The Requirement of Selenium for the Growth of Marine Coccolithophorids, Emiliania huxleyi, Gephyrocapsa oceanica and Helladosphaera sp. (Prymnesiophyceae)

Marine coccolithophorids, Emiliania huxleyi, Gephyrocapsa oceanicaand Helladosphaera sp. were found to require selenium for theirgrowth. The optimum concen trations were 1–10 nM for SeO₂and SeO₃^2– and above 1 µM for SeO₂^4– Duringthe depletion of selenium algal growth was strongly suppressedaccompanied with the decrease in net photosynthesis. (Received November 18, 1998; Accepted April 16, 1999)     

Identification and functional characterisation of genes encoding the omega-3 polyunsaturated fatty acid biosynthetic pathway from the coccolithophore Emiliania huxleyi

The Prymnesiophyceae coccolithophore Emiliania huxleyi is one of the most abundant alga in our oceans and therefore plays a central role in marine foodwebs. E. huxleyi is notable for the synthesis and accumulation of the omega-3 long chain polyunsaturated fatty acid docosahexaenoic acid (DHA; 22:6Δ^{4,7,10,13,16,19}, n − 3) which is accumulated in fish oils and known to have health-beneficial properties to humans, preventing cardiovascular disease and related pathologies. Here we describe the identification and functional characterisation of the five E. huxleyi genes which direct the synthesis of docosahexaenoic acid in this alga. Surprisingly, E. huxleyi does not use the conventional Δ6-pathway, instead using the alternative Δ8-desaturation route which has previously only been observed in a few unrelated microorganisms. Given that E. huxleyi accumulates significant levels of the Δ6-desaturated fatty acid stearidonic acid (18:4Δ^6,9,12,15, n − 3), we infer that the biosynthesis of DHA is likely to be metabolically compartmentalised from the synthesis of stearidonic acid.     

Characterization of Satellite DNA Sequences from the Commercially Important Marine Rotifers Brachionus rotundiformis and Brachionus plicatilis

This study was initiated to search for species-specific and strain-specific satellite DNA sequences for which oligonucleotide primers could be designed to differentiate between various commercially important strains of the marine monogonont rotifers Brachionus rotundiformis and Brachionus plicatilis. Two unrelated, highly reiterated satellite sequences were cloned and characterized. The eight sequenced monomers from B. rotundiformis and six from B. plicatilis had low intrarepeat variability and were similar in their overall lengths, A + T compositions, and high degrees of repeated motif substructure. However, hybridizations to 19 representative strains, sequence characterizations, and GenBank searches indicated that these two satellites are morphotype-specific and population-specific, respectively, and share little homology to each other or to other characterized sequences in the database. Primer pairs designed for the B. rotundiformis satellite confirmed hybridization specificities on polymerase chain reaction and could serve as a useful molecular diagnostic tool to identify strains belonging to the SS morphotype, which are gaining widespread usage as first feeds for marine fish in commercial production. Received April 12, 1999; accepted July 29, 1999.     

Construction of Plasmid Vectors and Transformation of the Marine Yeast Debaryomyces hansenii

We have constructed two plasmid vectors (pMR95 and pMR96) with selectable markers for the marine yeast Debaryomyces hansenii. Plasmid pMR95 contains an autonomously replicating sequence previously isolated from Debaryomyces and a hygromycin B resistance gene from the plasmid pLG90 under the control of the isocytochrome C1 promoter and terminator sequences, while pMR96 has, in addition, the Saccharomyces URA3 gene. Transformation in Debaryomyces was accomplished by electroporation. Plasmid pMR95 was capable of transforming both Saccharomyces cerevisiae and D. hansenii to hygromycin resistance at low frequencies; pMR96 transformed both yeasts at low frequencies when selected for hygromycin B resistance and at very high efficiencies when selected for uracil prototrophy. The presence of the plasmids in the transformed yeast was confirmed by polymerase chain reaction. The plasmids could be recovered back in Escherichia coli when transformed with total DNA from the yeast transformants, indicating at least a partial autonomous existence of the plasmids in the marine yeast. To our knowledge this is the first successful attempt to transform D. hansenii. Received April 16, 1998; accepted June 30, 1998.     

Antifouling Potential of Some Marine Organisms from India Against Species of Bacillus and Pseudomonas

Crude methanolic extracts of 37 marine organisms (16 species of flora, 21 species of fauna) were screened for antibacterial properties against 5 strains of bacteria isolated from marine environments. Of these, 10 plant and 9 animal extracts exhibited antibacterial activity against at least one bacterial strain. The extracts of 6 species were active against all the strains: i.e., Stoechospermum marginatum (brown algae), Cymodocea rotundata (seagrass), Petrosia sp. and Psammaplysilla purpurea (sponges), Sinularia compressa (soft coral), and Cassiopeia sp. (jellyfish). Among the plants, Padina tetrastromatica (brown algae) extract exhibited significant activity (9–11-mm inhibition zone at 500 μg per 6-mm disc) against Bacillus pumilus and Pseudomonas vesicularis, while the extracts of Petrosia, Psammaplysilla, and Cassiopeia were strongly active (11–13-mm inhibition zone at 500 μg per 6-mm disc) against B. circulans and P. putida. It was further confirmed that the attachment of bacterial strains on glass slides was inhibited remarkably with increasing concentrations of bioextracts of Petrosia sp. and Psammaplysilla purpurea. The present findings could form the basis for exploring the antibacterial potential of bioactive molecules from some of the marine organisms that exhibited moderate to strong antibacterial properties.     

Isolation and Culture of a Marine Bacterium Degrading the Sulfated Fucans from Marine Brown Algae

Fucoidans are matrix polysaccharides from marine brown algae, consisting of an α-l-fucose backbone substituted by sulfate-ester groups and masked with ramifications containing other monosaccharide residues. In spite of their interest as biologically active compounds in a number of homologous and heterologous systems, no convenient sources with fucanase activity are available yet for the degradation of the fucalean algae. We here report on the isolation, characterization, and culture conditions of a bacterial strain capable of degrading various brown algal fucoidans. This bacterium, a member of the family Flavobacteriaceae, was shown to secrete fucoidan endo-hydrolase activity. An extracellular enzyme preparation was used to degrade the fucoidan from the brown alga Pelvetia canaliculata. End products included a tetrasaccharide and a hexasaccharide made of the repetition of disaccharidic units consisting of α-1→3-l-fucopyranose-2-sulfate-α-1→4-l-fucopyranose-2,3-disulfate, with the 3-linked residues at the nonreducing end.     

抗肿瘤海绵放线菌的分离及菌株HA01184的鉴定

目的：对采自海南、湛江等海域的海绵样品进行放线菌选择性分离,采用其发酵液进行抗肿瘤活性筛选,并对活性较好的菌株进行鉴定。方法：用含50μg／mL重铬酸钾为抑制剂的海水高氏一号合成培养基分离培养海绵放线菌;以MTT法进行菌株的抗肿瘤活性筛选;通过培养特征、形态特征、生理生化特征、16S rDNA序列测定及系统发育分析,对菌株HA01184进行鉴定。结果与结论：海水高氏一号合成培养基用于海绵放线菌分离培养具有很好的选择性,从海绵样品中共分离得到放线菌165株,细胞毒活性达80％以上的阳性菌株有10株,其中菌株HA01184的发酵液细胞毒活性为90％。结合形态观察、生理生化特征和16S rDNA序列比对分析,将HA01184归于链霉菌属,可能是来自海洋环境的一个潜在新种。     

海洋无脊椎动物抗菌肽研究进展及其在食品保鲜中的应用

海洋无脊椎动物抗菌肽抑菌广谱,稳定性高,且对生物体本身无害,其应用日益引起大量研究者的关注。综述了抗菌肽的几种类型、抑菌机理,介绍了海洋无脊椎动物抗菌肽研究进展、存在的问题并分析其在食品保鲜中的应用前景。     

Ecological risk assessment of polycyclic aromatic hydrocarbons in surface sediments from the middle and lower reaches of the Yellow River,China

In this research, ecological risks for eight individual polycyclic aromatic hydrocarbons (PAHs) and ∑PAH₈ in surface sediments from middle and lower reaches of Yellow River are evaluated using overlapping areas of probability density curves and margin of safety (MOS), based on the toxicity data and the exposure concentrations of PAHs in sediments collected from 23 sites. In the overlapping areas of probability density curves, the risk of Ant and Pyr are the highest, then the risk level is in the order of Flua > Nap > Phe > BaP > Flu > Ace. The values of MOS₁₀ present that Pyr (4.62 × 10^?4), Ant (5.60 × 10^?3), and Flua (6.4 × 10^?3) have a significantly high ecological risk level, while Nap and Phe have middle-level ecological risk. As for Ace, BaP, and Flu, they pose limited risk to the ecological system with MOS₁₀ greater than 1.0. The ∑PAH₈ (2.66 × 10^?5) is a higher risk level than that of any individual PAHs, where the probabilities of ∑PAH₈ in excess of the 10th percentile of the toxicity data were 86%.     

Isolation and Structure Elucidation of Epipolasterol and 22,23-Dihydroepipolasterol from the Marine Sponge Epipolasis sp.

Two new sterols, epipolasterol and 22(23)-dihydroepipolasterol, have been isolated from the marine sponge Epipolasis sp. These are unusual metabolites as they both contain a t-butyl group in the sterol side chain. In addition, the presence of two degrees of unsaturation in the side chain of epipolasterol is rare. The known sterol, 22-dehydro-24-isopropylcholesterol was also found in this sponge.     

Comparative Characterization of Laminarinases from the Filamentous Marine Fungi Chaetomium Indicum Corda and Trichoderma Aureviride Rifai

Marine filamentous fungi (103 strains) isolated from various marine habitats were studied for their ability to produce extracellular O-glycosylhydrolases. Cultural filtrates of these strains were shown to contain a series of glycanases (laminarinases, amylases, cellulases, pustulanases) and glycosidases (β-glucosidases, N-acetyl-β-glucosaminidases, β-galactosidases, α-mannosidases).Two species of marine fungi from different habitats were chosen for isolation of laminarinases and detailed study on enzyme properties. The fungus Chaetomium indicum associated with the alga Fucus evanescens C. Agardh was collected near the Kuril Islands, and T. aureviride was sampled from bottom deposits of South China Sea. Properties of extracellular laminarinases were similar: temperature optimums (40–45 ^∘C), molecular masses (54–56 kDa), K _m (0.1–0.3 mg ml⁻¹). Temperature stability of laminarinase of C. indicum was significantly higher than those from T. aureveride. It is shown that these enzymes are specific to β-1,3-bonds in glucans, release predominantly glucose from laminaran and do not catalyze reaction of transglycosylation. Accoding to these data enzymes are exo-1,3-β-D-glucan-glucanohydrolases (EC 3.2.1.58). Inhibitor analysis demonstrated the significant role of tryptophan and tyrosine residues in the catalytic activity of enzymes. Molecules of T. aureviride laminarinase contained the functionally important thiol group.     

Physiological and Biochemical Characteristics of the Halophilic Bacteria Vibrio parahaemolyticus and V. alginolyticus Isolated from Marine Invertebrates of Peter the Great Bay, Sea of Japan

One hundred strains of halophilic vibrios were isolated from 16 species of marine invertebrates of Peter the Great Bay. Based on their morphological and biochemical characteristics, the bacteria were identified as Vibrio parahaemolyticus and Vibrio alginolyticus. Bacterial isolates possessed virulence enzymes (DNAase, lecithinase, catalase) and were characterized by a high enterotoxigenicity. It was determined that 76% of the V. parahaemolyticus strains and 43% of the V. alginolyticus strains were Kanagawa-positive. The isolates showed a high adhesive capability, the average adhesion index was 18.06 cells per erythrocyte for V. parahaemolyticus and 12.55 for V. alginolyticus. The results of this study suggest a high pathogenic potential of the isolated halophilic vibrios, which are an epidemic hazard to marine invertebrates and to humans.     

Purification and Characterization of an Alkaline Protease from the Marine Yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> for Bioactive Peptide Production from Different Sources

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45°C, respectively. The enzyme was activated by Cu²⁺ (at a concentration of 1.0 mM) and Mn²⁺ and inhibited by Hg²⁺, Fe²⁺, Fe³⁺, Zn²⁺, and Co²⁺. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 1–10-phenanthroline, and iodoacetic acid. The K _m and V _max values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 μmol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.     

Characterization of Psychrotrophic Bacteria in the Surface and Deep-Sea Waters from the Northwestern Pacific Ocean Based on 16S Ribosomal DNA Analysis

Seventy-eight 4°C-culturable bacteria were isolated using ZoBell 2216E medium from surface (0–200 m) and deep-sea (1000–9671 m) waters in the northwestern Pacific Ocean. Growth studies indicated that all 4°C-culturable bacteria were psychrotrophs. Six phylotypes were observed in the surface water samples and 8 phylotypes in the deep-sea waters. Phylogenetic characterization based on 16S ribosomal DNA sequence analysis of the representative phylotypes revealed that some bacterial genera, Pseudoalteromonas, Photobacterium, and Vibrio, were common to surface and deep-sea waters, and others, Pseudomonas and Halomonas, specifically occurred in surface water. Overall, the members of Vibrionaceae appear to be dominant in both habitats. Received June 13, 2000; accepted March 8, 2001.     

Cloning and Sequencing Analysis of Alginate Lyase Genes from the Marine Bacterium Vibrio sp. O2

We isolated a new marine bacteria, which displayed alginate-depolymerizing activity in plate assays, from seawater in Mihonoseki Harbor, Japan. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the genus Vibrio and it was named Vibrio sp. O2. The alginate lyase genes of Vibrio sp. O2 were cloned and expressed in Escherichia coli. Two alginate lyase-producing clones, pVOA-A4 and pVOA-B5, were obtained. The alginate lyase gene alyVOA from pVOA-A4 was composed of an 858-bp open reading frame (ORF) encoding 285 amino acid residues, while alyVOB from pVOA-B5 was composed of an 828-bp ORF encoding 275 amino acid residues. The degree of identity between the deduced amino acid sequences of AlyVOA or AlyVOB and Photobacterium sp. ATCC43367 alginate poly(ManA)lyase AlxM was 92.3% or 32.6%, respectively. Alginate lyase consensus regions corresponding to the sequences YFKAGXYXQ and RXELR were observed in all three of these sequences. AlyVOA and AlyVOB both degraded polymannuronate in plate assays and were therefore confirmed to be poly(β-D-mannuronate)lyases.