Inhibitory effects of a branched-chain fatty acid on larval settlement of the polychaete Hydroides elegans.

Eleven strains of Streptomyces isolated from deep-sea sediments were screened for anti-larval settlement activity and all were active. Among those strains, Streptomyces sp. UST040711-290 was chosen for the isolation of bioactive antifouling compounds through bioassay-guided isolation procedure. A branched-chain fatty acid, 12-methyltetradecanoid acid (12-MTA) was purified, and it strongly inhibited the larval settlement of the polychaete Hydroides elegans. Streptomyces sp. UST040711-290 produced the highest yield of 12-MTA when the bacterium was cultured at 30 degrees C and pH 7.0 in a modified MGY medium. To investigate the potential antifouling mechanism of 12-MTA in the larval settlement of Hydroides elegans, the expression level of four marker genes, namely, Ran GTPase activating protein (GAP), ATP synthase (AS), NADH dehydrogenase (ND), and cell division cycle protein (CDC), was compared among the untreated larvae (the control), isobutylmethylxanthine (an effective settlement inducer), and 12-MTA-treated larvae. The 12-MTA treatment down-regulated the expression of GAP and up-regulated the expression of AS in the H. elegans larvae, but did not affect the expression of ND and CDC. This study provides the first evidence that a branched-chain fatty acid produced by a marine bacterium isolated from deep-sea sediment effectively inhibited the larval settlement of the biofouling polychaete H. elegans and its effects on the expression of genes important for larval settlement.

Thalassococcus halodurans gen. nov., sp. nov., a novel halotolerant member of the Roseobacter clade isolated from the marine sponge Halichondria panicea at Friday Harbor, USA.

A Gram-negative, non-pigmented, ovoid-shaped, strictly aerobic, catalase- and oxidase-positive and highly halotolerant bacterial strain that was devoid of swimming and gliding motility, designated UST050418-052(T), was isolated from the surface of the marine sponge Halichondria panicea at Friday Harbor, WA, USA. Strain UST050418-052(T) required NaCl for growth and could tolerate salt concentrations of up to 18 %. The primary respiratory quinone was ubiquinone-10 and the DNA G+C content was 57.8 mol%. The predominant fatty acids were the saturated fatty acids 16 : 0 and 18 : 0 and the monounsaturated fatty acids 18 : 1 omega 7c and 18 : 1 omega 9c, altogether representing 82.9 % of the total. Phylogenetic analysis based on the 16S rRNA gene sequence placed UST050418-052(T) in a distinct lineage within the Roseobacter clade in the family Rhodobacteraceae, with 95.0-95.8 % sequence similarity to members of the nearest genus Thalassobius. The DNA-DNA relatedness between UST050418-052(T) and Thalassobius gelatinovorus IAM 12617(T) was 9 %. Strain UST050418-052(T) could be differentiated from closely related members of the Roseobacter clade by a number of chemotaxonomic and phenotypic characteristics such as its distinct fatty acid profile, ability to reduce nitrate to nitrite and inability to utilize citrate, succinate, L-arginine and pyruvate. Based on the phylogenetic, chemotaxonomic and phenotypic evidence presented in this study, we suggest that strain UST050418-052(T) represents a novel genus in the family Rhodobacteraceae. The name Thalassococcus halodurans gen. nov., sp. nov., is thus proposed. The type strain of Thalassococcus halodurans is UST050418-052(T) (=JCM 13833(T) =NRRL B-41465(T)).

Antibacterial and antilarval activity of deep-sea bacteria from sediments of the West Pacific Ocean.

Deep-sea microorganisms are a new source of bioactive compounds. In this study, crude ethyl acetate extracts of 176 strains of deep-sea bacteria, isolated from sediments of the West Pacific Ocean, were screened for their antibacterial activity against four test bacterial strains isolated from marine biofilms. Of these, 28 deep-sea bacterial strains exhibited antibacterial activity against one or more of the bacteria tested. Active deep-sea bacterial strains belonged mainly to the genera of Pseudomonas, Psychrobacter and Halomonas. Additionally, antilarval activity of 56 deep-sea bacterial strains was screened using Balanus amphitrite larvae. Seven bacterial strains produced metabolites that had strong inhibitive effects on larval settlement. None of these metabolites showed significant toxicity. The crude extract of one deep-sea Streptomyces strain could completely inhibit larval settlement at a concentration of 25 microg ml(-1).

Effect of culture conditions on antifouling compound production of a sponge-associated fungus.

Microorganisms associated with invertebrate hosts have long been suggested to be a source for bioactive metabolites. In this study, we reported that a sponge-associated fungus, Letendraea helminthicola, produced two antifouling compounds: 3-methyl-N-(2-phenylethyl) butanamide and cyclo(D-Pro-D-Phe). To optimize the production of these antifouling compounds, we then examined the production of compounds under different culture conditions (temperature, salinity, pH, and carbon and nitrogen sources). This fungus grew well and produced more compounds at temperatures between 18 and 30 degrees C; the fungus grew well at 75 parts per thousand (ppt) salinity but produced the highest amount of antifouling compounds at 30 and 45 ppt. The optimal initial pH value for mycelial growth was 5.5 to 6.5, whereas the production of the antifouling compounds was maximized at pH 3.5 and 4.5. Glucose and xylose (as carbon sources) increased the production of antifouling compounds. Yeast extract and peptone (as nitrogen sources) maximized the production of mycelial biomass and antifouling compounds. Our results indicate that culture conditions greatly affect the production of bioactive compounds from mycelial fungal cultures as exemplified by strain L. helminthicola and that the conditions favorable for fungal growth may not be the best conditions for bioactive compound production.

Shewanella irciniae sp. nov., a novel member of the family Shewanellaceae, isolated from the marine sponge Ircinia dendroides in the Bay of Villefranche, Mediterranean Sea.

Strain UST040317-058(T), comprising non-pigmented, rod-shaped, facultatively anaerobic, Gram-negative cells that are motile by means of single polar flagella, was isolated from the surface of a marine sponge (Ircinia dendroides) collected from the Mediterranean Sea. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a separate cluster with the recognized bacterium Shewanella algae IAM 14159(T), with which it showed a sequence similarity of 95.0 %. The sequence similarity between strain UST040317-058(T) and its other (six) closest relatives ranged from 91.6 to 93.8 %. Strain UST040317-058(T) showed oxidase, catalase and gelatinase activities. The typical respiratory quinones for shewanellas, menaquinone MK-7 and ubiquinones Q-7 and Q-8, were also detected. The predominant fatty acids in strain UST040317-058(T) were i15 : 0, 16 : 0, 17 : 1omega8c and summed feature 3 (comprising i15 : 0 2-OH and/or 16 : 1omega7c), altogether representing 56.9 % of the total. The DNA G+C content was 39.9 mol%. The strain could be differentiated from other Shewanella species by its inability to reduce nitrate or produce H(2)S and by 10-22 additional phenotypic characteristics. On the basis of the phylogenetic and phenotypic data presented in this study, strain UST040317-058(T) represents a novel species in the genus Shewanella, for which the name Shewanella irciniae sp. nov. is proposed. The type strain is UST040317-058(T) (=JCM 13528(T)=NRRL B-41466(T)).

Gillisia myxillae sp. nov., a novel member of the family Flavobacteriaceae, isolated from the marine sponge Myxilla incrustans.

A yellow-pigmented, Gram-negative, rod-shaped, strictly aerobic bacterium (strain UST050418-085(T)) was isolated from the surface of a marine sponge, Myxilla incrustans, at Friday Harbor, WA, USA. The DNA G+C content of this strain was 34.6 mol%. The predominant fatty acids were i15 : 0, a15 : 0, i15 : 1, i16 : 0, i17 : 0 3-OH, 17 : 0 2-OH and summed feature 3, comprising i15 : 0 2-OH and/or 16 : 1omega7c (altogether representing 69.0 % of the total fatty acids). MK-6 was the only respiratory quinone detected. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the closest relatives of UST050418-085(T) were members of the genus Gillisia, with sequence similarities of 93.2-96.6 %. Strain UST050418-085(T) differed from its closest relatives by 11 to 18 phenotypic traits. Molecular evidence and phenotypic characteristics suggest that strain UST050418-085(T) represents a novel species within the genus Gillisia. The name Gillisia myxillae sp. nov. is proposed, with UST050418-085(T) (=JCM 13564(T)=NRRL B-41416(T)) [corrected] as the type strain.

Novel antifouling and antimicrobial compound from a marine-derived fungus Ampelomyces sp.

In this study, using a bioassay-guided isolation and purification procedure, we obtained 3-chloro-2,5-dihydroxybenzyl alcohol from a marine-derived Ampelomyces species that effectively inhibited larval settlement of the tubeworm Hydroides elegans and of cyprids of the barnacle Balanus amphitrite. The inhibitive effect on larval settlement was nontoxic and the EC50 of 3-chloro-2,5-dihydroxybenzyl alcohol ranged from 3.19 microg ml-1 to 3.81 microg ml-1 while the LC50 was 266.68 microg ml-1 for B. amphitrite cyprids; EC50 ranged from 0.67 microg ml-1 to 0.78 microg ml-1, and LC50 was 2.64 microg ml-1 for competent larvae of H. elegans, indicating that inhibitive effect of this compound was nontoxic. At a concentration of 50 mug per disc, this compound showed strong inhibitive effects on the growth of 13 out of 15 marine bacterial species tested in disc diffusion bioassay. Overall, the high inhibitory activities against bacteria and larval settlement as well as the non- or low-toxic nature of this compound to the barnacle and polychaete larvae suggest this compound could be a potent antifoulant and/or antibiotic.

Description of Fabibacter halotolerans gen. nov., sp. nov. and Roseivirga spongicola sp. nov., and reclassification of [Marinicola] seohaensis as Roseivirga seohaensis comb. nov.

Bacterial strains UST030701-097T and UST030701-084T were isolated from a marine sponge in the Bahamas. Both strains were pink-pigmented, Gram-negative, strictly aerobic and chemo-organotrophic. Cells of strain UST030701-097T were short, curved rods with fast-gliding motility, whereas those of strain UST030701-084T were straight rods with a less rapid gliding motion. The two strains had MK-7 as the major respiratory quinone and did not produce flexirubin-type pigments. The DNA G+C contents of strains UST030701-097T and UST030701-084T were 42.5 and 43.7 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the two strains belonged to the family 'Flexibacteraceae' of the phylum Bacteroidetes. 16S rRNA gene sequence similarity between strains UST030701-097T and UST030701-084T was 95.0 %; their closest relative was [Marinicola] seohaensis, with 93.3 % and 96.0 % sequence similarity, respectively. Phylogenetic tree topology indicated that the two strains belonged to the same lineage, but were on separate branches. Whilst strain UST030701-084T and [Marinicola] seohaensis were found on one branch, strain UST030701-097T was in another branch that had no species with validly published names. Based on the polyphasic taxonomic data obtained in the present study, we propose that strain UST030701-097T represents a novel genus and that strain UST030701-084T represents a novel species in the phylum Bacteroidetes. The genus Fabibacter gen. nov. is proposed, with strain UST030701-097T (=NRRL B-41220T=JCM 13334T) as the type strain of the type species, Fabibacter halotolerans sp. nov. Strain UST030701-084T (=NRRL B-41219T=JCM 13337T) is proposed as the type strain of Roseivirga spongicola sp. nov. In an earlier study, it was suggested that the genus Marinicola is a later heterotypic synonym of the genus Roseivirga. However, a formal proposal to reclassify [Marinicola] seohaensis, the only member of the genus Marinicola, has not yet been made. The results of phylogenetic analyses in this study support the reclassification of [Marinicola] seohaensis as Roseivirga seohaensis comb. nov.

Stenothermobacter spongiae gen. nov., sp. nov., a novel member of the family Flavobacteriaceae isolated from a marine sponge in the Bahamas, and emended description of Nonlabens tegetincola.

A bacterial strain, UST030701-156T, was isolated from a marine sponge in the Bahamas. Strain UST030701-156T was orange-pigmented, Gram-negative, rod-shaped with tapered ends, slowly motile by gliding and strictly aerobic. The predominant fatty acids were a15 : 0, i15 : 0, i15 : 0 3-OH, i17 : 0 3-OH, i17 : 1omega9c and summed feature 3, comprising i15 : 0 2-OH and/or 16 : 1omega7c. MK-6 was the only respiratory quinone. Flexirubin-type pigments were not produced. Phylogenetic analysis based on 16S rRNA gene sequences placed UST030701-156T within a distinct lineage in the family Flavobacteriaceae, with 93.3 % sequence similarity to the nearest neighbour, Nonlabens tegetincola. The DNA G+C content of UST030701-156T was 41.0 mol% and was much higher than that of N. tegetincola (33.6 mol%). Strain UST030701-156T can be distinguished from other members of the Flavobacteriaceae by means of a number of chemotaxonomic and phenotypic characteristics. It is proposed, therefore, that UST030701-156T represents a novel taxon designated Stenothermobacter spongiae gen. nov., sp. nov. The type strain is UST030701-156T (= NRRL B-41138T = JCM 13191T). Carbon-source utilization by N. tegetincola was re-examined and an emended description is therefore included.

Antifouling diketopiperazines produced by a deep-sea bacterium, Streptomyces fungicidicus.

Modern antifouling coatings use heavy metals and toxic organic molecules to prevent biofouling, the undesirable growth of marine organisms on man-made substrata. In an ongoing survey of deep-sea microorganisms aimed at finding low toxic antifouling metabolites, an actinomycete bacterium was isolated from the Pacific sediment at the depth of about 5000 m. The bacterium was closely related to Streptomyces fungicidicus (99% similarity) according to 16S ribosomal RNA sequence information. The spent culture medium of this bacterium inhibited barnacle larval attachment. Bioassay-guided fractionation was employed to isolate antifouling compounds. The ethyl acetate extract was fractionated by using an open silica gel column. Active fractions were further purified on a HPLC C18 column. Five diketopiperazines, cyclo-(L-Leu-L-Pro), cyclo-(L-Phe-L-Pro), cyclo-(L-Val-L-Pro), cyclo-(L-Trp-L-Pro), and cyclo-(L-Leu-L-Val) were isolated for the first time from a deep sea bacterium, and the structures of the compounds were elucidated by nuclear magnetic resonance spectroscopy and mass spectrometry. The pure diketopiperazines were tested for antilarval activity using the barnacle Balanus amphitrite. Effective concentrations that inhibited 50% larval attachment (EC50) after 24 h ranged from 0.10- 0.27 mM. The data suggest that diketopiperazines and other compounds from deep-sea bacteria may be used as novel antifoulants.

Nonlabens tegetincola gen. nov., sp. nov., a novel member of the family Flavobacteriaceae isolated from a microbial mat in a subtropical estuary.

An orange-pigmented, Gram-negative, non-motile, rod-shaped, strictly aerobic bacterium (UST030701-324T) was isolated from a microbial mat in an estuary in the Bahamas. The DNA G+C content was 33.6 mol%. Predominant fatty acids were i15 : 0, i16 : 0, i17 : 0 3-OH, and summed feature 3, comprising i15 : 0 2-OH and/or 16 : 1omega7c. MK-6 was the only respiratory quinone. Flexirubin-type pigments were not produced. Phylogenetic analysis based on 16S rRNA gene sequences placed UST030701-324T in a distinct lineage in the family Flavobacteriaceae with less than 90.7 % sequence similarity to the members of the nearest genus, Psychroflexus. UST030701-324T can be distinguished from other members of Flavobacteriaceae by a number of chemotaxonomic and phenotypic characteristics. It is thus proposed that UST030701-324T represents a novel taxon designated Nonlabens tegetincola gen. nov., sp. nov. The type strain is UST030701-324T (=NRRL B-41136T=JCM 12886T).

Gramella portivictoriae sp. nov., a novel member of the family Flavobacteriaceae isolated from marine sediment.

A yellow-pigmented, Gram-negative, slowly gliding, rod-shaped, strictly aerobic bacterium (UST040801-001T) was isolated from marine sediment. The DNA G+C content was 39.9 mol%. The predominant fatty acids were a15 : 0, i15 : 0, i15 : 0 3-OH, i17 : 1omega9c, i17 : 0 3-OH and summed feature 3, comprising i15 : 0 2-OH and/or 16 : 1omega7c (altogether representing 76.2 % of the total). MK-6 was the only respiratory quinone. Flexirubin-type pigments were not produced. Phylogenetic analysis based on 16S rRNA gene sequences indicated that Gramella echinicola KMM 6050T (the only species in the genus) was the closest relative of UST040801-001T, sharing 98.0 % sequence similarity. The DNA-DNA relatedness between UST040801-001T and Gramella echinicola KMM 6050T was 13 %. Strain UST040801-001T can be distinguished from G. echinicola by means of 11 phenotypic traits. The results of molecular and phenotypic analyses suggested that UST040801-001T represents a novel species of Gramella. The name Gramella portivictoriae sp. nov. is proposed for this bacterium, with UST040801-001T (=NRRL B-41137T [corrected] =JCM 13192T) as the type strain.

Winogradskyella poriferorum sp. nov., a novel member of the family Flavobacteriaceae isolated from a sponge in the Bahamas.

A Gram-negative, rod-shaped bacterium (designated strain UST030701-295(T)) with fast gliding motility was isolated from the surface of the sponge Lissodendoryx isodictyalis in the Bahamas. Colonies of UST030701-295(T) were yellow in colour, 2-4 mm in diameter, convex with a smooth surface and entire margins. UST030701-295(T) was heterotrophic, strictly aerobic and required NaCl for growth (1.0-4.0%). Growth was observed at pH 6.0-10.0 and at 12-44 degrees C. Phylogenetic analysis of the 16S rRNA gene sequence placed UST030701-295(T) within the genus Winogradskyella of the family Flavobacteriaceae, sharing 94.7-95.8% similarity with the three recognized members of the genus. The G+C content of the DNA was 32.8 mol% and the predominant fatty acids were iso-C(15:1), iso-C(15:0), iso-C(15:0) 2-OH, iso-C(15:0) 3-OH, iso-C(16:0) 3-OH, C(16:1)omega7 and iso-C(17:0) 3-OH (together representing 75.4% of the total); these data supported the affiliation of UST030701-295(T) to the genus Winogradskyella. UST030701-295(T) differed from the three recognized species of Winogradskyella in 7-17 traits. Molecular evidence together with phenotypic characteristics suggests that UST030701-295(T) represents a novel species within the genus Winogradskyella, for which the name Winogradskyella poriferorum sp. nov. is proposed. The type strain is UST030701-295(T) (=NRRL B-41101(T)=JCM 12885(T)).

Pseudoalteromonas spongiae sp. nov., a novel member of the gamma-Proteobacteria isolated from the sponge Mycale adhaerens in Hong Kong waters.

A Gram-negative, non-spore-forming, short rod-shaped bacterium (UST010723-006(T)) was isolated from the surface of the sponge Mycale adhaerens in Hong Kong waters. Cells of UST010723-006(T) did not have flagella and were non-motile. Colonies were pale orange in colour, 2-4 mm in diameter, convex with a smooth surface and an entire translucent margin. Gas bubbles were observed in the colonies and also in the agar matrix underneath and adjacent to the colonies. UST010723-006(T) was heterotrophic, strictly aerobic and required NaCl for growth (2.0-6.0%). It grew at pH 5.0-10.0 and between 12 and 44 degrees C. Phylogenetic analysis of the 16S rRNA gene sequence placed UST010723-006(T) within the genus Pseudoalteromonas of the gamma-subclass of the Proteobacteria. The DNA G+C content is 40.6 mol% and the dominant fatty acids were 12:0 3-OH, 14:0, 15:0 iso 2-OH, 16:0, 16:1omega7, 17:1omega8 and 18:1omega7 (altogether representing 75.9% of the total). These data supported the affiliation of UST010723-006(T) to the genus Pseudoalteromonas. The closest relatives were Pseudoalteromonas luteviolacea, P. phenolica, P. rubra and P. ruthenica with similarity values ranging from 95.4 to 96.8%. UST010723-006(T) differed from these closest relatives by 9-19 traits. Molecular evidence, together with phenotypic characteristics, suggests that UST010723-006(T) constitutes a novel species within the genus Pseudoalteromonas. The name Pseudoalteromonas spongiae sp. nov. is proposed for this bacterium. The type strain is UST010723-006(T) (=NRRL B-41100(T)=JCM 12884(T)).

Loktanella hongkongensis sp. nov., a novel member of the alpha-Proteobacteria originating from marine biofilms in Hong Kong waters.

A Gram-negative, non-motile, non-spore-forming, short rod-shaped bacterium (UST950701-009P(T)) was isolated from a marine biofilm in Hong Kong waters. Colonies are pink in colour, convex with a smooth surface and entire edge. Brown diffusible pigment is produced. Whitish colonies, with otherwise identical morphology, emerge from every culture upon ageing. The white colonies can be maintained as separate cultures (UST950701-009W) without turning pink. UST950701-009P(T) and UST950701-009W have identical 16S rRNA gene sequences and similar G+C (65.9-66.2 mol%) and fatty acid (86.22-88.52 % 18 : 1omega7c) contents. Phylogenetic analysis of the 16S rRNA gene sequence places UST950701-009P(T) within the Rhodobacter group of the alpha-subclass of the Proteobacteria. The nearest neighbours belong to the genus Loktanella, with similarity values ranging from 94.5 to 95.5 %. Data on G+C and fatty acid contents support the affiliation to the genus Loktanella. UST950701-009P(T) and -009W are heterotrophic, strictly aerobic and require NaCl for growth (2.0-14.0 %). Both grow in pH 5.0-10.0 and at 8-44 degrees C. Both are positive in oxidase, catalase and beta-galactosidase tests, but they differ in the pattern of carbohydrate oxidation and assimilation. Molecular evidence together with phenotypic characteristics shows that UST950701-009P(T) constitutes a novel species within the genus Loktanella. The name Loktanella hongkongensis sp. nov. is proposed; the type strain is UST950701-009P(T) (=NRRL B-41039(T)=JCM 12479(T)) and a morphovar is UST950701-009W (=NRRL B-41040=JCM 12480).

Antibacterial bromophenols from the marine red alga Rhodomela confervoides.

Two bromophenols, together with three known compounds, were isolated from the methanolic extract of the marine alga, Rhodomela confervoides. By means of MS and NMR spectroscopic analyses, they were identified as 3-bromo-4-[2,3-dibromo-4,5-dihydroxyphenyl] methyl-5-(hydroxymethyl) 1,2-benzenediol (1) and 3-bromo-4-[2,3-dibromo-4,5-dihydroxyphenyl] methyl-5- (ethoxymethyl) 1,2-benzenediol (2). Three known compounds were also isolated, namely 3-bromo-4-[2,3-dibromo-4,5-dihydroxyphenyl] methyl-5-(methoxymethyl) 1,2-benzenediol (3), 4,4'- methylenebis [5,6-dibromo-1,2-benzenediol] (4) and bis (2,3-dibromo-4,5-dihydroxybenzyl) ether (5). Compound 5 was the most active against five strains of bacteria with the MIC less than 70 microg/ml, while compounds 2, 3 and 4 exhibited moderate activity.

Fatty acids of some algae from the Bohai Sea.

The fatty acid compositions of 22 species of marine macrophytes, belonging to the Ceramiales, Cryptonemiales, Nemalionales, Laminariales, Chordariales, Scytosiphonales, Desmarestiales, Dictyosiphonales, Fucales, Dictyotales and Ulvales and collected from the Bohai Sea, were determined by capillary gas chromatography. The contents of polyunsaturated fatty acids (FAs) in the Bohai Sea algae, in comparison with the same species from the Yellow Sea were found to be lower. Red algae had relatively high levels of the acids 16:0, 18:1(n-7), 18:1(n-9), 20:5(n-3) and 20:4(n-6), and those examined were rich in C(20) PUFAs, these chiefly being arachidonic and eicosapentaenoic acids. The major FAs encountered in the Phaeophyta were 14:0, 16:0, 18:1(n-9), 18:2(n-6), 18:3(n-3), 18:4(n-3), 20:4(n-6) and 20:5(n-3). C(18)PUFAs are of greater abundance in the brown algae than in the red algae examined. All three green algae from the Ulvales had similar fatty acid patterns with major components, 16:0, 16:4(n-3), 18:1(n-7), 18:2(n-6), 18:3(n-3), and 18:4(n-3). They contained 16:3(n-3) and more 16:4(n-3), were rich in C(18)PUFAs, chiefly 18:3(n-3) and 18:4(n-3) and had 18:1(n-7)/18:1(n-9) ratios higher than 1.