Takehiko Ogata’s research while affiliated with Kitasato University and other places

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.

Bacterial communities are important factors governing changes in river ecology. We evaluated the diversity of bacterial communities in sediment and size-fractionated water samples collected from the Tama River, Tokyo, Japan, to verify their taxonomy and functional classes. Samples were collected from three river areas, namely up-, mid- and downstream, and analyzed using MiSeq shotgun metagenomic sequencing. Bacterial diversity in the river water was highest midstream for the free-living bacterial communities. Dominant bacterial classes upstream were Alphaproteobacteria and Betaproteobacteria, and there was a gradual shift to dominance by Actinobacteria mid- and downstream. Potential pathogenic genera, such as Flavobacterium, Mycobacterium and Bacteroides, also dominated mid- and downstream. Interestingly, Mycobacterium was most abundant midstream, whereas Pseudomonas was observed upstream. The gene possibly associated with sulfur metabolism, sigB, and those associated with purine metabolism, purF, purD, purN, purH, guaB and guaA, dominated in the bacterial communities found mid- and downstream and are thought to ensure survival and virulence in their environments. NO -N, NH -N and PO -P were found to influence bacterial populations in water but not those residing in the sediment.

Feeding experiments with a natural microplankton assemblage and egg production experiments with the neritic copepod Acartia steueri were conducted simultaneously in an inlet on the Sanriku coast during autumn. Diapausing egg production status of A. steueri was also investigated. Further, A. steueri dominated during September to October and then gradually decreased until December, before disappearing after January. The dominant microplankton in natural waters during the study period were dinoflagellates, followed by centric and pennate diatoms and oligotrich ciliates. A. steueri fed only on dinoflagellates and oligotrich ciliates at rates of 87.0–309 and 17.7–71.5 cells ind⁻¹ d⁻¹, respectively. Egg production rate of A. steueri ranged from 1.6 to 15.0 eggs female⁻¹ d⁻¹ and significantly increased as ingestion rates of dinoflagellates and oligotrich ciliates increased. The copepod began producing diapausing eggs in October, and by late December, 86% of the eggs produced were diapausing. The population’s egg production rate was highest in September (18,961 eggs m⁻³ d⁻¹) and gradually decreased through the end of December. These dietary and diapausing egg production seasons of A. steueri in the Sanriku area are significantly different from the results of previous studies in the temperate zone, where the copepod mainly fed on diatoms and produced diapausing eggs during spring. A. steueri can alter its feeding habits and the timing of diapausing egg production in response to changes in habitat. The flexibility of this species to environmental change has likely allowed expansion of its geographical distribution.

In this article, methods or techniques of metagenomics including targeted 16S/18S rRNA analyses and shotgun sequencing will be discussed. It is sometimes difficult, especially for beginners, to follow the manufacturer’s recommendation as mentioned in the protocol and to go through different steps from the preparation of starting material (e.g., DNA), library preparation, and so on. We will try to explain all the steps in detail and share our experience here. It all starts with collection of samples and collection of ecological/environmental metadata followed by sample fractionation (optional), extraction of DNA, sequencing, and finally data analyses to interpret results. Sample collection has always been the most important part of a study as it requires proper planning, a good workforce to execute, permission(s) of sampling from appropriate authority, and precaution(s) about endangered species during sampling. Here, we first describe methodology for a shallow river and in the later section methodology for a deep marine bay. In either case, slight modifications can be made to succeed in sampling. Determination of physicochemical parameters as metadata simultaneously is also an important task. These samples are then processed to extract DNA which needs to be representative of all cells present in the sample. Finally, sequencing is done by a next-generation sequencer, and data analyses are completed. Through these methods, scientists are now able to overcome the unculturability problem of more than 99% of environmental microorganisms and uncovered functional gene diversity of environmental microorganisms.

Development of high-throughput DNA sequencing technologies has enabled scientists to generate vast amounts of genetic information that may provide a comprehensive understanding of key roles played by environmental microorganisms. Generally the microorganisms inhabit a particular niche and correlate well with environmental changes. It is accepted that the read counts obtained through metagenomic analyses correlate semi-quantitatively with the relative abundance of bacterial species. In our marine metagenomic study conducted on the Ofunato Bay, Iwate Prefecture, Japan, we observed such correlation which exists for bacterioplankton Candidatus Pelagibacter ubique, identified as the dominant bacterial species of the bay. Shotgun metagenomic analyses identified three strains of Ca. Pelagibacter in the bay, viz., dmdA-HTCC1062, dmdA-HTCC9022, and O19-dmdA, that showed a dynamic change throughout the year particularly in the 10-m depth zone. Interestingly, the total abundances of those strains that fall in the Ca. Pelagibacter genus were found to correlate well with the read counts per g seawater samples used for analyses. It is assumed that whole-genome sequence (WGS) reads for members of the metagenome would show similar trend provided that proper precautions are taken to ensure collection of representative sample from the environment.

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.

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.

Ofunato Bay, in Japan, is the home of buoy-and-rope-type oyster aquaculture activities. Since the oysters filter suspended materials and excrete organic matter 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.

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.