Marine seaweed endophytic fungi-derived active metabolites promote reactive oxygen species-induced cell cycle arrest and apoptosis in human breast cancer cells.

BACKGROUND: Endophytic fungi have an abundant sources rich source of rich bioactive molecules with pivotal pharmacological properties. Several studies have found that endophytic fungi-derived bioactive secondary metabolites have antiproliferative, anti-oxidant, and anti-inflammatory properties, but the molecular mechanism by which they induce cell cycle arrest and apoptosis pathways is unknown. This study aimed to determine the molecular mechanism underlying the anticancer property of the endophytic fungi derived active secondary metabolites on human breast cancer cells. METHODS: In this study, we identified four endophytic fungi from marine seaweeds and partially screened its phytochemical properties by Chromatography-Mass Spectrometry (GC-MS) analysis. Moreover, the molecular mechanism underlying the anticancer property of these active secondary metabolites (FA, FB, FC and FE) on human breast cancer cells were examined on MCF-7 cells by TT assay, Apoptotic assay by Acridine orang/Ethidium Bromide (Dual Staining), DNA Fragmentation by DAPI Staining, reactive oxygen species (ROS) determination by DCFH-DA assay, Cell cycle analysis was conducted Flow cytometry and the apoptotic signalling pathway was evaluated by westernblot analysis. Doxorubicin was used as a positive control drug for this experiment. RESULTS: The GC-MS analysis of ethyl acetate extract of endophytic fungi from the marine macro-algae revealed the different functional groups and bioactive secondary metabolites. From the library, we observed the FC (76%), FB (75%), FA (73%) and FE (71%) have high level of antioxidant activity which was assessed by DPPH scavenging assay. Further, we evaluated the cytotoxic potentials of these secondary metabolites on human breast cancer MCF-7 cells for 24 h and the IC50 value were calculated (FA:28.62 ± 0.3 µg/ml, FB:49.81 ± 2.5 µg/ml, FC:139.42 ± µg/ml and FE:22.47 ± 0.5 µg/ul) along with positive control Doxorubicin 15.64 ± 0.8 µg/ml respectively by MTT assay. The molecular mechanism by which the four active compound induced apoptosis via reactive oxygen species (ROS) and cell cycle arrest in MCF-7 cells was determined H2DCFDA staining, DAPI staining, Acridine orange and ethidium bromide (AO/EtBr) dual staining, flowcytometry analysis with PI staining and apoptotic key regulatory proteins expression levels measured by westernblot analysis. CONCLUSION: Our findings, revealed the anticancer potential of endophytic fungi from marine seaweed as a valuable source of bioactive compounds with anticancer properties and underscore the significance of exploring marine-derived endophytic fungi as a promising avenue for the development of novel anticancer agents. Further investigations are necessary to isolate and characterize specific bioactive compounds responsible for these effects and to validate their therapeutic potential in preclinical and clinical settings.

Ecology and evolution of algal-fungal symbioses.

Ecological interactions and symbiosis between algae and fungi are ancient, widespread, and diverse with many independent origins. The heterotrophic constraint on fungal nutrition drives fungal interactions with autotrophic organisms, including algae. While ancestors of modern fungi may have evolved as parasites of algae, there remains a latent ability in algae to detect and respond to fungi through a range of symbioses that are witnessed today in the astounding diversity of lichens, associations with corticoid and polypore fungi, and endophytic associations with macroalgae. Research into algal-fungal interactions and biotechnological innovation have the potential to improve our understanding of their diversity and functions in natural systems, and to harness this knowledge to develop sustainable and novel approaches for producing food, energy, and bioproducts.

Effects of UV-B radiation on epiphytic bacterial communities on male and female Sargassum thunbergii.

The effects of increased UV-B radiation on macroalgae have been widely studied, but knowledge concerning the response of communities of algal epiphytic bacteria to increased UV-B radiation and differences between male and female algae is still lacking. Via 16S rDNA high-throughput sequencing technology, changes in the epiphytic bacterial communities on male and female S. thunbergii under increased UV-B radiation were studied in the lab. Under different UV-B radiation intensities, although the α diversity and community composition of epiphytic bacteria changed little, the ß diversity indicated that the community structure of bacteria on S. thunbergii was obviously clustered, and the relative abundance of dominant bacteria and indicator species changed considerably. There were unique bacteria in each experimental group, and the bacteria whose abundance obviously changed were members of groups related to environmental resistance or adaptability. The variation in the abundance of epiphytic bacteria was different in male and female S. thunbergii, and the bacteria whose abundance greatly changed were mainly related to algal growth and metabolism. The abundance of genes with predicted functions related to metabolism, genetic information processing, environmental adaptation and infectious diseases changed with increased UV-B radiation, and those variations differed between epiphytic bacteria on male and female S. thunbergii. This study found that the algal epiphytic bacteria were influenced by the increase in UV-B radiation and underwent certain adaptations through adjustments to community structure and function, and this response was also affected by the sex of the macroalgae. These results are expected to serve as experimental basis and provide reference for further understanding of the response of algae epiphytic bacteria to enhanced UV-B radiation caused by the thinning of the ozone layer and the resulting changes in the relationship between algae and bacteria, which may change the community of the marine ecosystem and affect important marine ecological process.

Seaweed-associated heterotrophic Bacillus altitudinis MTCC13046: a promising marine bacterium for use against human hepatocellular adenocarcinoma.

Since the report of the antibiotic with anticancer properties, scientists have been focusing to isolate and characterize novel anti-microbial natural products possessing anticancer activities. The current study describes the production of seaweed-associated heterotrophic Bacillus altitudinis MTCC13046 with potential anticancer properties. The bacterium was screened for its capacity to diminish the cell proliferation of the human hepatocellular adenocarcinoma (HepG2) cell line, without upsetting the normal cells. The bacterial extract showed anticancer properties in a dose-reactive form against HepG2 (IC50, half maximal inhibitory concentration ~ 29.5 µg/ml) on tetrazolium bromide analysis with less significant cytotoxicity on common fibroblast (HDF) cells (IC50 ~ 77 µg/ml). The potential antioxidant ability of the organic extract of B. altitudinis MTCC13046 (IC90 133 µg/ml) could corroborate its capacity to attenuate the pathophysiology leading to carcinogenesis. The results of the apoptosis assay showed that the crude extracts of B. altitudinis maintained 68% viability in normal cells compared to 11% in the cancer cells (IC50 76.9 µg/ml). According to the findings, B. altitudinis MTCC13046 could be used to develop prospective anticancer agents.

Application of Feature-Based Molecular Networking for Comparative Metabolomics and Targeted Isolation of Stereoisomers from Algicolous Fungi.

Seaweed endophytic (algicolous) fungi are talented producers of bioactive natural products. We have previously isolated two strains of the endophytic fungus, Pyrenochaetopsis sp. FVE-001 and FVE-087, from the thalli of the brown alga Fucus vesiculosus. Initial chemical studies yielded four new decalinoylspirotetramic acid derivatives with antimelanoma activity, namely pyrenosetins A-C (1-3) from Pyrenochaetopsis sp. strain FVE-001, and pyrenosetin D (4) from strain FVE-087. In this study, we applied a comparative metabolomics study employing HRMS/MS based feature-based molecular networking (FB MN) on both Pyrenochaetopsis strains. A higher chemical capacity in production of decalin derivatives was observed in Pyrenochaetopsis sp. FVE-087. Notably, several decalins showed different retention times despite the same MS data and MS/MS fragmentation pattern with the previously isolated pyrenosetins, indicating they may be their stereoisomers. FB MN-based targeted isolation studies coupled with antimelanoma activity testing on the strain FVE-087 afforded two new stereoisomers, pyrenosetins E (5) and F (6). Extensive NMR spectroscopy including DFT computational studies, HR-ESIMS, and Mosher's ester method were used in the structure elucidation of compounds 5 and 6. The 3'R,5'R stereochemistry determined for compound 6 was identical to that previously reported for pyrenosetin C (3), whose stereochemistry was revised as 3'S,5'R in this study. Pyrenosetin E (5) inhibited the growth of human malignant melanoma cells (A-375) with an IC50 value of 40.9 µM, while 6 was inactive. This study points out significant variations in the chemical repertoire of two closely related fungal strains and the versatility of FB MN in identification and targeted isolation of stereoisomers. It also confirms that the little-known fungal genus Pyrenochaetopsis is a prolific source of complex decalinoylspirotetramic acid derivatives.

Seaweed-associated heterotrophic bacteria: new paradigm of prospective anti-infective and anticancer agents.

Ever since the development of the first antibiotic compound with anticancer potential, researchers focused on isolation and characterization of prospective microbial natural products with potential anti-infective and anticancer activities. The present work describes the production of bioactive metabolites by heterotrophic bacteria associated with intertidal seaweeds with potential anti-infective and anticancer activities. The bacteria were isolated in a culture-dependent method and were identified as Shewanella algae MTCC 12715 (KX272635) and Bacillus amyloliquefaciens MTCC 12716 (KX272634) based on combined phenotypic and genotypic methods. Further, the bacteria were screened for their ability to inhibit drug-resistant infectious pathogens and prevent cell proliferation of human liver carcinoma (HepG2) and breast cancer (MCF7) cell lines, without affecting the normal cells. Significant anti-infective activity was observed with bacterial cells and their organic extracts against broad-spectrum multidrug-resistant pathogens, such as vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia and Pseudomonas aeruginosa with minimum inhibitory concentration ≤ 3.0 µg mL-1 as compared to the antibiotic agents' chloramphenicol and ampicillin, which were active at ≥ 6.25 mg mL-1. The extracts also exhibited anticancer activity in a dose-responsive pattern against HepG2 (with IC50, half maximal inhibitory concentration ~ 78-83 µg mL-1) and MCF7 (IC50 ~ 45-48 µg mL-1) on tetrazolium bromide screening assay with lesser cytotoxic effects on normal fibroblast (L929) cell lines (IC50 > 100 µg mL-1). The results revealed that seaweed-associated heterotrophic bacteria could occupy a predominant role for a paradigm shift towards the development of prospective anti-infective and anticancer agents.

Pseudoalteropeptide A, a novel lipopeptide from the marine bacterium Pseudoalteromonas piscicida SWA4_PA4 isolated from marine seaweed.

A new lipopeptide, pseudoalteropeptide A (1) was isolated from the marine bacterium Pseudoalteromonas piscicida SWA4_PA4. The structure was elucidated by spectroscopic analyses including NMR and MSMS spectra. It showed moderate iron chelating activity as well as cytotoxic activity against Jurkat human T lymphocyte cells. isolation/marine bacterium/natural product/structure elucidation.

Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications.

BACKGROUND: Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost-effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization. RESULTS: Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum, Stemphylium lucomagnoense and Aspergillus nidulans. Among these five isolates, one T. asperellum strain (T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator. CONCLUSIONS: The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO4 as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

Hitherto Unknown Terpene Synthase Organization in Taxol-Producing Endophytic Bacteria Isolated from Marine Macroalgae.

Taxol is a successful anti-cancer drug, which extensively studied in Taxus spp. However, microbial endophytes also reported as taxol producers, and especially fungal endophytes extensively studied for the taxol biosynthesis pathway. Although it was well considered, the taxol biosynthesis pathway remains undisclosed since its discovery in bacteria. To decipher this gap, we isolated and identified the endophytic bacteria such as Bacillus flexus strain DMTMMB08, Bacillus licheniformis strain DMTMMB10, and Oceanobacillus picturae strain DMTMMB24, which are unprecedented for taxol production. Subsequently, the genome annotation of these bacteria exhibited the isoprene biosynthesis pathway and terpene synthase profile. Feasibly, this is the very first report on taxol-producing endophytic bacteria from the non-Taxus host and solitary investigation on its genome analysis. The genomic insight into the bacterial system for taxol biosynthesis leads to understanding the terpene synthesis and evolution. This piece of work could expand our perception of the diversity of terpenes and their related natural products.

Putridiphycobacter roseus gen. nov., sp. nov., isolated from Antarctic rotten seaweed.

A Gram-stain-negative, pink-pigmented, rod-shaped, non-flagellated, aerobic bacterium, designated strain SM1701T, was isolated from a rotten seaweed collected off Fildes Peninsula, King George Island, West Antarctica. The strain grew at 4-30 °C, pH 6.0-8.0 and with 0.5-5 % (w/v) NaCl. It hydrolysed gelatin and Tweens (40, 60 and 80), but did not reduce nitrates to nitrites. The major cellular fatty acids of strain SM1701T were iso-C15 : 0, iso-C15 : 1G, iso-C16 : 1G, C16 : 0 and iso-C17 : 0 3-OH. Polar lipids included phosphatidylethanolamine, one unidentified aminolipid, two unidentified glycolipids and one unidentified aminoglycolipid. The major respiratory quinone was MK-7. The genomic DNA G+C content of strain SM1701T was 34.1 mol%. It showed high 16S rRNA gene sequence similarities to Crocinitomix algicola (93.8 %) and Crocinitomix catalasitica (92.5 %) and less than 91 % sequence similarities to other known members in the family Crocinitomicaceae. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SM1701T constituted a distinct lineage within the family Crocinitomicaceae. The phylogenetic trees based on concatenated 261 protein sequences from genome sequences showed that strain SM1701T occupied a branch separated from those of known genera in the family of Crocinitomicaceae, indicating it may belong to a new genus. On the basis of the polyphasic characterization of strain SM1701T in this study, it is considered to represent a novel species in a new genus in the family Crocinitomicaceae, for which the name Putridiphycobacter roseus gen. nov., sp. nov. is proposed. The type strain is SM1701T (=KCTC 62302T=NBRC 113201T=CGMCC 1.16510T).

Aspermicrones A-C, novel dibenzospiroketals from the seaweed-derived endophytic fungus Aspergillus micronesiensis.

Chemical investigation of the Kappaphycus alvarezii-derived endophytic fungus Aspergillus micronesiensis lead to the isolation of three novel dibenzospiroketals, aspermicrones A-C (1-3). Their chemical structures were determined by extensive analysis of HR-ESI-MS and NMR spectral data. The absolute configurations of them were determined by experimental and TD-DFT theoretical calculated circular dichroism spectra. Compound 2 exhibited selective cytotoxic effect toward HepG2 cell line (IC50 = 9.9 µM). Additionally, both of compounds 2 and 3 displayed anti-microbial activity against Staphylococcus aureus (MIC = 123.2 µM for each compound). Compound 1 was inactivity in both cytotoxic and anti-microbial assays.

New Ophiobolin Derivatives from the Marine Fungus Aspergillus flocculosus and Their Cytotoxicities against Cancer Cells.

Five new sesterterpenes, 14,15-dehydro-6-epi-ophiobolin K (1), 14,15-dehydro- ophiobolin K (2), 14,15-dehydro-6-epi-ophiobolin G (3), 14,15-dehydro-ophiobolin G (4) and 14,15-dehydro-(Z)-14-ophiobolin G (5), together with four known ophiobolins (6-9) were isolated from the marine fungus Aspergillus flocculosus derived from the seaweed Padina sp. collected in Vietnam. The five new ophiobolins were first isolated as ophiobolin derivatives consisting of a fully unsaturated side chain. Their structures were elucidated via spectroscopic methods including 1D, 2D NMR and HR-ESIMS. The absolute configurations were determined by the comparison of chemical shifts and optical rotation values with those of known ophiobolins. All compounds (1-9) were then evaluated for their cytotoxicity against six cancer cell lines, HCT-15, NUGC-3, NCI-H23, ACHN, PC-3 and MDA-MB-231. All the compounds showed potent cytotoxicity with GI50 values ranging from 0.14 to 2.01 µM.

Alteromonas fortis sp. nov., a non-flagellated bacterium specialized in the degradation of iota-carrageenan, and emended description of the genus Alteromonas.

Strain 1T, isolated in the 1970s from the thallus of the carrageenophytic red algae, Eucheuma spinosum, collected in Hawaii, USA, was characterized using a polyphasic method. Cells were Gram-stain-negative, strictly aerobic, non-flagellated, ovoid or rod-shaped and grew optimally at 20-25 °C, at pH 6-9 and with 2-4 % NaCl. Strain 1T used the seaweed polysaccharides ι-carrageenan, laminarin and alginic acid as sole carbon sources. The major fatty acids were C16 : 0, C18 : 1 ω7c and summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2OH) with significant amounts (>6 %) of C16 : 0 N alcohol and 10 methyl C17 : 0. The respiratory quinone was Q-8 and major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and an unknown aminolipid. Phylogenetic analyses showed that the bacterium is affiliated to the genus Alteromonas (family Alteromonadaceae, class Gammaproteobacteria). Strain 1T exhibited 16S rRNA gene sequence similarity values of 98.8 and 99.2 % to the type strains of Alteromonas mediterranea and Alteromonas australica respectively, and of 95.2-98.6 % to other species of the genus Alteromonas. The DNA G+C content of strain 1T was determined to be 43.9 mol%. Digital DNA-DNA hybridization predictions by the ANI and GGDC methods between strain 1T and other members of the genus Alteromonas showed values below 83 % and 30 %, respectively. The phenotypic, phylogenetic and genomic analyses show that strain 1T is distinct from species of the genus Alteromonas with validly published names and that it represents a novel species of the genus Alteromonas, for which the name Alteromonasfortis sp. nov. is proposed. The type strain is 1T (=ATCC 43554T=RCC 5933T=CIP 111645T=DSM 106819T).

Influence of OSMAC-Based Cultivation in Metabolome and Anticancer Activity of Fungi Associated with the Brown Alga Fucus vesiculosus.

The fungi associated with marine algae are prolific sources of metabolites with high chemical diversity and bioactivity. In this study, we investigated culture-dependent fungal communities associated with the Baltic seaweed Fucus vesiculosus. Altogether, 55 epiphytic and endophytic fungi were isolated and identified. Twenty-six strains were selected for a small-scale One-Strain-Many-Compounds (OSMAC)-based fermentation in four media under solid and liquid culture regimes. In total, 208 fungal EtOAc extracts were tested for anticancer activity and general cytotoxicity. Ten most active strains (i.e., 80 extracts) were analyzed for their metabolome by molecular networking (MN), in-silico MS/MS fragmentation analysis (ISDB⁻UNPD), and manual dereplication. Thirty-six metabolites belonging to 25 chemical families were putatively annotated. The MN clearly distinguished the impact of culture conditions in chemical inventory and anticancer activity of the fungal extracts that was often associated with general toxicity. The bioactivity data were further mapped into MN to seek metabolites, exclusively expressed in the active extracts. This is the first report of cultivable fungi associated with the Baltic F. vesiculosus that combined an OSMAC and an integrated MN-based untargeted metabolomics approaches for efficient assessment and visualization of the impact of the culture conditions on chemical space and anticancer potential of the fungi.

Amycolatopsis antarctica sp. nov., isolated from the surface of an Antarctic brown macroalga.

Two moderately psychrophilic actinobacterial strains, designated AU-G6T and AU-A3.2, isolated from the surface of an Antarctic macroalga, Adenocystis utricularis (Bory) Skottsberg, was taxonomically characterized based on a polyphasic investigation. The two strains had nearly identical 16S rRNA gene sequences and formed a distinct phyletic line within the genus Amycolatopsis of the family Pseudonocardiaceae. They were phylogenetically close to Amycolatopsis nigrescens JCM 14717T, Amycolatopsis minnesotensis JCM 14545T and Amycolatopsis magusensis DSM 45510T, with 16S rRNA gene sequence similarities of 97.77, 97.20 and 97.19 %, respectively. Phylogenomic analysis based on the whole genome data supported that strain AU-G6T was distantly related to the Amycolatopsis species. The isolates shared a range of phenotypic markers typical of members of the genus Amycolatopsis, but also had a range of cultural, physiological and biochemical characteristics that separated them from related Amycolatopsis species. The isolates showed growth only in media supplemented with salt, indicating their marine origin. The cell wall of the isolates contained meso-diaminopimelic acid, and arabinose and galactose were detected as diagnostic sugars (type IV). The main menaquinone was MK-9(H4). The main polar lipids were phosphatidylethanolamine, hydroxy-phosphotidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol (type II). The fatty acid type was 3c. The combined genotypic and phenotypic data indicated that the two isolates represent a novel species of the genus Amycolatopsis. The name proposed for this species is Amycolatopsis antarctica sp. nov., with type strain AU-G6T (=CGMCC 4.7351T=NBRC 112404T).

Dokdonia aurantiaca sp. nov., isolated from seaweed Zostera marina.

A non-motile, orange-coloured and rod-shaped bacterial strain, designated strain ZOW29T, was isolated from a seaweed sample collected from the South Sea, Republic of Korea. Cells were Gram-stain-negative, aerobic and non-motile. The isolate required sea salts for growth. Carotenoid pigment was produced. A phylogenetic tree based on 16S rRNA gene sequences showed that strain ZOW29T forms an evolutionary lineage within the radiation enclosing the members of the genus Dokdonia with Dokdonia diaphoros MSKK-32T, Dokdonia eikasta PMA-26Tand Dokdonia donghaensis DSW-1T (97.1 % sequence similarity each) as its nearest neighbours. The DNA-DNA relatedness values between strain ZOW29T and these four type strains were 35-48 %. The major fatty acids were iso-C15:0, iso-C17 : 0 3-OH and iso-C15 : 1 G. Strain ZOW29T contained MK-6 and phosphatidylethanolamine, an unidentified aminolipid and an unidentified polar lipid as the only isoprenoid quinone and the major polar lipids, respectively. The DNA G+C content of strain ZOW29T was 38 mol%. On the basis of polyphasic characterization, it is suggested that the isolate represents a novel species of the genus Dokdonia, for which the name Dokdonia aurantiaca sp. nov. (type strain, ZOW29T=KCTC 52956T=JCM 32295T) is proposed.

Phylogeny and bioactivity of epiphytic Gram-positive bacteria isolated from three co-occurring antarctic macroalgae.

Marine macroalgae are emerging as an untapped source of novel microbial diversity and, therefore, of new bioactive secondary metabolites. This study was aimed at assessing the diversity and antimicrobial activity of the culturable Gram-positive bacteria associated with the surface of three co-occurring Antarctic macroalgae. Specimens of Adenocystis utricularis (brown alga), Iridaea cordata (red alga) and Monostroma hariotii (green alga) were collected from the intertidal zone of King George Island, Antarctica. Gram-positive bacteria were investigated by cultivation-based methods and 16S rRNA gene sequencing, and screened for antimicrobial activity against a panel of pathogenic microorganisms. Isolates were found to belong to 12 families, with a dominance of Microbacteriaceae and Micrococcaceae. Seventeen genera of Actinobacteria and 2 of Firmicutes were cultured from the three macroalgae, containing 29 phylotypes. Three phylotypes within Actinobacteria were regarded as potentially novel species. Sixteen isolates belonging to the genera Agrococcus, Arthrobacter, Micrococcus, Pseudarthrobacter, Pseudonocardia, Sanguibacter, Staphylococcus, Streptomyces and Tessaracoccus exhibited antibiotic activity against at least one of the indicator strains. The bacterial phylotype composition was distinct among the three macroalgae species, suggesting that these macroalgae host species-specific Gram-positive associates. The results highlight the importance of Antarctic macroalgae as a rich source of Gram-positive bacterial diversity and potentially novel species, and a reservoir of bacteria producing biologically active compounds with pharmacological potential.

Dokdonia flava sp. nov., isolated from the seaweed Zostera marina.

A non-motile, proteorhodopsin-containing, yellow and rod-shaped bacterial strain, designated ZODW10T, was isolated from the seaweed Zostera marina collected from the West Sea, Republic of Korea. Cells were Gram-stain-negative, aerobic and non-motile. The isolate required sea salts for growth. A carotenoid pigment was produced. A phylogenetic tree based on 16S rRNA gene sequences showed that strain ZODW10T forms an evolutionary lineage within the radiation enclosing members of the genus Dokdonia with Dokdoniadiaphoros CIP 108745T (96.7 % sequence similarity) as its nearest neighbour. The major fatty acids were iso-C15:0, iso-C17 : 0 3-OH and iso-C15 : 1 G. Strain ZODW10T contained menaquinone 6 (MK-6) and phosphatidylethanolamine, an unidentified aminolipid and an unidentified polar lipid as the only isoprenoid quinone and the major polar lipids, respectively. The DNA G+C content of strain ZODW10T was 36 mol%. On the basis of the present polyphasic characterization, it is suggested that the isolate represents a novel species of the genus Dokdonia, for which the name Dokdonia flava sp. nov. (type strain, ZODW10T=KCTC 52953T=JCM 32293T) is proposed.

Marinirhabdus citrea sp. nov., a marine bacterium isolated from a seaweed.

A gram-stain-negative, aerobic, rod-shaped (1.3-1.9×0.3-0.5 µm) and non-motile marine bacterium, designated MEBiC09412T, was isolated from seaweed collected at Yeonggwang County, South Korea. 16S rRNA gene sequence analysis demonstrated that strain MEBiC09412T shared high sequence similarity with Marinirhabdus gelatinilytica NH83T (95.4 %). Growth was observed at 17-38 °C (optimum 30 °C), at pH 4.0-8.5 (optimum pH 7.0) and with 0.5-6.0 % (w/v; optimum 2.5 %) NaCl. The predominant cellular fatty acids were iso-C15 : 0 (27.4 %), iso-C15 : 1 G (9.6 %), anteiso-C15 : 0 (14.6 %), iso-C16 : 0 (6.2 %), iso-C17 : 0 3OH (13.2 %) and summed feature 3 (comprising C16 : 1ω6c and/or C16 : 1ω7c; 7.4 %). The DNA G+C content was determined to be 43.1 mol%, while the major respiratory quinone was menaquinone-6. Several phenotypic characteristics such as indole production, the oxidizing patterns of several carbohydrtaes (of glucose, fructose, sucrose, maltose, mannose etc.) and organic acids, and the enzyme activities of α-chymotrypsin and α-glucosidase differentiated strain MEBiC09412T from M. gelatinilytica NH83T. On the basis of this polyphasic taxonomic data, strain MEBiC09412T should be classified as a novel species of the genus Marinirhabduswith the suggested name Marinirhabdus citrea sp. nov. The type strain is MEBiC09412T (=KCCM 43216T=JCM 31588T).

Flavivirga eckloniae sp. nov. and Flavivirga aquimarina sp. nov., isolated from seaweed Ecklonia cava.

Two Gram-stain-negative, non-spore-forming, rod-shaped, aerobic and yellow-coloured bacterial strains, designated strains ECD14T and EC2D5T, were isolated from a seaweed Eckloniacava. The isolates required sea salts for growth. Flexirubin-type and carotenoid pigment was produced. The 16S rRNA gene sequence similarity between the two new strains was 95.5 %. Flavivirga amylovorans JC2681T was the nearest neighbour of strains ECD14T and EC2D5T with 96.5 and 96.8 % 16S rRNA gene sequence similarity, respectively. The common major fatty acids and respiratory quinones were iso-C15 : 0, iso-C15 : 1 G and unknown 13.565 and menaquinone 6 (MK-6), respectively. The common major polar lipids were phosphatidylethanolamine, an unknown amino lipid and an unknown lipid. The DNA G+C contents of strains ECD14T and EC2D5T were 33 and 31 mol%, respectively. On the basis of the polyphasic characterization of the two strains, it is suggested that the two isolates represent novel species of the genus Flavivirga, for which the names Flavivirga eckloniae sp. nov. (type strain, ECD14T=KCTC 52352T=JCM 31797T) and Flavivirga aquimarina sp. nov. (type strain, EC2D5T=KCTC 52353T=JCM 31796T) are proposed.