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.

X-ray crystallographic analysis of IMP-1 metallo-ß-lactamase complexed with a 3-aminophthalic acid derivative, structure-based drug design, and synthesis of 3,6-disubstituted phthalic acid derivative inhibitors.

3-(4-Hydroxypiperidine-1-yl) phthalic acid 1 shows potent inhibitory activity against metallo-ß-lactamase, which is known to inactivate ß-lactam antibiotics such as carbapenems. Here, the structure of co-crystals of the metallo-ß-lactamase IMP-1 and 1 was first analyzed by X-ray crystallography, and then used for structure-based drug design. Four novel compounds bearing substituents at the 6-position were synthesized to produce 3,6-disubstituted phthalic acid derivatives, and their IMP-1 inhibitory activity and synergistic effect with the carbapenem biapenem (BIPM) were evaluated. 3,6-Disubstituted phthalic acid derivatives showed potent IMP-1 inhibitory activity. In particular, compound 13 showed 10-fold higher IMP-1 inhibitory activity as compared with the parent derivative 1.

Identification of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid in steroid degradation by Comamonas testosteroni TA441 and its conversion to the corresponding 6-en-5-oyl coenzyme A (CoA) involving open reading frame 28 (ORF28)- and ORF30-encoded acyl-CoA dehydrogenases.

Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30.

Draft Genome Sequences of Geomicrobium sp. Strains JCM 19037, JCM 19038, JCM 19039, and JCM 19055, Isolated from Aquatic Samples.

Haloalkaliphilic strains JCM 19037, JCM 19038, JCM 19039, and JCM 19055, closely related to Geomicrobium sediminis, were isolated from aquatic samples, and their draft genome sequences were determined. The genome information of these four strains will be useful for studies of their physiology and ecology.

Draft Genome Sequences of Vibrio sp. Strains Isolated from Tetrodotoxin-Bearing Scavenging Gastropod.

Vibrio sp. strains JCM 18905 and JCM 19053 were isolated from a tetrodotoxin (TTX)-bearing scavenging gastropod, and Vibrio sp. strain JCM 18904 was isolated from a sea cucumber. All these are closely related to Vibrio alginolyticus. Their comparative genome information is useful for studies of TTX production in bacteria.

Identification of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid and ß-oxidation products of the C-17 side chain in cholic acid degradation by Comamonas testosteroni TA441.

Comamonas testosteroni degrades testosterone into 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and 2-hydroxyhexa-2,4-dienoic acid via aromatization of the A-ring. The former compound is suggested to be degraded further by ß-oxidation, but the details of the process remain unclear. In this study, we identified 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid as an intermediate compound in the ß-oxidation of this compound. ORF32, located in one of the two main steroid degradation gene clusters, was shown to be indispensable for the conversion of this compound. A homology search indicated that ORF32 encodes a hydratase for the CoA-ester, suggesting that ORF32 encodes a hydratase that adds a water molecule to a double bond at C-6 of the CoA-ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. From the culture of an ORF32-disrupted mutant incubated with cholic acid for a short period (around two days, when a considerable number of intermediate compounds were detected by HPLC), 7α,12α-dihydroxy-3-oxochola-1,4-dien-24-oic acid, 7α,12α-dihydroxy-3-oxochol-4-en-24-oic acid, 12α-hydroxy-3-oxochola-4,6-dien-24-oic acid, 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylic acid, 12α-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid, 7α,12α-dihydroxy-3-oxopregn-4-ene-20-carboxylic acid, 12α-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid, 7α-hydroxy-3-oxopregna-4,17(20)-diene-20-carboxylic acid, and 3-oxopregna-4,6,17(20)-triene-20-carboxylic acid were isolated as intermediate compounds of C-17 side-chain degradation. The presence of these compounds implies that the process of degradation of the C-17 side chain in C. testosteroni will be similar to the process in Pseudomonas. The final two compounds, which have a double bond at the C-17(20) position, are here identified for the first time, to the best of our knowledge, as intermediate compounds in bacterial steroid degradation; their composition suggests that the remaining three carbons at the C-17 position would be removed oxidatively as a propionic acid derivative.

Draft Genome Sequences of Psychrobacter Strains JCM 18900, JCM 18901, JCM 18902, and JCM 18903, Isolated Preferentially from Frozen Aquatic Organisms.

Four Psychrobacter strains, JCM 18900, JCM 18901, JCM 18902, and JCM 18903, related to either Psychrobacter nivimaris or Psychrobacter cibarius, were isolated from frozen marine animals. The genome information of these four strains will be useful for studies of their physiology and adaptation properties to frozen conditions.

Draft Genome Sequences of Cyclodextrin-Producing Alkaliphilic Bacillus Strains JCM 19045, JCM 19046, and JCM 19047.

Bacillus strains JCM 19045, JCM 19046, and JCM 19047 are alkaliphiles that produce ß-cyclodextrin from starch. They are related to Bacillus xiaoxiensis and Bacillus lehensis. The genome information for these three strains will be useful for studies of the physiological role of cyclodextrin and cyclodextrin production.

Metallo-ß-lactamase inhibitory activity of 3-alkyloxy and 3-amino phthalic acid derivatives and their combination effect with carbapenem.

3-Alkyloxy and 3-amino phthalic acid derivatives were found to have metallo-ß-lactamase inhibitory activity. Among them, 3-amino phthalic acid derivatives showed both potent activity against metallo-ß-lactamase, IMP-1 inhibitory activity and a strong combination effect with biapenem (BIPM), carbapenem antibiotic. In particular, the 4'-hydroxy-piperidine derivative showed strong IMP-1 inhibitory activity and a combination effect with various antibiotics.

Physiological characterization of aerobic culturable bacteria in the intestine of the sea cucumber Apostichopus japonicus.

Various aerobic culturable bacteria (1,133 isolates) were isolated from the gut of Apostichopus japonicus (black adult, green adult, black small, green small, black juvenile, and green juvenile sea cucumbers) and from the sea sediment and the seawater using different culture conditions and without enrichment culture. By molecular analysis of partial 16S rRNA gene sequences of 231 isolates, they were tentatively affiliated with 53 described species in the phyla Firmicutes (42 species), Proteobacteria (9 species) and Actinobacteria (2 species). Eighteen species were often found among the intestines and the sea sediment. High diversity was observed in the genus Bacillus (20 species), Oceanobacillus and Virgibaillus but there were no isolates affiliated to members of the genus Vibrio, well-known sea pathogens. There were no clear differences in the bacterial communities among the hosts varied in size and color. Most isolates showed various polysaccharide degradation activities, suggesting their possible contributions in the digestion of organic matters in the gut.

Heterologous expression and characterization of a glycoside hydrolase family 45 endo-ß-1,4-glucanase from a symbiotic protist of the lower termite, Reticulitermes speratus.

The termite symbiotic system is one of the efficient lignocellulose degradation systems. We tried to express and characterize a novel cellulolytic enzyme from this system. Here, we report the isolation of an endo-ß-1,4-glucanase gene homolog of glycoside hydrolase family 45 from a symbiotic protistan community of Reticulitermes speratus. Heterologous expression of this gene was performed using the expression system of Aspergillus oryzae. Analysis of enzymatic properties revealed 786 µmol/min/mg protein in specific activity, a V max of 833.0 units/mg protein, and a K m value of 2.58 mg/ml with carboxymethyl cellulose as the substrate. Thin-layer chromatography analysis showed that RsSymEG2 produces cellobiose from cellodextrins larger than cellohexaose. This enzyme showed high specific activity like other endo-ß-1,4-glucanases from the symbiotic system of termites. It means that the termite symbiotic system is a good resource for highly active endo-ß-1,4-glucanases.

Characterization of Halomonas sp. strain H11 α-glucosidase activated by monovalent cations and its application for efficient synthesis of α-D-glucosylglycerol.

An α-glucosidase (HaG) with the following unique properties was isolated from Halomonas sp. strain H11: (i) high transglucosylation activity, (ii) activation by monovalent cations, and (iii) very narrow substrate specificity. The molecular mass of the purified HaG was estimated to be 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HaG showed high hydrolytic activities toward maltose, sucrose, and p-nitrophenyl α-D-glucoside (pNPG) but to almost no other disaccharides or malto-oligosaccharides higher than trisaccharides. HaG showed optimum activity to maltose at 30°C and pH 6.5. Monovalent cations such as K(+), Rb(+), Cs(+), and NH(4)(+) increased the enzymatic activity to 2- to 9-fold of the original activity. These ions shifted the activity-pH profile to the alkaline side. The optimum temperature rose to 40°C in the presence of 10 mM NH(4)(+), although temperature stability was not affected. The apparent K(m) and k(cat) values for maltose and pNPG were significantly improved by monovalent cations. Surprisingly, k(cat)/K(m) for pNPG increased 372- to 969-fold in their presence. HaG used some alcohols as acceptor substrates in transglucosylation and was useful for efficient synthesis of α-d-glucosylglycerol. The efficiency of the production level was superior to that of the previously reported enzyme Aspergillus niger α-glucosidase in terms of small amounts of by-products. Sequence analysis of HaG revealed that it was classified in glycoside hydrolase family 13. Its amino acid sequence showed high identities, 60%, 58%, 57%, and 56%, to Xanthomonas campestris WU-9701 α-glucosidase, Xanthomonas campestris pv. raphani 756C oligo-1,6-glucosidase, Pseudomonas stutzeri DSM 4166 oligo-1,6-glucosidase, and Agrobacterium tumefaciens F2 α-glucosidase, respectively.

Diversity and function of aerobic culturable bacteria in the intestine of the sea cucumber Holothuria leucospilota.

Sea cucumbers play an important role in nutrient cycling of marine ecosystems by consuming sediments and moving sand, thus occupying a similar niche to earthworms in terrestrial ecosystems. However, our understanding of microbial diversity and functions associated with sea cucumbers is meager. Here, we isolated 141 bacterial strains under aerobic conditions using various media from the intestine of Holothuria leucospilota, a common sea cucumber in Japanese warm waters. By partial 16S rRNA gene sequences of the isolates, the isolates were tentatively affiliated with 55 described species. Among them, 23 species were common between 2 individuals of H. leucospilota. High diversity was observed in the genera Bacillus and Vibrio, which are often found in marine sediments, marine animals and other various environments. Most isolates showed various polysaccharide degradation activities and were able to grow under or were tolerant of anaerobic condition. We suggest that these aerobically isolated bacteria can play a role in digestion of detritus in aerobic and/or anaerobic regions of the intestine.