Novel aerobic anoxygenic phototrophic bacterium Jannaschia pagri sp. nov., isolated from seawater around a fish farm.

The genus Jannaschia is one of the representatives of aerobic anoxygenic phototrophic (AAP) bacteria, which is a strictly aerobic bacterium, producing a photosynthetic pigment bacteriochlorophyll (BChl) a. However, a part of the genus Jannaschia members have not been confirmed the photosynthetic ability. The partly presence of the ability in the genus Jannaschia could suggest the complexity of evolutionary history for anoxygenic photosynthesis in the genus, which is expected as gene loss and/or horizontal gene transfer. Here a novel AAP bacterium designated as strain AI_62T (= DSM 115720 T = NBRC 115938 T), was isolated from coastal seawater around a fish farm in the Uwa Sea, Japan. Its closest relatives were identified as Jannaschia seohaensis SMK-146 T (95.6% identity) and J. formosa 12N15T (94.6% identity), which have been reported to produce BChl a. The genomic characteristic of strain AI_62T clearly showed the possession of the anoxygenic photosynthesis related gene sets. This could be a useful model organism to approach the evolutionary mystery of anoxygenic photosynthesis in the genus Jannaschia. Based on a comprehensive consideration of both phylogenetic and phenotypic characteristics, we propose the classification of a novel species within the genus Jannaschia, designated as Jannaschia pagri sp. nov. The type strain for this newly proposed species is AI_62T (= DSM 115720 T = NBRC 115938 T).

Distribution and survival strategies of endemic and cosmopolitan diazotrophs in the Arctic Ocean.

Dinitrogen (N2) fixation is the major source of reactive nitrogen in the ocean and has been considered to occur specifically in low-latitude oligotrophic oceans. Recent studies have shown that N2 fixation also occurs in the polar regions and thus is a global process, although the physiological and ecological characteristics of polar diazotrophs are not yet known. Here, we successfully reconstructed diazotroph genomes, including that of cyanobacterium UCYN-A (Candidatus 'Atelocyanobacterium thalassa'), from metagenome data corresponding to 111 samples isolated from the Arctic Ocean. These diazotrophs were highly abundant in the Arctic Ocean (max., 1.28% of the total microbial community), suggesting that they have important roles in the Arctic ecosystem and biogeochemical cycles. Further, we show that diazotrophs within genera Arcobacter, Psychromonas, and Oceanobacter are prevalent in the <0.2 µm fraction in the Arctic Ocean, indicating that current methods cannot capture their N2 fixation. Diazotrophs in the Arctic Ocean were either Arctic-endemic or cosmopolitan species from their global distribution patterns. Arctic-endemic diazotrophs, including Arctic UCYN-A, were similar to low-latitude-endemic and cosmopolitan diazotrophs in genome-wide function, however, they had unique gene sets (e.g., diverse aromatics degradation genes), suggesting adaptations to Arctic-specific conditions. Cosmopolitan diazotrophs were generally non-cyanobacteria and commonly had the gene that encodes the cold-inducible RNA chaperone, which presumably makes their survival possible even in deep, cold waters of global ocean and polar surface waters. This study shows global distribution pattern of diazotrophs with their genomes and provides clues to answering the question of how diazotrophs can inhabit polar waters.

Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primers.

Many studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.

Draft Genome Sequence of the Aerobic Anoxygenic Phototrophic Bacterium Roseobacter sp. Strain OBYS 0001, Isolated from Coastal Seawater in Otsuchi Bay, Japan.

Here, we report the draft genome sequence of the aerobic anoxygenic phototrophic bacterium Roseobacter sp. strain OBYS 0001, isolated from coastal seawater in Ostuchi Bay, Japan. This genome sequence could be useful for our understanding of the variation in photosynthesis-related genes among aerobic anoxygenic phototrophs.

Distribution of Dimethylsulfoniopropionate Degradation Genes Reflects Strong Water Current Dependencies in the Sanriku Coastal Region in Japan: From Mesocosm to Field Study.

Dimethyl sulfide (DMS) is an important component of the global sulfur cycle as it is the most abundant sulfur compound that is emitted via the ocean surface to the atmosphere. Dimethylsulfoniopropionate (DMSP), the precursor of DMS, is mainly produced by phytoplankton and is degraded by marine bacteria. To reveal the role of bacteria in the regulation of DMSP degradation and DMS production, mesocosm and field studies were performed in the Sanriku Coast on the Pacific Ocean in northeast Japan. The responsible bacteria for the transformation of DMSP to DMS and the assimilation of DMSP were monitored, and the genes encoding DMSP lyase (dddD and dddP) and DMSP demethylase (dmdA) were analyzed. The mesocosm study showed that the dmdA subclade D was the dominant DMSP degradation gene in the free-living (FL) and particle-associated (PA) fractions. The dddD gene was found in higher abundance than the dddP gene in all the tested samples. Most importantly, DMS concentration was positively correlated with the abundance of the dddD gene. These results indicated that bacteria possessing dmdA and dddD genes were the major contributors to the DMSP degradation and DMS production, respectively. The genes dmdA subclade D and dddP were abundant in the Tsugaru Warm (TW) Current, while the dmdA subclade C/2 and dddD genes were dominant in the Oyashio (OY) Current. Functional gene network analysis also showed that the DMSP degradation genes were divided into OY and TW Current-related modules, and genes sharing similar functions were clustered in the same module. Our data suggest that environmental fluctuations resulted in habitat filtering and niche partitioning of bacteria possessing DMSP degradation genes. Overall, our findings provide novel insights into the distribution and abundance of DMSP degradation genes in a coastal region with different water current systems.

Eukaryotic Phytoplankton Contributing to a Seasonal Bloom and Carbon Export Revealed by Tracking Sequence Variants in the Western North Pacific.

Greater diversity of eukaryotic phytoplankton than expected has been revealed recently through molecular techniques, but little is known about their temporal dynamics or fate in the open ocean. Here, we examined size-fractionated eukaryotic phytoplankton communities from the surface to abyssopelagic zone (5,000 m) throughout the year, by tracking sequence variants of the 18S rRNA gene in the western subtropical North Pacific. The oceanographic conditions were divided into two periods, stratification and mixing, between which the surface phytoplankton community differed. During the mixing period, the abundance of large phytoplankton (≥3 µm) increased, with diatoms and putative Pseudoscourfieldia marina dominating this fraction. Picophytoplankton (<3 µm) also increased during the mixing period and were dominated by Mamiellophyceae. Taxa belonging to prasinophytes (including Ps. marina and Mamiellophyceae) were observed in the epipelagic zone throughout the year, and thus likely seeded the seasonal bloom that occurred during the mixing period. In contrast, diatoms observed during the mixing period mostly represented taxa unique to that period, including coastal species. Numerical particle backtracking experiments indicated that water masses in the surface layer could be transported from coastal areas to the study site. Gene sequences of coastal diatoms were present in the abyssopelagic zone. Therefore, allochthonous species drove the seasonal bloom and could be transported to deep waters. In the abyssopelagic zone, the relative abundance of Ps. marina in deep waters was similar to or higher than that of diatoms during the mixing period. Among picophytoplankton, Mamiellophyceae made up a significant fraction in the abyssopelagic zone, suggesting that prasinophytes are also involved in carbon export. Our molecular survey showed that these previously overlooked phytoplankton species could contribute significantly to the seasonal bloom and biological pump in the subtropical open ocean.

Vertical distribution of particle-associated and free-living ammonia-oxidizing archaea in Suruga Bay, a deep coastal embayment of Japan.

We analyzed the vertical distributions of ammonia-oxidizing archaea (AOA) in terms of abundance in Suruga Bay, Japan. We distinguished particle-associated (PA) from free-living (FL) assemblages. According to quantitative PCR measurements of the ammonia monooxygenase subunit A gene (amoA), most marine AOA were in an FL state. The vertical distributions of PA AOA ecotypes differed from the general trend; the Shallow Marine clade was dominant in both the surface and deep layers. Thus, although PA AOA account for a small percentage of AOA abundance, they have a community structure distinct from that of FL AOA in planktonic environments. Marine particles should be investigated further as an unexplored niche of AOA in the ocean.

Genome analysis of Rubritalea profundi SAORIC-165T, the first deep-sea verrucomicrobial isolate, from the northwestern Pacific Ocean.

Although culture-independent studies have shown the presence of Verrucomicrobia in the deep sea, verrucomicrobial strains from deep-sea environments have been rarely cultured and characterized. Recently, Rubritalea profundi SAORIC-165T, a psychrophilic bacterium of the phylum Verrucomicrobia, was isolated from a depth of 2,000 m in the northwestern Pacific Ocean. In this study, the genome sequence of R. profundi SAORIC-165T, the first deep-sea verrucomicrobial isolate, is reported with description of the genome properties and comparison to surface-borne Rubritalea genomes. The draft genome consisted of four contigs with an entire size of 4,167,407 bp and G+C content of 47.5%. The SAORIC-165T genome was predicted to have 3,844 proteincoding genes and 45 non-coding RNA genes. The genome contained a repertoire of metabolic pathways, including the Embden-Meyerhof-Parnas pathway, pentose phosphate pathway, tricarboxylic acid cycle, assimilatory sulfate reduction, and biosynthesis of nicotinate/nicotinamide, pantothenate/coenzyme A, folate, and lycopene. The comparative genomic analyses with two surface-derived Rubritalea genomes showed that the SAORIC-165T genome was enriched in genes involved in transposition of mobile elements, signal transduction, and carbohydrate metabolism, some of which might be related to bacterial enhancement of ecological fitness in the deep-sea environment. Amplicon sequencing of 16S rRNA genes from the water column revealed that R. profundi-related phylotypes were relatively abundant at 2,000 m and preferred a particle-associated life style in the deep sea. These findings suggest that R. profundi represents a genetically unique and ecologically relevant verrucomicrobial group well adapted to the deep-sea environment.

High temperature accelerates growth of aerobic anoxygenic phototrophic bacteria in seawater.

Temperature is an important controlling factor in the growth activity of all microorganisms. Aerobic anoxygenic phototrophic (AAP) bacteria actively grow in the ocean and are known as one of the main driving forces in organic matter cycling in surface seawater environments. Whether temperature change affects AAP bacteria activity from an ecological viewpoint remains an open question. To date, no known studies have reported the effect of temperature change on AAP bacteria growth in the ocean. We here show that the growth rate of AAP bacteria exceeded that of other bacterial types at high water temperatures in the absence of grazers. The slope of the regression line of the net growth rate of AAP bacteria as a function of water temperature was the same as that for non-AAP bacteria at all temperatures (10, 20, and 30°C); however, when grazers were eliminated, it was 4.7 times higher than that of non-AAP bacteria. This result suggests that AAP bacteria are more responsive to water temperature increases than other bacteria and that AAP bacteria might become more dominant than other bacteria under elevated water temperatures.

Ammonia oxidizers in the sea-surface microlayer of a coastal marine inlet.

Planktonic archaea are thought to play an important role in ammonia oxidation in marine environments. Data on the distribution, abundance, and diversity of ammonia oxidizers in the coastal sea-surface microlayer (SML) are lacking, despite previous reports of high abundance of Thaumarchaeota in the SML of estuaries and freshwater lakes. Here, we failed to detect the presence of ammonia-oxidizing bacteria in any of our samples taken from a semi-enclosed marine inlet in Japan. Therefore, we shifted our focus to examine the archaeal community composition as well as the Thaumarchaeota marine group I (MG-I) and ammonia monooxygenase subunit A (amoA) gene copy numbers and composition in the SML and corresponding underlying water (UW, 20 cm). amoA gene copy numbers obtained by quantitative PCR were consistent with the typical values observed in the surface waters of oceanic and coastal environments where nitrification activity has been detected, but the copy numbers were two- to three-fold less than those reported from the surface layers and UW of high mountain lakes. Both amoA and MG-I 16S rRNA gene copy numbers were significantly negatively correlated with chlorophyll-a and transparent exopolymer particle concentrations in the SML. Communities of archaea and ammonia-oxidizing archaea in SML samples collected during low wind conditions (≤5 m s-1) differed the most from those in UW samples, whereas the communities in SML samples collected during high wind conditions were similar to the UW communities. In the SML, low ratios of amoA to MG-I 16S rRNA genes were observed, implying that most of the SML Thaumarchaeota lacked amoA. To our knowledge, our results provide the first comparison of ammonia-oxidizing communities in the coastal SML with those in the UW.

Amylibacter kogurei sp. nov., a novel marine alphaproteobacterium isolated from the coastal sea surface microlayer of a marine inlet.

A novel Gram-negative bacterium, designated 4G11T, was isolated from the sea surface microlayer of a marine inlet. On the basis of 16S rRNA gene sequence analysis, the strain showed the closest similarity to Amylibacter ulvae KCTC 32465T (99.0 %). However, DNA-DNA hybridization values showed low DNA relatedness between strain 4G11T and its close phylogenetic neighbours, Amylibacter marinus NBRC 110140T (8.0±0.4 %) and Amylibacter ulvae KCTC 32465T (52.9±0.9 %). Strain 4G11T had C18 : 1, C16 : 0 and C18 : 2 as the major fatty acids. The only isoprenoid quinone detected for strain 4G11T was ubiquinone-10. The major polar lipids were phosphatidylglycerol, phosphatidylcholine, one unidentified polar lipid, one unidentified phospholipid and one unidentified aminolipid. The DNA G+C content of strain 4G11T was 50.0 mol%. Based on phenotypic and chemotaxonomic characteristics and analysis of the 16S rRNA gene sequence, the novel strain should be assigned to a novel species, for which the name Amylibacter kogurei sp. nov. is proposed. The type strain of Amylibacter kogurei is 4G11T (KY463497=KCTC 52506T=NBRC 112428T).

Bacterioplankton drawdown of coral mass-spawned organic matter.

Coral reef ecosystems are highly sensitive to microbial activities that result from dissolved organic matter (DOM) enrichment of their surrounding seawater. However, the response to particulate organic matter (POM) enrichment is less studied. In a microcosm experiment, we tested the response of bacterioplankton to a pulse of POM from the mass-spawning of Orbicella franksi coral off the Caribbean coast of Panama. Particulate organic carbon (POC), a proxy measurement for POM, increased by 40-fold in seawater samples collected during spawning; 68% degraded within 66 h. The elevation of multiple hydrolases presumably solubilized the spawn-derived POM into DOM. A carbon budget constructed for the 275 µM of degraded POC showed negligible change to the concentration of dissolved organic carbon (DOC), indicating that the DOM was readily utilized. Fourier transform ion cyclotron resonance mass spectrometry shows that the DOM pool became enriched with heteroatom-containing molecules, a trend that suggests microbial alteration of organic matter. Our sensitivity analysis demonstrates that bacterial carbon demand could have accounted for a large proportion of the POC degradation. Further, using bromodeoxyuridine immunocapture in combination with 454 pyrosequencing of the 16S ribosomal RNA gene, we surmise that actively growing bacterial groups were the primary degraders. We conclude that coral gametes are highly labile to bacteria and that such large capacity for bacterial degradation and alteration of organic matter has implications for coral reef health and coastal marine biogeochemistry.

Algibacter aquaticus sp. nov., a slightly alkaliphilic marine Flavobacterium isolated from coastal surface water.

A rod-shaped, pale yellow-pigmented, aerobic, Gram-staining-negative strain with gliding motility, designated as strain SK-16T, was isolated from the coastal surface water of a semi-enclosed coastal inlet in Misaki, Japan. Analysis of 16S rRNA gene sequences revealed that SK-16T represented a member of the family Flavobacteriaceae and was closely related to the genus Algibacter, with sequence similarities ranging from 95.9 to 94.3 % to the type strains of species of the genus Algibacter. The major cellular fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C15 : 0 G and iso-C15 : 0 3-OH. Major polar lipids were phosphatidylethanolamine, an aminophospholipid and an unidentified phospholipid. The DNA G+C content of SK-16T was 32.3 mol% and MK-6 was the only predominant isoprenoid quinone. On the basis of the results of phenotypic, genotypic, chemotaxonomic and phylogenetic studies, it was suggested that SK-16T represents a novel species within the genus Algibacter, with the newly proposed name Algibacteraquaticus. The type strain is SK-16T (=NBRC 110220T=KCTC 32974T).

Abundance of Common Aerobic Anoxygenic Phototrophic Bacteria in a Coastal Aquaculture Area.

Aerobic anoxygenic phototrophic bacteria (AAnPB) rely on not only heterotrophic but also phototrophic energy gain. AAnPB are known to have high abundance in oligotrophic waters and are the major portion of the bacterial carbon stock in the environment. In a yearlong study in an aquaculture area in the Uwa Sea, Japan, AAnPB, accounted for 4.7 to 24% of the total bacteria by count. Since the cell volume of AAnPB is 2.23 ± 0.674 times larger than the mean for total bacteria, AAnPB biomass is estimated to account for 10-53% of the total bacterial assemblage. By examining pufM gene sequence, a common phylogenetic AAnPB species was found in all sampling sites through the year. The common species and other season-specific species were phylogenetically close to unculturable clones recorded in the Sargasso Sea and Pacific Ocean. The present study suggests that the common species may be a cosmopolitan species with worldwide distribution that is abundant not only in the oligotrophic open ocean but also in eutrophic aquaculture areas.

Draft Genome Sequences of Two Fabibacter sp. Strains Isolated from Coastal Surface Water of Aburatsubo Inlet, Japan.

Here, we report the draft genome sequences of Fabibacter sp. strain 4D4 and F. misakiensis strain SK-8T, isolated from surface seawater of a semienclosed inlet.

Draft Genome Sequence of Lewinella sp. Strain 4G2 Isolated from the Coastal Sea Surface Microlayer.

We report here the draft genome of Lewinella sp. strain 4G2, isolated from the sea surface microlayer (SML) of a coastal marine inlet. The genome sequence of strain 4G2 should contribute to understanding the lifestyles of bacteria living in the SML.

Possible association of diazotrophs with marine zooplankton in the Pacific Ocean.

Dinitrogen fixation, the biological reduction in N2 gas to ammonia contributes to the supply of new nitrogen in the surface ocean. To understand the diversity and abundance of potentially diazotrophic (N2 fixing) microorganisms associated with marine zooplankton, especially copepods, the nifH gene was studied using zooplankton samples collected in the Pacific Ocean. In total, 257 nifH sequences were recovered from 23 nifH-positive DNA extracts out of 90 copepod samples. The nifH genes derived from cyanobacteria related to Trichodesmium, α- and γ-subdivisions of proteobacteria, and anaerobic euryarchaeota related to Methanosaeta concilii were detected. Our results indicated that Pleuromamma, Pontella, and Euchaeta were the major copepod genera hosting dinitrogen fixers, though we found no species-specific association between copepods and dinitrogen fixers. Also, the digital PCR provided novel data on the number of copies of the nifH gene in individual copepods, which we report the range from 30 to 1666 copies per copepod. This study is the first systematic study of zooplankton-associated diazotrophs, covering a large area of the open ocean, which provide a clue to further study of a possible new hotspot of N2 fixation.

Nitrification and its influence on biogeochemical cycles from the equatorial Pacific to the Arctic Ocean.

We examined nitrification in the euphotic zone, its impact on the nitrogen cycles, and the controlling factors along a 7500 km transect from the equatorial Pacific Ocean to the Arctic Ocean. Ammonia oxidation occurred in the euphotic zone at most of the stations. The gene and transcript abundances for ammonia oxidation indicated that the shallow clade archaea were the major ammonia oxidizers throughout the study regions. Ammonia oxidation accounted for up to 87.4% (average 55.6%) of the rate of nitrate assimilation in the subtropical oligotrophic region. However, in the shallow Bering and Chukchi sea shelves (bottom ⩽67 m), the percentage was small (0-4.74%) because ammonia oxidation and the abundance of ammonia oxidizers were low, the light environment being one possible explanation for the low activity. With the exception of the shallow bottom stations, depth-integrated ammonia oxidation was positively correlated with depth-integrated primary production. Ammonia oxidation was low in the high-nutrient low-chlorophyll subarctic region and high in the Bering Sea Green Belt, and primary production in both was influenced by micronutrient supply. An ammonium kinetics experiment demonstrated that ammonia oxidation did not increase significantly with the addition of 31-1560 nm ammonium at most stations except in the Bering Sea Green Belt. Thus, the relationship between ammonia oxidation and primary production does not simply indicate that ammonia oxidation increased with ammonium supply through decomposition of organic matter produced by primary production but that ammonia oxidation might also be controlled by micronutrient availability as with primary production.

Active populations of rare microbes in oceanic environments as revealed by bromodeoxyuridine incorporation and 454 tag sequencing.

The "rare biosphere" consisting of thousands of low-abundance microbial taxa is important as a seed bank or a gene pool to maintain microbial functional redundancy and robustness of the ecosystem. Here we investigated contemporaneous growth of diverse microbial taxa including rare taxa and determined their variability in environmentally distinctive locations along a north-south transect in the Pacific Ocean in order to assess which taxa were actively growing and how environmental factors influenced bacterial community structures. A bromodeoxyuridine-labeling technique in combination with PCR amplicon pyrosequencing of 16S rRNA genes gave 215-793 OTUs from 1200 to 3500 unique sequences in the total communities and 175-299 OTUs nearly 860 to 1800 sequences in the active communities. Unexpectedly, many of the active OTUs were not detected in the total fractions. Among these active but rare OTUs, some taxa (2-4% of rare OTUs) showed much higher abundance (>0.10% of total reads) in the active fraction than in the total fraction, suggesting that their contribution to bacterial community productivity or growth was much larger than that expected from their standing stocks at each location. An ordination plot by the principal component analysis presented that bacterial community compositions among 4 sampling locations and between total and active fractions were distinctive with each other. A redundancy analysis revealed that the variability of community compositions significantly correlated to seawater temperature and dissolved oxygen concentration. Also, a variation partitioning analysis showed that the environmental factors explained 49% of the variability of community compositions and the distance only explained 4.0% of its variability. These results implied very dynamic change of community structures due to environmental filtering. The active bacterial populations are more diverse and spread further in rare biosphere than we have ever seen. This study implied that rare microbes are important as an active part of microbial communities functioning ecosystems.