Enzymatic degradation of alginate by marine fungi

A total of 72 pre-selected strains of 19 species of marine fungi were tested for their ability to decompose sodium alginate, calcium alginate or freshly prepared calcium alginate gel. Active alginate decomposition was evident in 18 strains (25% of total tested). These belong to only three different species: Asteromyces cruciatus, Corollospora intermedia, and Dendryphiella salina. In broth culture, decomposition of sodium alginate by the two deuteromycetes was followed by gravimetric, electrometric, viscometric, photometric and chromatographic methods in order to characterize the alginase enzyme system and its degradation products. The alginase enzyme complex consisted of at least two different enzyme components: the already known alginate lyase (eliminase) and a new endo-alginate hydrolase. In summary, a model is presented on the alginase-mediated structural and molecular decomposition of sodium alginate by marine fungi.     

Niche overlap and trophic resource partitioning of two sympatric batoids co‐inhabiting an estuarine system in southeast Australia

Elasmobranchs play an important role within the trophic structure of marine ecosystems, but there are relatively few studies published on the feeding ecology of these species. Reported herein is the feeding ecology and trophic resource partitioning of two sympatric batoid species, Urolophus cruciatus and Narcine tasmaniensis from southeast Australia. The diet of males and females of both species was similar, suggesting no sex‐specific dietary preferences. Ontogenetic changes in diet were observed from the diets of both species: as the body size increased, the proportion consumed of crustacea to polychaeta decreased. A relatively high degree of niche overlap (70%) was detected between the trophic resources of the two species. The way in which the predators partitioned the resources, however, was significantly different. U. cruciatus fed predominately on small benthic crustaceans (amphipods and decapods), while N. tasmaniensis displayed a preference towards Maldanidae polychaetes. Therefore, although U. cruciatus and N. tasmaniensis both feed predominately on benthic invertebrates, they specialise on different taxa. This trophic resource partitioning contributes to the biodiversity of the region by facilitating the coexistence of these sympatric species.     

Isolation of gliovictin from the marine deuteromycete Asteromyces cruciatus

(−)Gliovictin, a diketopiperazine isolated from terrestrial fungi of the genera Helminthosporium and Penicillium, has been isolated from culture broths of the marine deuteromycete Astermyces cruciatus.     

Biodiversity of freshwater fungi

There are more than 600 species of freshwater fungi with more known from temperate, as compared to tropical regions. These includeca 340 ascomycetes, 300 deuteromycetes, and a number of lower fungi which are not discussed here.Aniptodera, Annulatascus, Massarina, Ophioceras andPseudohalonectria are common freshwater ascomycetes, which appear to be well adapted for this lifestyle either in their ascospore types or their competitive-degradative characters. The most common genera of wood-inhabiting deuteromycetes includeCancellidium, Dactylaria, Dictyosporium andHelicomyces. They are categorized into four groups depending on their form and life style: the ingoldian hyphomycetes; the aero-aquatic hyphomycetes; the terrestrial-aquatic hyphomycetes; and the submerged-aquatic hyphomycetes. The adaptations of aquatic fungi for their dispersal and subsequent attachment to new substrates are discussed.     

Physiological responses of marine Dendryphiella species from different geographical locations

The saprobic, cosmopolitan, marine fungi Dendryphiella arenaria and Dendryphiella salina, isolated from various plant and algal substrates from different geographical locations and climatic zones, were studied for their adaptations to the abiotic and biotic parameters commonly found in their natural marine habitats. All the tested strains of D. arenaria and D. salina grew optimally on culture media with added marine salts, at pH values between 6.5 and 8.0 and at an incubation temperature of 25°C. The D. arenaria strains had faster mean colony extension rates under all conditions of culture. All strains exhibited an increased salt optimum with increasing incubation temperature. The TLC profiles of strains of the two species were similar. The culture extracts were antimicrobial, though production of the biologically active metabolites was strain-specific. There were no significant correlations between source of origin and responses to the investigated parameters. These results demonstrate phenotypic plasticity and the ability of each isolate to adapt to diverse biotopes.     

Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus

Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2^T was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into l-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.

     

Monomer composition and sequence of sodium alginate extracted at pilot plant scale from three commercially important seaweeds from Mexico

The marine waters of the Baja California peninsula (Mexico) are a rich source of brown seaweeds with a great potential for exploitation. For that reason, Sargassum sinicola, Eisenia arborea, and Macrocystis pyrifera collected from different locations were subjected to extraction of sodium alginate using a pilot-plant scale process developed in our facilities. The composition and sequence parameters of the recovered alginate were studied by infrared and nuclear magnetic resonance spectroscopy. The spectral analysis of the products revealed that sodium alginate from S. sinicola contains a greater proportion of guluronate monomers (64%) than that from E. arborea (48%), and M. pyrifera (38%). Computation of the frequencies of diads and triads indicated that the alginate from S. sinicola was constructed by intercalated guluronate-blocks of 14 residues in length. In contrast, the length of the G-block in the alginates from E. arborea and M. pyrifera were 7 and 4 residues, respectively. The results show that S. sinicola, E. arborea, and M. pyrifera are sources of sodium alginate with different mannuronate/guluronate ratios, as well as a varied building-block length. In consequence, aqueous dispersions of sodium alginate from the three studied species are expected to exhibit different physical properties.     

Vertical distribution of marine fungi onRhizophora apiculata at Morib mangrove, Selangor, Malaysia

Studies on the vertical distribution of marine fungi in aRhizophora apiculata mangrove stand in Morib, Selangor were carried out in June 1993 and June to November 1997. Prop roots, subterranean roots and overhanging branches ofR. apiculata were collected from three intertidal levels namely upper (high water mark), middle and lower. Fifty-three species were recorded including 39 ascomycetes, 13 deuteromycetes and one basidiomycete. The most common fungi wereHalocyphina villosa (frequency occurrence 21%),Kallichroma tethys (20%),Lulworthia grandispora (18%),Leptosphaeria australiensis (16%),Julella avicenniae (15%) andMassarina ramunculicola (13%). The fungi were found to be vertically zoned, some were limited to the upper level such asPyrenographa xylographoides, Julella avicenniae andAigialus grandis or lower level such asTrichocladium achrasporum andT. alopallonellum, while only five species showed a broader distribution, being present at all levels:Leptosphaeria australiensis, Halocyphina villosa, Cryptovalsa sp.,Lulworthia grandispora andLulworthia sp. The greatest diversity of marine fungi were collected from the middle level with a Shannon Diversity Index of 5.9 while the Jaccard Similarity Index of 2.25 indicated that the upper and middle levels were the most similar in terms of species composition. Fungi with certain characteristics were also limited to particular levels, for example, carbonaceous and superficial ascomata were confined above mean tide while membranous walls and immersed ascomata were common below mean tide level.     

Cloning and Characterization of a Novel Oligoalginate Lyase from a Newly Isolated Bacterium Sphingomonas sp. MJ-3

A bacterium possessing alginate-degrading activity was isolated from marine brown seaweed soup liquefied by salted and fermented anchovy. The isolated strain was designated as Sphingomonas sp. MJ-3 based on the analyses of 16S ribosomal DNA sequences, 16S-23S internal transcribed spacer region sequences, biochemical characteristics, and cellular fatty acid composition. A novel alginate lyase gene was cloned from genomic DNA library and then expressed in Escherichia coli. When the deduced amino acid sequence was compared with the sequences on the databases, interestingly, the cloned gene product was predicted to consist of AlgL (alginate lyase L)-like and heparinase-like protein domain. The MJ-3 alginate lyase gene shared below 27.0% sequence identity with exolytic alginate lyase of Sphingomonas sp. A1. The optimal pH and temperature for the recombinant MJ-3 alginate lyase were 6.5 and 50°C, respectively. The final degradation products of alginate oligosaccharides were analyzed by electrospray ionization mass spectrometry and proved to be alginate monosaccharides. Based on the results, the recombinant alginate lyase from Sphingomonas sp. MJ-3 is regarded as an oligoalginate lyase that can degrade oligoalginate and alginate into alginate monosaccharides.     

Toxigenic saprophytic fungi in marine shellfish farming areas

Toxigenic saprophytic fungi were isolated from samples of shellfish, sediment and seawater obtained from marine shellfish farming areas. The 456 strains identified included 12 different genera, with a clear predominance (68%) of Penicillium, Aspergillus, Trichoderma and Cladosporium. To assess the risk of poisoning due to the presence of these fungi in shellfish farming areas, the strains were cultured in liquid medium, filtered, and tested on larvae of Artemia salina, a small crustacean highly sensitive to mycotoxins. Thirty-five point five percent of the strains proved active with this test. This study confirms the existence of fungi in shellfish farming areas, as suggested by our earlier work showing that filter-feeding shellfish accumulate toxic metabolites of fungal origin. The presence of fungi in the marine environment represents a real risk of poisoning through the consumption of contaminated shellfish.This revised version was published online in October 2005 with corrections to the Cover Date.     

海水来源的548株真菌的近海及远海分布模式及其抑菌活性分析

[目的]海洋真菌是新颖天然产物的理想来源.本研究旨在加深对可培养海洋真菌多样性的了解,以及挖掘具有应用潜力的海洋真菌.[方法]采用膜过滤法分离近海和远海海水样品中的真菌,通过菌株分离纯化、ITS基因序列测序,分析浅海带、半深海带和深海带海水样品中可培养真菌的多样性.在此基础上,采用固体平板法筛选具有抑菌活性的菌株.[结...     

Diet-induced variations in fatty acid content and composition of two on-grown stages of Artemia Salina

Three species of microalgae, the freshwater Euglena gracilis and themarine Dunaliella salina and Tetraselmis suecica, were fed tothe brine shrimp Artemia salina in order to compare their suitabilityin terms of fatty acid enrichment, and their effect on the biometric parametersof the zooplankter. The fatty acid content and composition were analyzed for the post-larval and pre-adult stages of Artemia fed the algae and theresults compared to the initial content of unfed 24-hour post-hatch nauplii.Differences in the total fatty acid content occurred between the three stages,the fatty acid profile being determined by the composition of the diet. A decreasing trend for almost all the individual fatty acids occurred throughdevelopment from post-larva to pre-adult with each of the three algal diets.Biometrical differences between Artemia fed the marine algae and that fed Euglena were not consistent in the post-larval stage, but became considerable in the pre-adult stage. Artemia fed with Euglena achieved twice the weight of animals fed the marine algae and showed thehighest length. The implications for the use of on-grown Artemia as afeed in larviculture of marine and freshwater fish and crustaceans are considered.     

Identification of a Unique Azaphilone Produced by Chaetomium globosum Isolated from Polygonatum sibiricum

A new azaphilone, chaephilone E, eight azaphilone derivatives, and three chaetoglobosins were isolated from endophytic fungi Chaetomium globosum. The structures of the compounds were elucidated by 1D and 2D NMR as well as HR‐ESI‐MS data, and the absolute configuration of chaephilone E was established on the basis of electronic circular dichroism and NOESY spectrum. The activity of chaephilone E was evaluated via the cytotoxic assay (human hepatoma cell lines HepG‐2) and brine shrimp (Artemia salina) bioassay.     

Mixotrophy in the sand-dwelling dinoflagellate Thecadinium kofoidii

Thecadinium kofoidii is a marine sand-dwelling dinoflagellate that sometimes forms dense blooms. This species was previously thought to be an exclusively autotrophic dinoflagellate, and its mixotrophic ability has not been explored yet. By investigating its ecophysiology, its trophic mode should be revealed. We explored the mixotrophic ability of T. kofoidii by examining its protoplasm under light and transmission electron microscopes with diverse algal prey species. Furthermore, the feeding mechanism of T. kofoidii and prey species on which it feeds were investigated. In addition, the growth and ingestion rates of T. kofoidii as a function of prey concentration were determined when feeding on the benthic cryptophyte Rhodomonas salina. Thecadinium kofoidii was able to feed on R. salina and the dinoflagellate Symbiodinium voratum, which had equivalent spherical diameters (ESDs) ≤ 10.1?µm, while it did not feed on the benthic dinoflagellates Levanderina fissa, Prorocentrum concavum or Ostreopsis cf. ovata, which had ESDs ≥ 15?µm. Thecadinium kofoidii fed on the edible prey cells using the peduncle. The maximum ingestion rate of T. kofoidii on R. salina was 1.3 cells predator^?1 d^?1. However, feeding on R. salina did not significantly increase the growth rate of T. kofoidii. The low ingestion rate of T. kofoidii on R. salina may have partially resulted in the lack of significant increase in its growth rate due to mixotrophy. The present study discovered predator–prey relationships between T. kofoidii and R. salina and S. voratum, which may change our view of the energy flow and carbon cycling in marine benthic food webs.     

微泡菌属菌株YPW1和YPW16多糖降解特性分析

【背景】海洋环境中分离到的微泡菌属菌株具有多糖降解能力,在环境中可以作为糖类代谢的重要执行者参与海洋碳循环过程。【目的】测定2株微泡菌属菌株的多糖降解活性,通过与微泡菌属其他菌株基因组比较分析2株菌的多糖降解基因特征。【方法】通过3,5-dinitrosalicylicacid(DNS)定糖法测定多糖降解活性,同时利用高通量测序技术对菌株基因组序列进行测定与组装,并与其他基因组注释结果进行比较分析。【结果】分离得到2株微泡菌属菌株YPW1和YPW16,二者均为潜在新种。结果表明,菌株YPW1能够降解琼胶、褐藻胶、果胶、几丁质、木聚糖、淀粉、普鲁兰等7种多糖,而菌株YPW16仅可降解淀粉和普鲁兰。基因组分析表明,YPW1具有上述7种多糖的降解酶基因,但菌株YPW16只具有淀粉酶与普鲁兰酶降解基因。相较于其他微泡菌属菌株,菌株YPW1多糖降解范围、多糖降解酶基因种类与丰度较高,但菌株YPW16多糖降解范围却较为狭窄。由此可知,多糖降解酶基因在微泡菌属基因组中的分布差异性较大。【结论】本研究为微泡菌属提供了2株潜在的新型菌株资源,为生物多糖降解提供了生化工具,也为研究微泡菌属菌株中多糖降解基...     

Bioactive natural products from marine sponges and fungal endophytes

This review highlights recent findings of our group on bioactive marine natural products isolated from marine sponges and marine derived fungi. The activated chemical defence of the Mediterranean sponge Aplysina aerophoba is introduced as an example of a dynamic response of marine sponges to wounding. Following tissue disrupture preformed brominated isoxazoline alkaloids are enzymatically cleaved and thereby give rise to aeroplysinin-1 which is believed to protect sponges from invasion of pathogenic bacteria. A preliminary characterization of the membrane bound enzyme(s) involved in the cleavage reaction is presented. Bromotyrosine derived, oxime group bearing peptides, the so called bastadins, obtained from the sponge Ianthella basta and some of their synthetic derivatives were shown to exhibit pronounced antifouling activity against larvae of the barnacle Balanus improvisus. The antifouling activity could be traced to the oxime group as an important pharmacophore that was also found to be present in other sponge derived natural products exhibiting antifouling activity. Marine derived fungi that reside within invertebrates such as sponges or inside Mangrove plants are emerging as a new source of bioactive metabolites as demonstrated for Aspergillus ustus and Alternaria sp. that were isolated from the sponge Suberites domuncula or from the Mangrove plant Sonneratia alba, respectively. The former fungus yielded new moderately cytotoxic sesquiterpenoids of the drimane type whereas the latter was found to produce polyketides such as alternariol that exhibited strong and selective inhibitory activity against several protein kinases, for instance Aurora A and B which are targets for anticancer chemotherapy.     

Selective alginate degradation by marine bacteria associated with the algal genusSargassum

Summary Alginase-secreting bacteria associated with actively growing tissues of the marine Phaeophyta speciesSargassum fluitans andS. natans have been isolated and evaluated for their ability to degrade alginate (ALG), carboxymethylcellulose, and agar. Of seven isolates selected for their ability to grow on 2% agar containing 1% sodium alginate, none were able to grow on either 2% agar or 2% agar supplemented with 0.1% carboxymethylcellulose. Two of these with fermentative potential, i.e., ALG-A and ALG-G, showed selective activities with respect to their ability to degrade native alginate and/or take up the products resulting from alginate degradation. The ALG-A isolate was able to rapidly degrade native alginate with the generation of a stable polymer fraction and small oligouronides, most of which were dissimilated for growth. The ALG-G isolate was able to completely degrade native alginate with the accumulation of significant quantities of unsaturated dimeric and trimeric oligouronides. A limit polymer was generated from the action of a polymannuronan-specific extracellular alginate lyase purified from exponential cultures of the ALG-A organism. This product proved to be an effective substrate for the alginate lyase activity obtained from the medium of exponential phase cultures of the ALG-G isolate, and upon incubation with concentrated and dialyzed ALG-G medium was converted to the products that were observed to accumulate in the medium of the ALG-G isolate grown on native alginate. These organisms represent examples of the microflora associated with actively growingSargassum tissues, each with a selective ability to degrade and dissimilate the biomass of the marine brown algae.     

Highly diverse and spatially heterogeneous mycorrhizal symbiosis in a rare epiphyte is unrelated to broad biogeographic or environmental features

Symbiotic interactions are common in nature. In dynamic or degraded environments, the ability to associate with multiple partners (i.e. broad specificity) may enable species to persist through fluctuations in the availability of any particular partner. Understanding how species interactions vary across landscapes is necessary to anticipate direct and indirect consequences of environmental degradation on species conservation. We asked whether mycorrhizal symbiosis by populations of a rare epiphytic orchid (Epidendrum firmum) is related to geographic or environmental heterogeneity. The latter would suggest that interactions are governed by environmental conditions rather than historic isolation of populations and/or mycorrhizal fungi. We used DNA-based methods to identify mycorrhizal fungi from eleven E. firmum populations in Costa Rica. We used molecular and phylogenetic analyses to compare associations. Epidendrum firmum exhibited broad specificity, associating with diverse mycorrhizal fungi, including six Tulasnellaceae molecular operational taxonomic units (MOTUs), five Sebacinales MOTUs and others. Notably, diverse mycorrhizal symbioses formed in disturbed pasture and roadside habitats. Mycorrhizal fungi exhibited significant similarity within populations (spatial and phylogenetic autocorrelation) and significant differences among populations (phylogenetic community dissimilarity). However, mycorrhizal symbioses were not significantly associated with biogeographic or environmental features. Such unexpected heterogeneity among populations may result from complex combinations of fine-scale environmental factors and macro-evolutionary patterns of change in mycorrhizal specificity. Thus, E. firmum exhibits broad specificity and the potential for opportunistic associations with diverse fungi. We suggest that these characteristics could confer symbiotic assurance when mycorrhizal fungi are stochastically available, which may be crucial in dynamic or disturbed habitats such as tropical forest canopies.     

Ecology of tropical marine fungi

Lignicolous material was collected from 5 marine locations in Brunei: a rocky headland, a sandy beach, a man-made brackish lake, a healthy mangrove and an oil polluted mangrove. Higher marine fungi present were identified and their percentage occurrence noted. The common marine fungi varied from habitat to habitat. Antennospora quadricornuta was most common at the rocky headland, Corollospora pulchella at the sandy beach and Halosarpheia marina at the brackish lake. In the mangroves the most common species were Halocyphina villosa (healthy) and Cirrenalia pygmea and Lulworthia grandispora (oil polluted). Differences in species composition from one habitat to the next were observed, although some fungi occurred throughout. There were significantly less diversity and numbers of fungi in the oil polluted mangrove when compared to the healthy mangrove. Observations concerning the ecology of tropical marine fungi are made.     

Marine mycoflora of south India with special emphasis on lignicolous marine fungi

A study dealing with the marine fungi associated with decaying wood samples in the brackish water mangrove ecosystem and shoreline ecosystem was carried out in south India. A total of 19 marine fungi were isolated from the brackish water mangrove ecosystem. They included 13 Ascomycetes, one Basidiomycete and five Mitosporic fungi. In terms of percent frequency of occurrence, the most frequent species obtained from the brackishwater were the Lignincola longirostris (16.60%) and Savoryella lignicola (12.09%). Nine species were found frequently. Five species were occasionally encountered. Aigialus mangrovei, Aniptodera mangrovei and Halosarpheia marina were the rare species recorded. The average number of isolates per wood sample was 1.53. A total of 27 marine fungi including 15 ascomycetes, one basidiomycete and ten mitosporic fungi were recorded from the shoreline ecosystem. In terms of percent frequency of occurrence, the most frequent species obtained from Kanyakumari were the Arenariomyces trifurcates (13.66%), Corollospora maritima (12.44%), and Cirrenalia pygmea (10.98%). Seven species were found frequently. Fourteen species were occasionally encountered. Three species were found to be rare in occurrence. The average number of isolates per wood sample was 1.21.