PUFA synthase-independent DHA synthesis pathway in Parietichytrium sp. and its modification to produce EPA and n-3DPA.

The demand for n-3 long-chain polyunsaturated fatty acids (n-3LC-PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), will exceed their supply in the near future, and a sustainable source of n-3LC-PUFAs is needed. Thraustochytrids are marine protists characterized by anaerobic biosynthesis of DHA via polyunsaturated fatty acid synthase (PUFA-S). Analysis of a homemade draft genome database suggested that Parietichytrium sp. lacks PUFA-S but possesses all fatty acid elongase (ELO) and desaturase (DES) genes required for DHA synthesis. The reverse genetic approach and a tracing experiment using stable isotope-labeled fatty acids revealed that the ELO/DES pathway is the only DHA synthesis pathway in Parietichytrium sp. Disruption of the C20 fatty acid ELO (C20ELO) and ∆4 fatty acid DES (∆4DES) genes with expression of ω3 fatty acid DES in this thraustochytrid allowed the production of EPA and n-3docosapentaenoic acid (n-3DPA), respectively, at the highest level among known microbial sources using fed-batch culture.

Draft Genome Sequence of Sicyoidochytrium minutum DNA Virus Strain 001.

Sicyoidochytrium minutum DNA virus strain 001 (SmDNAV 001) is a double-stranded DNA (dsDNA) virus that infects the marine fungoid protist Sicyoidochytrium minutum. We report the draft genome sequence of SmDNAV 001. The 236,345-bp genome contained 358 coding sequences (CDSs) and 3 tRNA-coding sequences.

Taming chlorophylls by early eukaryotes underpinned algal interactions and the diversification of the eukaryotes on the oxygenated Earth.

Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll must become particularly prominent upon dynamic cellular interactions that potentially disrupt the mechanisms that are designed to quench photoexcited chlorophylls in the phototrophic cells. Extensive examination of a wide variety of phagotrophic, parasitic, and phototrophic microeukaryotes demonstrates that a catabolic process that converts chlorophylls into nonphotosensitive 132,173-cyclopheophorbide enols (CPEs) is phylogenetically ubiquitous among extant eukaryotes. The accumulation of CPEs is identified in phagotrophic algivores belonging to virtually all major eukaryotic assemblages with the exception of Archaeplastida, in which no algivorous species have been reported. In addition, accumulation of CPEs is revealed to be common among phototrophic microeukaryotes (i.e., microalgae) along with dismantling of their secondary chloroplasts. Thus, we infer that CPE-accumulating chlorophyll catabolism (CACC) primarily evolved among algivorous microeukaryotes to detoxify chlorophylls in an early stage of their evolution. Subsequently, it also underpinned photosynthetic endosymbiosis by securing close interactions with photosynthetic machinery containing abundant chlorophylls, which led to the acquisition of secondary chloroplasts. Our results strongly suggest that CACC, which allowed the consumption of oxygenic primary producers, ultimately permitted the successful radiation of the eukaryotes throughout and after the late Proterozoic global oxygenation.

Nutritional intake of Aplanochytrium (Labyrinthulea, Stramenopiles) from living diatoms revealed by culture experiments suggesting the new prey-predator interactions in the grazing food web of the marine ecosystem.

Labyrinthuleans (Labyrinthulea, Stramenopiles) are recognized as decomposers in marine ecosystems but their nutrient sources are not fully understood. We conducted two-membered culture experiments with labyrinthuleans and diatoms to discover where labyrinthuleans obtain their nutrients from. The results showed that Aplanochytrium strains obtained nutrients by consuming living diatoms. Aplanochytrium cells did not release digestive enzymes into the medium, but adhered to diatom cells via the tip of their characteristic ectoplasmic net system to obtain nutrients from them. The chloroplast and cell contents of the diatoms shrank and were absorbed, and then the number of Aplanochytrium cells rapidly increased as multiple aplanospores were released. To estimate the effect of labyrinthulean organisms including Aplanochytrium on marine ecosystem, we explored the dataset generated by the Tara Oceans Project from a wide range of oceanic regions. The average proportion of all labyrinthulean sequences to diatom sequences at each station was about 10%, and labyrinthulids, oblongichytrids, and aplanochytrids were the major constituent genera, accounting for more than 80% of labyrinthuleans. Therefore, these groups are suggested to greatly affect the marine ecosystem. There were positive correlations between aplanochytrids and phototrophs, green algae, and diatoms. At many stations, relatively large proportions of aplanochytrid sequences were detected in the size fraction larger than their cell size. This implied that Aplanochytrium cells increased their particle size by adhering to each other and forming aggregates with diatoms that are captured by larger zooplankton in the environment, thereby bypassing the food web pathway via aplanochytrids to higher predators. The intake of nutrients from diatoms by aplanochytrids represents a newly recognized pathway in the grazing food chain in the marine ecosystem.

Elemental composition and ultrafine structure of the skeleton in shell-bearing protists-A case study of phaeodarians and radiolarians.

Cross-sections were prepared by ultramicrotome (UM) and focused ion beam (FIB) system in order to examine the skeletal structure of ecologically and geologically important shell-bearing protists: phaeodarians and radiolarians. The elemental composition of the skeleton was clarified by the energy dispersive X-ray spectroscopy, suggesting that the skeletons of both groups are mainly made of amorphous silica (SiO2·nH2O) with other minor elements (Na, Mg, Al, Cl, K, Ca and Fe) and that these two groups have similar elemental composition, compared with other siliceous organisms (diatoms and sponges). However, the structural difference among the two groups was confirmed: phaeodarian skeletons are porous, unlike radiolarians with solid skeletons. It was also revealed that the phaeodarian skeleton contains concentric layered structure with spaces, presumably related to the ontogenetic skeleton formation. The distinction in the skeletal ultrafine structure (porous/solid and non-dense/dense) would reflect the ecological difference among the two groups and could be an effective criterion to determine whether microfossils belong to Radiolaria or Phaeodaria. The UM and FIB combined method presented in this study could be a useful approach to examine the chemical and structural characteristics of unculturable and/or rare microorganisms.

Nutritional Intake by Ectoplasmic Nets of Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles).

Thraustochytrid cells attach to their food via ectoplasmic nets (ENs). Here, we analyzed the cause and effect relationship between the various forms and functions of ENs of Schizochytrium aggregatum. The ENs spread out over a large area forming a fine network to efficiently search for the experimental food source. After recognizing the experimental food source, the ENs that attached to the food source became thicker, and net elements developed. The thick ENs on the surface at the attachment site were enveloped in dense materials (fibrous materials), which were visualized as fibrous layers under a transmission electron microscope. Experiments using fluorescein diacetate and the fluorescent glucose analog 2-NBDG showed that the production rate of hydrolytic enzymes and the absorption rate of glucose by ENs of S. aggregatum increased in the presence of an experimental food source. Our results reveal that ENs change their shape and function according to the presence/absence of a food source.

Correction: Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine.

[This corrects the article DOI: 10.1371/journal.pone.0102377.].

Bothrosome Formation in Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles) during Zoospore Settlement.

Labyrinthulomycetes are characterized by the presence of ectoplasmic nets originating from an organelle known as the bothrosome, whose evolutionary origin is unclear. To address this issue, we investigated the developmental process from a zoospore to a vegetative cell in Schizochytrium aggregatum. After disappearance of the flagellum during zoospore settlement, the bothrosome emerged at the anterior-ventral pole of the cells. A new Golgi body also appeared at this stage, and the bothrosome was positioned close to both the new and the old Golgi bodies. This observation suggested that the Golgi body is related to the formation of the bothrosome. Actin appeared as a spot in the same location as the newly appeared bothrosome, as determined by immunofluorescence labeling. An immunoelectron microscopic analysis revealed that actin was present in the ectoplasmic nets and in the cytoplasm around the bothrosome, indicating that the electron-dense materials of the bothrosome are not the polar center of F-actin. This suggests that actin filaments pull the endoplasmic reticulum to the bothrosome and induce the membrane to become evaginated within ectoplasmic nets.

Ecological Dynamics of Two Distinct Viruses Infecting Marine Eukaryotic Decomposer Thraustochytrids (Labyrinthulomycetes, Stramenopiles).

Thraustochytrids are cosmopolitan osmotrophic or heterotrophic microorganisms that are considered as important decomposers in coastal ecosystems. However, because of a lack of estimation method for each genus or systematic group of them, relatively little is known about their ecology in situ. Previously, we reported two distinct types of virus infecting thraustochytrids (AuRNAV: reported as SssRNAV, and SmDNAV) suggesting they have wide distributions in the host-virus systems of coastal environments. Here we conducted a field survey from 2004 through 2005 to show the fluctuation pattern of thraustochytrids and their viruses in Hiroshima Bay, Japan. During the field survey, we monitored the dynamics of the two types of thraustochytrid-infecting virus: small viruses causing lysis of Aurantiochytrium sp. NIBH N1-27 (identified as AuRNAV) and the large viruses of Sicyoidochytrium minutum NBRC 102975 (similar to SmDNAV in physiology and morphology). Fluctuation patterns of the two distinct types of virus were different from each other. This may reflect the difference in the preference of organic substrates; i.e., it may be likely the host of AuRNAV (Aurantiochytrium sp.) increases utilizing algal dead bodies or feeble cells as the virus shows a large increase in abundance following raphidophyte blooms; whereas, the trophic nutrient supply for S. minutum may primarily depend on other constantly-supplied organic compounds because it did not show any significant change in abundance throughout the survey. Further study concerning the population composition of thraustochytrids and their viruses may demonstrate the microbial ecology (especially concerning the detrital food web) of marine environments.

Novel lysophospholipid acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b responsible for generation of palmitate-docosahexaenoate-phosphatidylcholine and phosphatidylethanolamine.

N-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, 22:6n-3), have been reported to play roles in preventing cardiovascular diseases. The major source of DHA is fish oils but a recent increase in the global demand of DHA and decrease in fish stocks require a substitute. Thraustochytrids, unicellular marine protists belonging to the Chromista kingdom, can synthesize large amounts of DHA, and, thus, are expected to be an alternative to fish oils. DHA is found in the acyl chain(s) of phospholipids as well as triacylglycerols in thraustochytrids; however, how thraustochytrids incorporate DHA into phospholipids remains unknown. We report here a novel lysophospholipid acyltransferase (PLAT1), which is responsible for the generation of DHA-containing phosphatidylcholine and phosphatidylethanolamine in thraustochytrids. The PLAT1 gene, which was isolated from the genomic DNA of Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, and the FLAG-tagged recombinant enzyme was characterized after purification with anti-FLAG affinity gel. PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates. In contrast to the in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-knockout mutants, resulting in a decrease of 16:0-DHA-phosphatidylcholine (PC) [PC(38:6)] and 16:0-DHA-phosphatidylethanolamine (PE) [PE(38:6)], which are two major DHA-containing phospholipids in A. limacinum F26-b. However, the amounts of other phospholipid species including DHA-DHA-PC [PC(44:12)] and DHA-DHA-PE [PE(44:12)] were almost the same in plat-knockout mutants and the wild-type. These results indicate that PLAT1 is the enzyme responsible for the generation of 16:0-DHA-PC and 16:0-DHA-PE in the thraustochytrid.

Bacillus ligniniphilus sp. nov., an alkaliphilic and halotolerant bacterium isolated from sediments of the South China Sea.

An alkaliphilic and halotolerant Gram-stain-positive bacterium, which was isolated from sediment samples from the South China Sea, was subjected to a taxonomic study. The isolate, strain L1T, grew well at a wide range of temperatures and pH values, 10.0-45.0 °C and pH 6-11, with optima at 30 °C and pH 9.0, respectively. The growth of strain L1T occurred at total salt concentrations of 0-10% (w/v) with an optimum at 2% (w/v). Phylogenetic analysis based on 16S rRNA sequence comparison indicated that the isolate represented a member of the genus Bacillus. The strains most closely related to strain L1T were Bacillus nanhaiisediminis JCM 16507T, Bacillus halodurans DSM 497T and Bacillus pseudofirmus DSM 8715T, with 16S rRNA similarities of 96.5%, 95.9% and 95.7%, respectively. DNA-DNA hybridization of strain L1T with the type strains of the most closely related species, B. nanhaiisediminis JCM 16507T, B. halodurans DSM 497T and B. pseudofirmus DSM 8715T, showed reassociation values of about 21.7%, 14.3% and 13.9%, respectively. The DNA G+C content of strain L1T was 40.76 mol%. The predominant isoprenoid quinone was menaquinone 7 (MK-7). The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant cellular fatty acids of strain L1T were iso-C14 : 0 and anteiso-C15:0. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Based on the phenotypic and phylogenetic characteristics, it is proposed that strain L1T (=JCM 18543T=DSM 26145T) should be classified as the type strain of Bacillus ligniniphilus sp. nov.

Effect of trace elements on growth of marine eukaryotes, tharaustochytrids.

We determined the effect of trace elements on the growth of thraustochytrids. The growth of the strains cultured with the trace elements was much higher than that of the strains cultured without any trace element. Iron and zinc were particularly important to obtaining the optimum growth of thraustochytrids.

Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for Thraustochytrids.

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo(r)), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo(r) marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo(r) mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C(20:3n-6)) and eicosatetraenoic acid (C(20:4n-3)), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C(20:4n-6)) and eicosapentaenoic acid (C(20:5n-3)), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.

Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304.

Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des-disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des-disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways.

Optimization of culture conditions of the thraustochytrid Aurantiochytrium sp. strain 18W-13a for squalene production.

Optimum conditions of temperature, salinity and glucose concentration were investigated for squalene production of the strain of Aurantiochytrium sp. 18 W-13a, with a high content of squalene. Squalene production by this strain was optimum at 25 °C, 25-50% seawater concentration and 2-6% glucose concentration. When this strain was grown in the optimum condition, the squalene content and production of approximately 171 mg/g dry weight and 0.9 g/L were much higher than that previously reported in thraustochytrids, plants and yeasts, respectively. Therefore, 18 W-13a could be used as an alternative source of commercial squalene.

Detection of genes involved in fatty acid elongation and desaturation in thraustochytrid marine eukaryotes.

Heterotrophic marine protists known as thraustochytrids can synthesize polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The biosynthetic pathways of PUFAs in thraustochytrids are poorly understood, however. In this study, we attempted to reveal the enzymes involved in DHA synthesis in thraustochytrids. Nine thraustochytrid strains representing 3 genera (Aurantiochytrium, Schizochytrium, and Thraustochytrium) were used for PCR-based detection of the genes encoding Δ5-elongase and Δ4-desaturase and for fatty acid analysis. The degenerate primers were designed to amplify the Δ5-elongase and Δ4-desaturase genes, and the partial sequences of the enzymes were obtained from the genera Thraustochytrium and Schizochytrium. These fragments were identical to those of known Δ5-elongase and Δ4-desaturase. Neither Δ5-elongase nor Δ4-desaturase was detected in the strains belonging to the genus Aurantiochytrium, however, suggesting that this group likely synthesizes DHA not via the elongation/desaturation pathway but via an alternate pathway such as the polyketide synthase pathway. The fatty acid profiles of thraustochytrids were consistent with the presence of genes involved in PUFA biosynthesis in thraustochytrid genera. Thus, our findings suggest that two biosynthetic pathways for PUFAs exist in these organisms.

Increase of eicosapentaenoic acid in thraustochytrids through thraustochytrid ubiquitin promoter-driven expression of a fatty acid {delta}5 desaturase gene.

Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C(16:0)), n - 6 docosapentaenoic acid (DPA) (C(22:5)(n) (- 6)), and docosahexaenoic acid (DHA) (C(22:6)(n) (- 3)), with eicosapentaenoic acid (EPA) (C(20:5)(n) (- 3)) and arachidonic acid (AA) (C(20:4)(n) (- 6)) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid Δ5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C(20:4)(n) (- 3)) by the Δ5 desaturase. The levels of EPA and AA were also increased by 4.6- and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-γ-linolenic acid (DGLA) (C(20:3)(n) (- 6)) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the Δ5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs.

The distribution of extracellular cellulase activity in marine Eukaryotes, thraustochytrids.

Cellulolytic ability was evaluated in 19 strains of thraustochytrids, representing nine genera, using carboxymethylcellulose (CMC) as a substrate. Extracellular cellulolytic enzyme activity was determined in the culture supernatants during cell growth. CMC hydrolysis was observed in 14 out of the 19 strains examined. These belonged to the genera Aplanochytrium, Botryochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium and Ulkenia. On the other hand, cellulolytic enzyme activity was not detected in any strains belonging to the genus Aurantiochytrium.

Labyrinthulomycetes phylogeny and its implications for the evolutionary loss of chloroplasts and gain of ectoplasmic gliding.

The labyrinthulomycetes, also known as the 'Labyrinthulomycota' are saprotrophic or less frequently parasitic stramenopilan protists, usually in marine ecosystems. Their distinguishing feature is an 'ectoplasmic net,' an external cytoplasmic network secreted by a specialized organelle that attaches the cell to its substrate and secretes digestive enzymes for absorptive nutrition. In this study, one of our aims was to infer the phylogenetic position of the labyrinthulomycetes relative to the non-photosynthetic bicoeceans and oomycetes and the photosynthetic ochrophytes and thereby evaluate patterns of change from photosynthesis to saprotrophism among the stramenopiles. For the labyrinthulomycetes, we determined sequences of the actin, beta-tubulin, and elongation factor 1-alpha gene fragments and where necessary, ribosomal small subunit (SSU) genes. Multilocus analysis using standard tree construction techniques not only strongly supported the oomycetes as the sister group to the phototrophic stramenopiles, but also, for the first time with moderate statistical support, showed that the labyrinthulomycetes and the bicoecean as sister groups. The paraphyly of the non-photosynthetic groups was consistent with independent loss of photosynthesis in labyrinthulomycetes and oomycetes. We also wished to develop a phylogenetically based hypothesis for the origin of the gliding cell bodies and the ectoplasmic net found in some labyrinthulomycetes. The cells of species in Labyrinthula and Aplanochytrium share a specialized form of motility involving gliding on ectoplasmic tracks. Before our study, only ribosomal DNA genes had been determined for these genera and their phylogenetic position in the labyrinthulomycetes was equivocal. Multilocus phylogenies applying our newly determined protein-coding sequences divided the labyrinthulomycetes between sister clades 'A' and 'B' and showed that the monophyletic group containing all of the gliding species was nested among non-gliding species in clade B. This phylogeny suggested that species that glide via an ectoplasm evolved from species that had used the ectoplasm mainly for anchorage and assimilation rather than motility.