria spongiae sp. nov. is proposed. The type strain is 2012CJ41-6T (=KACC 22645T=LMG 32585T).

Computational Metabolomics Tools Reveal Subarmigerides, Unprecedented Linear Peptides from the Marine Sponge Holobiont Callyspongia subarmigera.

A detailed examination of a unique molecular family, restricted to the Callyspongia genus, in a molecular network obtained from an in-house Haplosclerida marine sponge collection (including Haliclona, Callyspongia, Xestospongia, and Petrosia species) led to the discovery of subarmigerides, a series of rare linear peptides from Callyspongia subarmigera, a genus mainly known for polyacetylenes and lipids. The structure of the sole isolated peptide, subarmigeride A (1) was elucidated through extensive 1D and 2D NMR spectroscopy, HRMS/MS, and Marfey's method to assign its absolute configuration. The putative structures of seven additional linear peptides were proposed by an analysis of their respective MS/MS spectra and a comparison of their fragmentation patterns with the heptapeptide 1. Surprisingly, several structurally related analogues of subarmigeride A (1) occurred in one distinct cluster from the molecular network of the cyanobacteria strains of the Guadeloupe mangroves, suggesting that the true producer of this peptide family might be the microbial sponge-associated community, i.e., the sponge-associated cyanobacteria.

Antimicrobial Chlorinated 3-Phenylpropanoic Acid Derivatives from the Red Sea Marine Actinomycete Streptomyces coelicolor LY001.

The actinomycete strain Streptomyces coelicolor LY001 was purified from the sponge Callyspongia siphonella. Fractionation of the antimicrobial extract of the culture of the actinomycete afforded three new natural chlorinated derivatives of 3-phenylpropanoic acid, 3-(3,5-dichloro-4-hydroxyphenyl)propanoic acid (1), 3-(3,5-dichloro-4-hydroxyphenyl)propanoic acid methyl ester (2), and 3-(3-chloro-4-hydroxyphenyl)propanoic acid (3), together with 3-phenylpropanoic acid (4), E-cinnamic acid (5), and the diketopiperazine alkaloids cyclo(l-Phe-trans-4-OH-l-Pro) (6) and cyclo(l-Phe-cis-4-OH-d-Pro) (7) were isolated. Interpretation of nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS) data of 1-7 supported their assignments. Compounds 1-3 are first candidates of the natural chlorinated phenylpropanoic acid derivatives. The production of the chlorinated derivatives of 3-phenylpropionic acid (1-3) by S. coelicolor provides insight into the biosynthetic capabilities of the marine-derived actinomycetes. Compounds 1-3 demonstrated significant and selective activities towards Escherichia. coli and Staphylococcus aureus, while Candida albicans displayed more sensitivity towards compounds 6 and 7, suggesting a selectivity effect of these compounds against C. albicans.

Reactive oxygen species generation and mitochondrial dysfunction for the initiation of apoptotic cell death in human hepatocellular carcinoma HepG2 cells by a cyclic dipeptide Cyclo(-Pro-Tyr).

Cyclic dipeptides are increasingly gaining importance as considering its significant biological and pharmacological activities. This study was aimed to investigate the anticancer activity of a dipeptide Cyclo(-Pro-Tyr) (DP) identified from marine sponge Callyspongia fistularis symbiont Bacillus pumilus AMK1 and the underlying apoptotic mechanisms in the liver cancer HepG2 cell lines. MTT assay was done to demonstrate the cytotoxic effect of DP in HepG2 cells and mouse Fibroblast McCoy cells. Initially, apoptosis inducing activity of DP was identified using propidium iodide (PI) and acridine orange/ethidium bromide (AO/EB) dual staining, then it was confirmed by DNA fragmentation assay and western blotting analysis of apoptosis related markers Bax, Bcl-2, cytochrome c, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Rhodamine 123 staining was performed to observe DP effects on the mitochondrial membrane potential (MMP) and DCFH-DA (Dichloro-dihydro-fluorescein diacetate) staining was done to measure the intracellular reactive oxygen species (ROS) levels. The MTT results revealed that DP initiated dose-dependent cytotoxicity in HepG2 cells, but no significant toxicity in mouse Fibroblast McCoy cells treated with DP at the specified concentrations. DP induced apoptosis, which is confirmed by the appearance of apoptotic bodies with PI and AO/EB dual staining, and DNA fragmentation. DP significantly elevated the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), enhanced cytochrome c release from mitochondria, increased caspase-3 activation, the cleavage of PARP and increased intracellular reactive oxygen species (ROS) levels. Besides this, DP successfully inhibited the phosphorylation of PI3K, AKT and increased PTEN expression. These results suggested DP might have anti-cancer effect by initiating apoptosis through mitochondrial dysfunction and downregulating PI3K/Akt signaling pathway in HepG2 cells with no toxicity effect on normal fibroblast cells. Therefore, DP may be developed as a potential alternative therapeutic agent for treating hepatocellular carcinoma.

Labrenzia callyspongiae sp. nov., Isolated from Marine Sponge Callyspongia elegans in Jeju Island.

A Gram-staining-negative, aerobic, light brown pigment bacterium, designated strain CE80T was isolated from marine sponge Callyspongia elegans in Jeju Island, Republic of Korea. Strain CE80T grew optimally at 25°C, in the range of pH 5.0-11.0 (optimum 7.0-8.0), and with 1.0-5.0% NaCl (optimum 1-3% (w/v)). Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain CE80T belonged to the genus Labrenzia and was closely related to L. suaedae YC6927T (98.3%), L. alexandrii DFL-11T (96.6%), L. aggregata IAM 12614T (96.6%) L. marina mano18T (96.5%) and L. alba CECT 5094T (96.2%). The major fatty acids of strain CE80T were C18:1 ω7c, and summed feature. The polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamin, one unidentified aminolipid, one phospholipid and four unidentified lipids. The DNA G+C content of strain CE80T was 55.9 mol%. The major respiratory quinone was Q-10. DNA-DNA relatedness between strain CE80T and L. suaedae YC6927T was 56.1±2.8%. On the basis of physiological and biochemical characterization and phylogenetic and chemotaxonomic analysis, strain CE80T represents a novel species of the Labrenzia, for which the name Labrenzia callyspongiae sp. nov., is proposed. The type strain is CE80T (=KCTC 42849T =JCM 31309T).

New Cytotoxic Cyclic Peptide from the Marine Sponge-Associated Nocardiopsis sp. UR67.

A new cyclic hexapeptide, nocardiotide A (1), together with three known compounds-tryptophan (2), kynurenic acid (3), and 4-amino-3-methoxy benzoic acid (4)-were isolated and identified from the broth culture of Nocardiopsis sp. UR67 strain associated with the marine sponge Callyspongia sp. from the Red Sea. The structure elucidation of the isolated compounds were determined based on detailed spectroscopic data including ¹D and ²D nuclear magnetic resonance (NMR) experimental analyses in combination with high resolution electrospray ionization mass spectrometry (HR-ESI-MS), while the absolute stereochemistry of all amino acids components of nocardiotide A (1) was deduced using Marfey's method. Additionally, ten known metabolites were dereplicated using HR-ESI-MS analysis. Nocardiotide A (1) displayed significant cytotoxic effects towards the murine CT26 colon carcinoma, human HeLa cervix carcinoma, and human MM.1S multiple myeloma cell lines. The results obtained revealed sponge-associated Nocardiopsis as a substantial source of lead natural products with pronounced pharmacological activities.

Volatiles from the fungal microbiome of the marine sponge Callyspongia cf. flammea.

The volatiles emitted by five fungal strains previously isolated from the marine sponge Callyspongia cf. flammea were captured with a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Besides several widespread compounds, a series of metabolites with interesting bioactivities were found, including the quorum sensing inhibitor protoanemonin, the fungal phytotoxin 3,4-dimethylpentan-4-olide, and the insect attractant 1,2,4-trimethoxybenzene. In addition, the aromatic polyketides isotorquatone and chartabomone that are both known from Eucalyptus and a new O-desmethyl derivative were identified. The biosynthesis of isotorquatone was studied by feeding experiments with isotopically labeled precursors and its absolute configuration was determined by enantioselective synthesis of a reference compound. Bioactivity testings showed algicidal activity for some of the identified compounds, suggesting a potential ecological function in sponge defence.

Epipolythiodiketopiperazines from the Marine Derived Fungus Dichotomomyces cejpii with NF-κB Inhibitory Potential.

The Ascomycota Dichotomomyces cejpii was isolated from the marine sponge Callyspongia cf. C. flammea. A new gliotoxin derivative, 6-acetylmonodethiogliotoxin (1) was obtained from fungal extracts. Compounds 2 and 3, methylthio-gliotoxin derivatives were formerly only known as semi-synthetic compounds and are here described as natural products. Additionally the polyketide heveadride (4) was isolated. Compounds 1, 2 and 4 dose-dependently down-regulated TNFα-induced NF-κB activity in human chronic myeloid leukemia cells with IC50s of 38.5 ± 1.2 µM, 65.7 ± 2.0 µM and 82.7 ± 11.3 µM, respectively. The molecular mechanism was studied with the most potent compound 1 and results indicate downstream inhibitory effects targeting binding of NF-κB to DNA. Compound 1 thus demonstrates potential of epimonothiodiketopiperazine-derived compounds for the development of NF-κB inhibitors.

A statistical approach for optimization of polyhydroxybutyrate production by marine Bacillus subtilis MSBN17.

The important biological macromolecule polyhydroxybutyrate (PHB) producing Bacillus subtilis was isolated from the marine sponge Callyspongia diffusa and identified by means of 16S rRNA analysis. The central composite design (CCD) was used to optimize the PHB production using cheap raw materials such as pulp industry waste (PIW), tamarind kernel powder (TKP), palm jaggery (PJ) and green gram flour (GGF). The extracted polymer was characterized by (1)H NMR analysis. The PIW was fed at three different intervals and the maximum production of PHB (19.08g/L) was attained after a period of 40h of incubation of B. subtilis. Dissolved oxygen, sodium chloride and nitrogen source were found to be the critical control factors that affected the PHB polymer production. The present investigation demonstrates an inexpensive model of producing PHB green thermoplastics in vitro for biomedical applications.

6-Hydroxymethyl-1-phenazine-carboxamide and 1,6-phenazinedimethanol from a marine bacterium, Brevibacterium sp. KMD 003, associated with marine purple vase sponge.

Two new antibacterial phenazines were isolated from the culture broth of Brevibacterium sp. KMD 003 obtained from a marine purple vase sponge of the genus Callyspongia, collected in Kyeongpo, Gangwondo, Korea. The structures of these compounds were determined to be 6-hydroxymethyl-1-phenazine-carboxamide (1) and 1,6-phenazinedimethanol (2) through analyses of HR-EI-MS and NMR data. Compounds 1 and 2 showed antibacterial activities against Enterococcus hirae and Micrococcus luteus with 5 microM MIC values.