Diversity of culturable bacteria in the mucus of the Red Sea coral Fungia scutaria

Coral reefs are the most biodiverse of all marine ecosystems. Bacteria are known to be abundant and active in seawater around corals, inside coral tissues, and within their surface microlayer. Very little is known, however, about the structure, composition and maintenance of these bacterial communities. In the current study we characterize the culturable bacterial community within the mucus of healthy specimens of the Red Sea solitary coral Fungia scutaria. This was achieved using culture-based methods and molecular techniques for the identification of the bacterial isolates. More than 30% of the isolated bacteria were novel species and a new genus. The culturable heterotrophic bacterial community of the mucus of this coral is composed mainly of the bacterial groups Gammaproteobacteria, Alphaproteobacteria and of Actinobacteria. This study provides the first evidence of actinomycetes isolated from corals.     

Preliminary studies of sperm cryopreservation in the mushroom coral, Fungia scutaria

Coral species throughout the world are facing severe environmental pressures. Because of this, we began cryobiological studies on the sperm of the mushroom coral, Fungia scutaria. We determined that F. scutaria sperm had a mean length of 56 microm and head diameter of 2.5 microm, and a mean spontaneous ice nucleation temperature of -37.2 +/- 1.7 degrees C. When the sperm were exposed to the cryoprotectant glycerol for 5 or 20 min (at 10% v/v), no fertilized larvae were produced. However, when sperm were exposed for 20 min to propylene glycol (10% v/v), fertilizations were produced at the same rate as untreated control eggs and sperm (P > 0.05), but slightly less for dimethyl sulfoxide (10% v/v) (P < 0.05). Regardless, dimethyl sulfoxide caused less osmotic damage to the sperm membrane than did propylene glycol. Therefore, we used the dimethyl sulfoxide (10% v/v) to develop cryopreservation protocols that yielded good post-thaw morphology and motility (>95%) for coral sperm.     

Structural determination of the oligosaccharide side chains from a glycoprotein isolated from the mucus of the coral Acropora formosa

An extracellular mucous glycoprotein has been isolated from the hard coral Acropora formosa. The glycoprotein contains sulfated oligosaccharide side chains attached through O-glycosidic linkages to serine and threonine, the principal amino acids (77%) in the polypeptide. The oligosaccharide side chains consist of D-arabinose, D-mannose, and N-acetyl-D-glucosamine with smaller amounts of D-galactose, L-fucose, and N-acetyl-D-galactosamine, but no sialic or uronic acids. Alkaline borohydride reductive cleavage resulted in a mixture of oligosaccharide alditols. Six oligosaccharides were purified by high performance liquid chromatography. The structures of these oligosaccharides, which do not resemble those of any other glycoprotein so far examined, were determined by a combination of gas chromatography/mass spectrometry analysis of methylation products and NMR spectroscopy. All oligosaccharides contain a reducing terminal mannitol residue with N-acetylglucosamine linked to carbon 2, 4, or 6 of the mannitol. There is no evidence for linkage of N-acetylglucosamine to any other glycoses in the glycoprotein. Galactose was detected in two oligosaccharides linked to the 4-position of mannitol. Arabinose (Ara) was found in only one oligosaccharide. This was probably due to hydrolysis of the labile arabino-furanoside linkages. Evidence is presented which indicates the arabinose occurs primarily at the terminal position of oligosaccharide side chains. The structures of the oligosaccharides isolated from the glycoprotein were: (Formula: see text).     

Characterization of FSH-regulated genes isolated by mRNA differential display from pig ovarian granulosa cells

The present authors have isolated FSH-regulated genes from primary granulosa cell cultures with or without Follicle Stimulating Hormone (FSH) treatment using mRNA differential display. mRNA differential display consists of amplification of partial sequences of cDNAs (150–400 bp) corresponding to 3' ends of cellular messenger RNAs, and thus, generates 3' expressed sequence tags (3' ESTs). Five thousand cDNA bands were examined, among which the present authors have isolated and sequenced 16 different FSH-regulated products. These sequences were compared with those available in databases. Three of the sequences showed similarity to identified genes from other species (bovine NADH dehydrogenase subunit 4, Xenopus chromosome sequence-associated polypeptide E and transformation-sensitive protein IEF SSP) and four others with human ESTs. Regulation of the corresponding genes has been checked by RT-PCR since most of these are expressed at a low level. FSH-regulation was confirmed for 12 mRNAs (four down- and eight up-regulated). The present authors have also mapped 12 of these ESTs on porcine chromosomes regions using a somatic cell hybrid panel.     

A new strain of Desulfovibrio gigas isolated from a sewage plant

Summary A new strain of Desulfovibrio gigas was isolated from sludge of a sewage plant. The medium contained ethanol, sulfate, minerals, some vitamins but no source of combined nitrogen. Several enrichment cultures of sulfate reducers could be obtained by employing the method to sludge samples from various sewage plants. With respect to their morphology, the dominating bacteria were of the Desulfovibrio gigas type.     

Isolation and characterization of rabbit ovarian surface epithelium,granulosa cells,and peritoneal mesothelium in primary culture

Summary Mammalian ovarian surface epithelial (OSE) cells and peritoneal mesothelial (PM) cells have a common embryologic origin, yet certain morphologic and histochemical characteristics are different in the adult. In this study, a two-step culture method was developed to examine the characteristics of these two cell types in vitro. OSE, PM, and ovarian granulosa (GC) cells were isolated from estrous rabbits and cultured for 6 d in 5% serum-supplementedd-valine medium (to inhibit fibroblast growth), then incubated for a further 2 d in serum-free McCoy's 5A medium. This study showed that rabbit OSE and PM cells in vitro maintained certain in vivo morphologic characteristics; OSE cells exhibited distinct cell borders and abundant microvilli of homogeneous size and shape, whereas PM cells were characterized by obscure cell borders and abundant microvilli of heterogeneous form. GC in vitro exhibited overlapping cell borders and sparse microvilli of homogeneous structure. This study showed for the first time that cultured rabbit OSE and PM cells, but not GC, contain distinct filaments of cytokeratin 18. In addition, rabbit OSE cells and GC, but not PM cells, contained 17β-hydroxysteroid dehydrogenase. However, only GC contained delta 5-3β hydroxysteroid dehydrogenase. OSE, PM, and GC maintained their ultrastructural and histochemical characteristics in serum-free medium. These results suggest that rabbit OSE cells in vitro could be distinguished from PM cells by histochemical and ultrastructural differences. Furthermore, because these characteristics were not altered in serum-free medium, the two-step culture method will be valuable in further hormonal studies of these cells in vitro. This work was supported in part by Grant No. 202-3192 from the University of South Dakota Parsons Fund     

Microsatellite loci isolated from the scleractinian coral, Acropora nobilis

We report the isolation and characterization of eight microsatellite loci from the scleractinian coral, Acropora nobilis. The microsatellite loci were obtained using compound SSR primers or an enrichment protocol. All the loci were polymorphic with four to eight alleles per locus and observed heterozygosities ranging from 0.22 to 0.76. Some of the primers developed for the two congeners, Acropora palmata and Acropora millepora were applicable to A. nobilis. These loci are useful for studying the connectivity among A. nobilis populations in Okinawa, southern Japan.     

A new bacteriophage, VHML, isolated from a toxin-producing strain of Vibrio harveyi in tropical Australia

Some strains of Vibrio harveyi are known to be pathogenic for fish and many invertebrates including crustaceans. Despite their importance, their modes of virulence have yet to be fully elucidated. Here, we present a previously unreported bacteriophage extracted from a toxin-producing strain of V. harveyi isolated from moribund prawn larvae in tropical Australia. Classification into the family Myoviridae was based upon morphological characteristics (an icosahedral head, a neck/collar region and a sheathed rigid tail) and nucleic acid characteristics (double-stranded linear DNA). We have termed the bacteriophage VHML (Vibrio Harveyi Myovirus Like). VHML is a temperate bacteriophage that has a narrow host range and shows an apparent preference for V. harveyi above other vibrios (63 Vibrio isolates tested) and other genera (10 other genera were tested). The conventional methods for phage concentration and extraction of nucleic acids from phage particles were not efficient and the alternative methods that were used are discussed.     

Milleporin-1, a new phospholipase A2 active protein from the fire coral Millepora platyphylla nematocysts

Stings of fire corals, potent hydroids common in the Red Sea, are known to cause severe pain and they develop burns and itching that lasts few hours after contact. Nematocyst venom of Millepora platyphylla (Mp-TX) was isolated according to a recent method developed in our laboratory to conduct a previous investigation on the nematocyst toxicity of Millepora dichotoma and M. platyphylla. In this study, Mp-TX was fractionated by using both gel filtration and ion exchange chromatography. Simultaneous biological and biochemical assays were performed to monitor the hemolytic (using washed human red blood cells, RBCs) and phospholipase A2 (using radiolabeled sn-2 C14-arachidonyl phosphatidylcholine as a substrate) active venom fractions. The magnitude of both hemolysis and phospholipase A2 activity was found in a fraction rich of proteins of molecular masses approximately 30,000-34,000 Daltons. The former fraction was purified by ion exchange chromatography, and a major bioactive protein factor (approx. 32,500 Daltons , here named milleporin-1) was recovered. Milleporin-1 enzymatic activity showed a significant contribution to the overall hemolysis of human RBCs. This activity, however, could not be completely inhibited using phospholipid substrates. Melliporin-1 fraction retained about 30% hemolysis, until totally rendered inactive when boiled for 3 min. The overall mechanism of action of milleporin-1 to impact the cellular membrane was discussed; however, it is pending more biochemical and pharmacological future studies.     

Ruminococcin A, a new lantibiotic produced by a Ruminococcus gnavus strain isolated from human feces

When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to R. gnavus. This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.     

A new hydroid coral from the Famennian stage of the Chernyshev Uplift, Polar Urals

A new stromatoporate species of the unusual genus Pararosenella Vassiljuk et Bogoyavlenskaya, 1990, P. olimpiadae sp. nov., is described; it is found for the first time in the Middle Famennian Substage of the Devonian in the Chernyshev Uplift, Polar Urals.     

A novel Bacillus thuringiensis strain LLB6, isolated from bryophytes, and its new cry2Ac-type gene

AIMS: To isolate and characterize the novel Bacillus thuringiensis strains from bryophytes collected from Wuyi Mountain, Fujian Province of China, and identify new B. thuringiensis strains and toxins active against mosquitoes. METHODS AND RESULTS: Twelve novel B. thuringiensis strains were isolated from 76 bryophyte samples. According to the results of this preliminary screening, LLB6 was the most toxic to Aedes albopictus. Then phase-contrast as well as scanning electron microscopy, bioassays, cloning, sequencing and expression were performed to characterize the novel isolate LLB6 and its new gene cry2Ac5. CONCLUSIONS: Bacillus thuringiensis occurred naturally on bryophytes. LLB6 isolated from Physcomitrium japonicum was toxic to A. albopictus. A new cry2Ac5 gene of LLB6 was detected, cloned and expressed successfully. Bioassays on A. albopictus showed that the expressed Cry2Ac5 was also toxic to the third instar larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of B. thuringiensis strains isolated from bryophytes. It represents a specific source of new B. thuringiensis strains and is of great importance for the knowledge of the ecology of B. thuringiensis. Novel LLB6 harboring the new gene cry2Ac5 and its expressed Cry2Ac5 protein revealed activity against A. albopictus and became a new member of B. thuringiensis toxins.     

Restriction mapping of plasmid pSa1, isolated from Streptomyces albus G strain

A novel plasmid designated pSa1 has been isolated from Streptomyces albus G strain producing SalGI restriction endonuclease. Molecular weight of the plasmid is 3.4 +/- 0.2 mD. The action of 12 restriction endonucleases on the plasmid DNA was studied. Restriction map of pSa1 DNA was established for SmaI, HindII, XbaI and KpnI endonucleases.     

Three new chlorinated marine steroids, yonarasterols G, H and I, isolated from the okinawan soft coral, Clavularia viridis

Three new chlorinated marine steroids, yonarasterols G, H and I, were isolated from the Okinawan soft coral, Clavularia viridis. Their structures including the absolute configuration were determined based on the results of spectroscopic analysis and chemical conversion.     

Durancin L28-1A, a new bacteriocin from Enterococcus durans L28-1, isolated from soil

AIMS: To isolate, characterize and identify bacteriocins from lactic acid bacteria in soil. METHODS AND RESULTS: Thirty-four acid-producing bacteria were isolated from 87 soil samples. Antibacterial activities were detected, and one strain, L28-1 produced a bacteriocin that was active against some Gram-positive bacteria. L28-1 was identified as Enterococcus durans by 16S rDNA sequence analysis and API50CHL. This bacteriocin did not lose its activity after autoclaving (121 degrees C for 15 min), but was inactivated by protease K. The bacteriocin was purified by hydrophobic column chromatography, and Sep-Pak C(18). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the partially purified bacteriocin contained numerous protein bands. Two bands that displayed antibacterial activities were c. 3.4 and 2.5 kDa in size. In this work, the 3.4-kDa bacteriocin was analysed with N-terminal amino acid and DNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results indicated that the 3.4-kDa bacteriocin of Ent. durans L28-1 is a new natural enterocin variant. CONCLUSIONS: Enterococcus durans L28-1 produced a new bacteriocin. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a novel bacteriocin that is produced by Ent. durans that has potential for use as a food preservative.     

A new species of Tylophora from coral reef areas in Hengchun Peninsula, Taiwan, China

Tylophora sui Y. H. Tseng & C. T. Chao, sp. nov. 苏氏欧蔓(S3-3. Fig. 1, S3-4. Fig. 2) Type: China. Taiwan: Pintung County, Hengchun Town, Longpan, coral reef areas near seashore, 21 55 20.6 N, 120 51 14.25 E, ca. 76 m, 2009-05-25, Yen-Hsueh Tseng 4668 (holotype, TCF).     

Trochelioid A and B,new cembranoid diterpenes from the Red Sea soft coral Sarcophyton trocheliophorum

Chemical investigations of the soft coral Sarcophyton trocheliophorum, has led to the isolation of six cembranoids, two of which are new, Trochelioid A (1) and B (2), and one, 16-oxosarcophytonin E (3) isolated from nature for the first time. Additionally, two have been isolated from S. trocheliophorum for the first time (4 and 6). Structures were elucidated by employing extensive NMR and HR-FAB-MS experimentation.