Seasonal and annual changes in the microbial communities of Ofunato Bay, Japan, based on metagenomics.

Five years of datasets from 2015 to 2019 of whole genome shotgun sequencing for cells trapped on 0.2-µm filters of seawater collected monthly from Ofunato Bay, an enclosed bay in Japan, were analysed, which included the 2015 data that we had reported previously. Nucleotide sequences were determined for extracted DNA from three locations for both the upper (1 m) and deeper (8 or 10 m) depths. The biotic communities analysed at the domain level comprised bacteria, eukaryotes, archaea and viruses. The relative abundance of bacteria was over 60% in most months for the five years. The relative abundance of the SAR86 cluster was highest in the bacterial group, followed by Candidatus Pelagibacter and Planktomarina. The relative abundance of Ca. Pelagibacter showed no relationship with environmental factors, and those of SAR86 and Planktomarina showed positive correlations with salinity and dissolved oxygen, respectively. The bacterial community diversity showed seasonal changes, with high diversity around September and low diversity around January for all five years. Nonmetric multidimensional scaling analysis also revealed that the bacterial communities in the bay were grouped in a season-dependent manner and linked with environmental variables such as seawater temperature, salinity and dissolved oxygen.

Seasonal changes in the communities of photosynthetic picoeukaryotes in Ofunato Bay as revealed by shotgun metagenomic sequencing.

Small photosynthetic eukaryotes play important roles in oceanic food webs in coastal regions. We investigated seasonal changes in the communities of photosynthetic picoeukaryotes (PPEs) of the class Mamiellophyceae, including the genera Bathycoccus, Micromonas and Ostreococcus, in Ofunato Bay, which is located in northeastern Japan and faces the Pacific Ocean. The abundances of PPEs were assessed over a period of one year in 2015 at three sampling stations, KSt. 1 (innermost bay area), KSt. 2 (middle bay area) and KSt. 3 (bay entrance area) at depths of 1 m (KSt. 1, KSt. 2 and KSt. 3), 8 m (KSt. 1) or 10 m (KSt. 2 and KSt. 3) by employing MiSeq shotgun metagenomic sequencing. The total abundances of Bathycoccus, Ostreococcus and Micromonas were in the ranges of 42-49%, 35-49% and 13-17%, respectively. Considering all assayed sampling stations and depths, seasonal changes revealed high abundances of PPEs during the winter and summer and low abundances during late winter to early spring and late summer to early autumn. Bathycoccus was most abundant in the winter, and Ostreococcus showed a high abundance during the summer. Another genus, Micromonas, was relatively low in abundance throughout the study period. Taken together with previously suggested blooming periods of phytoplankton, as revealed by chlorophyll a concentrations in Ofunato Bay during spring and autumn, these results for PPEs suggest that greater phytoplankton blooming has a negative influence on the seasonal occurrences of PPEs in the bay.

Metagenome-based diversity analyses suggest a strong locality signal for bacterial communities associated with oyster aquaculture farms in Ofunato Bay.

Ofunato Bay, in Japan, is the home of buoy-and-rope-type oyster aquaculture activities. Since the oysters filter suspended materials and excrete organic matters into the seawater, bacterial communities residing in its vicinity may show dynamic changes depending on the oyster culture activities. We employed a shotgun metagenomic technique to study bacterial communities near oyster aquaculture facilities at the center of the bay (KSt. 2) and compared the results with those of two other localities far from the station, one to the northeast (innermost bay, KSt. 1) and the other to the southwest (bay entrance, KSt. 3). Seawater samples were collected every month from January to December 2015 from the surface (1 m) and deeper (8 or 10 m) layers of the three locations, and the sequentially filtered fraction on 0.2-µm membranes was sequenced on an Illumina MiSeq system. The acquired reads were uploaded to MG-RAST for KEGG functional abundance analysis, while taxonomic analyses at the phylum and genus levels were performed using MEGAN after parsing the BLAST output. Discrimination analyses were then performed using the ROC-AUC value of the cross validation, targeting the depth (shallow or deep), locality [(KSt. 1 + KSt. 2) vs. KSt 3; (KSt. 1 + KSt. 3) vs. KSt. 2 or the (KSt. 2 + KSt. 3) vs. KSt. 1] and seasonality (12 months). The matrix discrimination analysis on the adjacent 2 continuous seasons by ROC-AUC, which was based on the datasets that originated from different depths, localities and months, showed the strongest discrimination signal on the taxonomy matrix at the phylum level for the datasets from July to August compared with those from September to June, while the KEGG matrix showed the strongest signal for the datasets from March to June compared with those from July to February. Then, the locality combination was subjected to the same ROC-AUC discrimination analysis, resulting in significant differences between KSt. 2 and KSt. 1 + KSt. 3 on the KEGG matrix. These results suggest that aquaculture activities markedly affect bacterial functions.

Taxonomic profiles in metagenomic analyses of free-living microbial communities in the Ofunato Bay.

The Ofunato Bay in Iwate Prefecture, Japan is a deep coastal bay located at the center of the Sanriku Rias Coast and considered an economically and environmentally important asset. Here, we describe the first whole genome sequencing (WGS) study on the microbial community of the bay, where surface water samples were collected from three stations along its length to cover the entire bay; we preliminarily sequenced a 0.2 µm filter fraction among sequentially size-fractionated samples of 20.0, 5.0, 0.8 and 0.2 µm filters, targeting the free-living fraction only. From the 0.27-0.34 Gb WGS library, 0.9 × 106-1.2 × 106 reads from three sampling stations revealed 29 bacterial phyla (~80% of assigned reads), 3 archaeal phyla (~4%) and 59 eukaryotic phyla (~15%). Microbial diversity obtained from the WGS approach was compared with 16S rRNA gene results by mining WGS metagenomes, and we found similar estimates. The most frequently recovered bacterial sequences were Proteobacteria, predominantly comprised of 18.0-19.6% Planktomarina (Family Rhodobacteraceae) and 13.7-17.5% Candidatus Pelagibacter (Family Pelagibacterales). Other dominant bacterial genera, including Polaribacter (3.5-6.1%), Flavobacterium (1.8-2.6%), Sphingobacterium (1.4-1.6%) and Cellulophaga (1.4-2.0%), were members of Bacteroidetes and likely associated with the degradation and turnover of organic matter. The Marine Group I Archaea Nitrosopumilus was also detected. Remarkably, eukaryotic green alga Bathycoccus, Ostreococcus and Micromonas accounted for 8.8-15.2%, 3.6-4.9% and 2.1-3.1% of total read counts, respectively, highlighting their potential roles in the phytoplankton bloom after winter mixing.

Basin-scale seasonal changes in marine free-living bacterioplankton community in the Ofunato Bay.

The Ofunato Bay in the northeastern Pacific Ocean area of Japan possesses the highest biodiversity of marine organisms in the world and has attracted much attention due to its economic and environmental importance. We report here a shotgun metagenomic analysis of the year-round variation in free-living bacterioplankton collected across the entire length of the bay. Phylogenetic differences among spring, summer, autumn and winter bacterioplankton suggested that members of Proteobacteria tended to decrease at high water temperatures and increase at low temperatures. It was revealed that Candidatus Pelagibacter varied seasonally, reaching as much as 60% of all sequences at the genus level in the surface waters during winter. This increase was more evident in the deeper waters, where they reached up to 75%. The relative abundance of Planktomarina also rose during winter and fell during summer. A significant component of the winter bacterioplankton community was Archaea (mainly represented by Nitrosopumilus), as their relative abundance was very low during spring and summer but high during winter. In contrast, Actinobacteria and Cyanobacteria appeared to be higher in abundance during high-temperature periods. It was also revealed that Bacteroidetes constituted a significant component of the summer bacterioplankton community, being the second largest bacterial phylum detected in the Ofunato Bay. Its members, notably Polaribacter and Flavobacterium, were found to be high in abundance during spring and summer, particularly in the surface waters. Principal component analysis and hierarchal clustering analyses showed that the bacterial communities in the Ofunato Bay changed seasonally, likely caused by the levels of organic matter, which would be deeply mixed with surface runoff in the winter.

Seasonal changes in the abundance of bacterial genes related to dimethylsulfoniopropionate catabolism in seawater from Ofunato Bay revealed by metagenomic analysis.

Ofunato Bay is located in the northeastern Pacific Ocean area of Japan, and it has the highest biodiversity of marine organisms in the world, primarily due to tidal influences from the cold Oyashio and warm Kuroshio Currents. Our previous results from performing shotgun metagenomics indicated that Candidatus Pelagibacter ubique and Planktomarina temperata were the dominant bacteria (Reza et al., 2018a, 2018b). These bacteria are reportedly able to catabolize dimethylsulfoniopropionate (DMSP) produced from phytoplankton into dimethyl sulfide (DMS) or methanethiol (MeSH). This study was focused on seasonal changes in the abundances of bacterial genes (dddP, dmdA) related to DMSP catabolism in the seawater of Ofunato Bay by BLAST+ analysis using shotgun metagenomic datasets. We found seasonal changes among the Candidatus Pelagibacter ubique strains, including those of the HTCC1062 type and the Red Sea type. A good correlation was observed between the chlorophyll a concentrations and the abundances of the catabolic genes, suggesting that the bacteria directly interact with phytoplankton in the marine material cycle system and play important roles in producing DMS and MeSH from DMSP as signaling molecules for the possible formation of the scent of the tidewater or as fish attractants.

Repercussions of the Great East Japan Earthquake tsunami on ellipsoidal Alexandrium cysts (Dinophyceae) in Ofunato Bay, Japan.

Shellfish aquaculture in Ofunato Bay, Northeast Japan, was seriously damaged by a tsunami generated by the Great East Japan Earthquake, March 11th, 2011, accompanied by paralytic shellfish poisoning (PSP) outbreaks caused by Alexandrium tamarense (Dinophyceae). To understand longer future trends of PSP, an investigation was made of the historical occurrence and causes of Alexandrium outbreaks after the tsunami. Vertical distributions of Alexandrium cysts in two sediment-cores from Ofunato Bay revealed that the sediments above ca. 25 cm were eroded, re-suspended and re-deposited, and they included unusually abundant Alexandrium cysts. This abundance of cysts was due to re-deposition of older sediments by the tsunami. The first Ofunato Bay PSP incident was in 1961 after the Chilean Earthquake tsunami and was probably caused by similar unusual blooms of Alexandrium germinated from older sediments as the Great East Japan tsunami, together with nutrient enrichment because of population increase at the start of shellfish aquaculture.

Differential DNA rearrangements of plastid genes, psbA and psbD, in two species of the dinoflagellate Alexandrium.

Plastomes of the peridinin-containing dinoflagellates are composed of a limited number of genes, which are carried individually on small circular molecules, termed 'minicircles'. Although the prevalent plastid chromosome of most algae and plants has only a single copy of each gene, our previous study showed that low copy numbers of multiple variants of the gene psbA co-exist with the 'ordinary' gene encoding the D1 protein in minicircles of Alexandrium tamarense. Although none of the psbA variants encoded the entire protein, they persisted in culture. In this study, we compared the distribution and structure of psbA and psbD variants in two species of Alexandrium to characterize DNA rearrangement within these genes. In addition to four previously reported psbA variants, three psbD variants were found in A. tamarense minicircles. The ordinary psbA and psbD genes also co-existed with variants in another species, A. catenella. The sequences of the ordinary genes were virtually identical in the two species. All the variants comprised insertion or deletion mutations, with no base substitutions being identified. Duplicated parts of the coding sequences were contained in most of the insertions. Short direct repeats (4-14 bp) and/or adenine + thymine-rich motifs were present in all mutation regions, although the position and/or the sequence of each DNA rearrangement was unique to each variant. The results indicated that replication-based repeat-mediated recombination was responsible for generation of the variants.

Identification of transcribed and persistent variants of the psbA gene carried by plastid minicircles in a dinoflagellate.

Plastid genomes of the peridinin-containing dinoflagellates are composed of a limited number of genes that are contained separately on small circular molecules (minicircles). It has been shown that occasionally aberrant minicircles are generated, but they are usually small and contain little coding information. In this study, we discovered multiple variants, a putative "gene family", of the gene psbA in the plastid minicircles of the dinoflagellate Alexandrium tamarense, which have persisted for almost 3 years in culture. Each variant, like the ordinary psbA, existed on distinct minicircles of similar size (5-6 kb). These psbA variants retained all, or almost all, the coding sequence of the ordinary gene, and all four were transcribed and edited after transcription, even though they could not encode the entire protein due to intervening or translocated sequences. Repeat elements were generally found in the relatively large non-coding region of these minicircles. Each psbA variant might have been generated by DNA recombination and/or replication slippage, as for previously reported aberrant minicircles. The fact that these minicircles are transcribed, individually edited and maintained in the genome suggests that they are functionally important, although their precise roles remain unclear.

Characterization of cyanobacterial carotenoid ketolase CrtW and hydroxylase CrtR by complementation analysis in Escherichia coli.

The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW148) can convert not only beta-carotene but also zeaxanthin into their 4,4'-ketolated products, canthaxanthin and astaxanthin, respectively. In contrast, the Anabaena CrtR enzymes were very poor in accepting either beta-carotene or canthaxanthin as substrates. By comparison, the Synechocystis sp. PCC 6803 CrtR converted beta-carotene into zeaxanthin efficiently. We could assign the catalytic functions of the gene products involved in ketocarotenoid biosynthetic pathways in Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, based on the present and previous findings. This explains why these cyanobacteria cannot produce astaxanthin and why only Synechocystis sp. PCC 6803 can produce zeaxanthin.

The D1 and D2 proteins of dinoflagellates: unusually accumulated mutations which influence on PSII photoreaction.

Plastid encoded genes of the dinoflagellates are rapidly evolving and most divergent. The importance of unusually accumulated mutations on structure of PSII core protein and photosynthetic function was examined in the dinoflagellates, Symbiodinium sp. and Alexandrium tamarense. Full-length cDNA sequences of psbA (D1 protein) and psbD (D2 protein) were obtained and compared with the other oxygen-evolving photoautotrophs. Twenty-three amino acid positions (7%) for the D1 protein and 34 positions (10%) for the D2 were mutated in the dinoflagellates, although amino acid residues at these positions were conserved in cyanobacteria, the other algae, and plant. Many mutations were likely to distribute in the N-terminus and the D-E interhelical loop of the D1 protein and helix B of D2 protein, while the remaining regions were well conserved. The different structural properties in these mutated regions were supported by hydropathy profiles. The chlorophyll fluorescence kinetics of the dinoflagellates was compared with Synechocystis sp. PCC6803 in relation to the altered protein structure.

A novel type of kleptoplastidy in Dinophysis (Dinophyceae): presence of haptophyte-type plastid in Dinophysis mitra.

Red-fluorescent, non-phycobilin-containing plastids were found in the heterotrophic dinoflagellate, Dinophysis mitra. Transmission electron microscopy showed that they contained a three-layer thylakoid, the absence of girdle lamella, and an embedded pyrenoid with thylakoid intrusions. These characteristics all coincide with haptophyte plastids. Phylogenetic analysis of the plastid small-subunit ribosomal DNA (SSU rDNA) revealed that the Dinophysis mitra sequences are distantly related to those of phycobilin-containing Dinophysis species and are positioned within a lineage of haptophytes belonging to Prymnesiophyceae. Because the plastid SSU rDNA sequences of Dinophysis mitra showed significant heterogeneity, despite being derived from a single species, it is highly likely that they were not established as plastids through an evolutionary process but are "kleptoplastids" (temporally stolen plastids) from multiple sources of haptophytes in the environment. We deduced that Dinophysis mitra takes up haptophytes myzocytotically and selectively retains the plastid with surrounding plastidal membranes, whereas other haptophyte cell components are degraded. This represents another type of kleptoplastidy in the Dinophysis species, which mostly harbor cryptophyte plastids, and is the first evidence of kleptoplastidy originating from haptophytes.

Salinity effect on growth and toxin production of four tropical Alexandrium species (Dinophyceae).

Four tropical PSP toxins-producing dinoflagellates, Alexandrium minutum, Alexandrium tamiyavanichii, Alexandrium tamarense and Alexandrium peruvianum from Malaysian waters were studied to investigate the influences of salinity on growth and toxin production. Experiments were conducted on constant temperature 25 degrees C, 140 microE mol m(-2) s(-1) and under 14:10 light:dark photo-cycle with salinity ranged from 2 to 30 psu. The PSP-toxin congeners, GTX 1-6, STX, dcSTX, NEO and C1-C2 were analysed by high performance liquid chromatography. Salinity tolerance of the four species in decreasing order is A. minutum>A. peruvianum>A. tamarense>A. tamiyavanichii. Specific growth rates and maximum densities varied among these species with A. minutum recorded as the highest, 0.5 day(-1) and 6 x 10(4) cells L(-1). Toxin content decreased with elevated salinities in A. minutum, the highest toxin content was about 12 fmole cell(-1) at 5 psu. In A. tamiyavanichii, toxin content peaked at optimal growth salinity (20 and 25 psu). Toxin content of A. tamarense, somehow peaked at sub-optimal growth salinity (15 and 30 psu). Results of this study implied that salinity fluctuation not only influenced the growth physiology but also toxin production of these species.

Development of molecular probes for dinophysis (dinophyceae) plastid: a tool to predict blooming and explore plastid origin.

Dinophysis are species of dinoflagellates that cause diarrhetic shellfish poisoning. We have previously reported that they probably acquire plastids from cryptophytes in the environment, after which they bloom. Thus monitoring the intracellular plastid density in Dinophysis and the source cryptophytes occurring in the field should allow prediction of Dinophysis blooming. In this study the nucleotide sequences of the plastid-encoded small subunit ribosomal RNA gene and rbcL (encoding the large subunit of RuBisCO) from Dinophysis spp. were compared with those of cryptophytes, and genetic probes specific for the Dinophysis plastid were designed. Fluorescent in situ hybridization (FISH) showed that the probes bound specifically to Dinophysis plastids. Also, FISH on collected nanoplankton showed the presence of probe-hybridized eukaryotes, possibly cryptophytes with plastids identical to those of Dinophysis. These probes are useful not only as markers for plastid density and activity of Dinophysis, but also as tools for monitoring cryptophytes that may be sources of Dinophysis plastids.

Presence of free D-amino acids in microalgae.

Several species of microalgae (phytoplankton), 4 species of freshwater algae and 4 species of marine diatoms, were cultured germ-free in the laboratory. The presence of free D-amino acids was verified in these species by a reversed-phase HPLC analysis. D-Aspartate was detected in all the microalgae examined, but D-alanine was only present in the marine diatoms. The D-amino acid content in Asterionella sp. of the marine diatoms increased from the exponential phase to the stationary phase and then decreased to the phase of decline.

Molecular evidence for plastid robbery (Kleptoplastidy) in Dinophysis, a dinoflagellate causing diarrhetic shellfish poisoning.

The dinoflagellate genus Dinophysis contains species known to cause diarrhetic shellfish poisoning. Although most photosynthetic dinoflagellates have plastids with peridinin, photosynthetic Dinophysis species have cryptophyte-like plastids containing phycobilin rather than peridinin. We sequenced nuclear- and plastid-encoded SSU rDNA from three photosynthetic species of Dinophysis for phylogenetic analyses. In the tree of nuclear SSU rDNA, Dinophysis was a monophyletic group nested with peridinin-containing dinoflagellates. However, in the tree of plastid SSU rDNA, the Dinophysis plastid lineage was within the radiation of cryptophytes and was closely related to Geminigera cryophila. These analyses indicate that an ancestor of Dinophysis, which may have originally possessed peridinin-type plastid and lost it subsequently, adopted a new plastid from a cryptophyte. Unlike dinoflagellates with fully integrated plastids, the Dinophysis plastid SSU rDNA sequences were identical among the three species examined, while there were species-specific base substitutions in their nuclear SSU rDNA sequences. Queries of the DNA database showed that the plastid SSU rDNA sequence of Dinophysis is almost identical to that of an environmental DNA clone of a <10 pm sized plankter, possibly a cryptophyte and a likely source of the Dinophysis plastid. The present findings suggest that these Dinophysis species engulfed and temporarily retained plastids from a cryptophyte.