Seriniquinone was isolated as a melanoma-selective anti-cancer agent from a culture broth of the marine-derived bacterium Serinicoccus marinus CNJ927 in 2014. It targets the unique small protein, dermcidin, which affects the drug resistance of cancer cells. Due to its significant activity against cancer cells, particularly melanoma, and its unique target, seriniquinone has been developed as a new pharmacophore. However, it has the disadvantage of poor solubility in drug discovery research, which needs to be resolved. A new seriniquinone glycoside (1) was synthesized by the biological transformation of seriniquinone using the deep sea-derived bacterium Bacillus licheniformis KDM612. Compound 1 exhibited selective anti-cancer activity against melanoma, similar to seriniquinone, and was 50-fold more soluble in DMSO than seriniquinone.

A direct relationship exists between diverse corals and fish farming in Keten Bay, Amami-Oshima, Japan. The release of coral mucus has a significant impact on the microbial activity of surrounding seawater. To obtain a more detailed understanding of biogeochemical cycles in this environment, the effects of coral mucus on the community structure and function of bacteria in surrounding seawater need to be elucidated. We herein used a bromodeoxyuridine approach to investigate the structures and functions of bacterial communities growing close to mucus derived from two different Acropora corals, AC1 and AC2. The alpha diversities of actively growing bacteria (AGB) were lower in mucus-containing seawater than in control seawater and their community structures significantly differed, suggesting that the growth of specific bacteria was modulated by coral mucus. Rhodobacteraceae and Cryomorphaceae species were the most dominant AGB in response to the mucus of Acropora AC1 and AC2, respectively. In contrast, the growth of Actinomarinaceae, Alteromonadaceae, Flavobacteriaceae, and SAR86 clade bacteria was inhibited by coral mucus. The results of a Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) ana-lysis suggested that the predicted functions of AGB in mucus-containing seawater differed from those in seawater. These functions were related to the biosynthesis and degradation of the constituents of coral mucus, such as polysaccharides, sugar acids, and aromatic compounds. The present study demonstrated that complex bacterial community structures and functions may be shaped by coral mucus, suggesting that corals foster diverse bacterial communities that enhance the ecological resilience of this fish farming area.

We investigated the succession process of intestinal bacteria during seed production in full-cycle aquaculture of Pacific bluefin tuna (Thunnus orientalis). During the seed production, eggs, healthy fish, rearing water, and feeds from three experimental rounds in 2012 and 2013 were collected before transferring to offshore net cages and subjected to a fragment analysis of the bacterial community structure. We identified a clear succession of intestinal bacteria in bluefin tuna during seed production. While community structures of intestinal bacteria in the early stage of larvae were relatively similar to those of rearing water and feed, the bacterial community structures seen 17 days after hatching were different. Moreover, although intestinal bacteria in the late stage of larvae were less diverse than those in the early stage of larvae, the specific bacteria were predominant, suggesting that the developed intestinal environment of the host puts selection pressure on the bacteria in the late stage. The specific bacteria in the late stage of larvae, which likely composed ‘core microbiota’, were also found on the egg surface. The present study highlights that proper management of the seed production process, including the preparation of rearing water, feeds, and fish eggs, is important for the aquaculture of healthy fish.

In fish-farming areas, copious amounts of organic matter are released into the surrounding environment. Although it is well-known that bacterial community structures and activities are tightly coupled with organic conditions in the environment, actively growing bacteria (AGB) species that are responsible are still largely unknown. Here, we determined seasonal variations in the community structures of free-living and particle-attached AGB in surface and bottom seawater, and also in the easily resuspendable sediment boundary layer. Accordingly, we used bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) analysis. Whereas overall bacterial communities in the resuspendable sediment were quite different from those of the free-living and particle-attached bacteria, the AGB community structures were similar among them. This result suggests that sediment resuspension in aquaculture environments functions as an organic source for bacteria in the water column, and that bacterial species contributing to the environmental capacity and carbon cycle are limited. We identified 25 AGB phylotypes, belonging to Alphaproteobacteria (Roseobacterclade, nine phylotypes), Gammaproteobacteria (five phylotypes), Deltaproteobacteria (one phylotype), Bacteroidetes (seven phylotypes), and Actinobacteria (three phylotypes). Among them, some AGB phylotypes appeared throughout the year with high frequency and were also identified in other coastal environments. This result suggests that these species are responsible for the environmental capacity and carbon cycle, and are key species in this fish-farming area, as well as other coastal environments.

合流式下水道越流水（Combined Sewer Overflow: CSO）が都市河川に及ぼす影響と，大規模雨水貯留管（平成の太閤下水）の供用による改善効果の評価を目的に，降雨時の東横堀川（大阪市）にて細菌叢を指標とした水質調査を実施した．東堀橋地点では，4.0 mm/hを越える降雨時にCSOが原因と思われる懸濁態物質濃度の上昇と，それに伴う細菌叢の変化が確認された．しかしこのとき確認された河川中の細菌叢は，下水処理場で入手した処理前下水の細菌叢とは異なるものであった．雨水貯留管供用後も降雨時に河川水の電気伝導度が増加したが，それに伴った河川中の細菌叢の変化は確認されなかった．雨水貯留管の供用は，CSO流入を抑制することで，河川水質の改善や河川微生物群集攪乱の緩和に効果があると考えられた．

A major concern in larvae production is a mass mortality caused by fish diseases. In larvae production, pumped-up natural seawater filtered through a sand filter system is used for fish rearing, and microalgae and rotifer cultures. Here, we investigated the community structures of eukaryotic microbes, as well as total bacteria and vibrios, in various processed waters' used in a larvae production site. We observed that ultraviolet irradiation of seawater was effective to reduce not only total bacteria and vibrios but also eukaryotic microbes. Moreover, the community structures of total bacteria and vibrios in rearing waters for fish larvae were different from those in rotifer cultures fed with Chlorella, but rather similar to those in natural seawater and microalgae cultures. These results suggest that the bacterial community in rearing waters may originate mainly from natural seawater and then be selected by microalgae in rearing water. Overall, this study provides useful information for avoiding the risk of fish disease outbreaks in a larvae production site.

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. (C) 2015 The Authors. Published by Elsevier B.V.

Coral mucus influences the composition and abundance of bacteria in the surrounding seawater. In this study, the phylogenetic affiliations of actively growing bacteria (AGB) in seawater supplemented with the mucus of Acropora sp. during a 24-h incubation period were determined. For this purpose, bromodeoxyuridine magnetic-beads immunocapture and PCR-DGGE (BUMP-DGGE) analysis was used. The coral mucus contained higher concentrations of particulate organic carbon and nitrogen and exhibited higher bacterial abundance than seawater did, and the organic matter and bacteria varied dramatically during the incubation. BUMP-DGGE analysis showed that the AGB stimulated by the coral mucus also varied during the incubation. The bulk of the active growers originated from seawater and not the mucus. However, not all of the bacterial phylotypes in seawater could use the coral mucus as a suitable growth substrate. Cluster analysis revealed that the AGB community structures identified across the incubation period were considerably different in seawater supplemented with coral mucus as compared with those in coral mucus. In total, 12 phylotypes of AGB were identified, and these belonged to Alphaproteobacteria (Rhodobacterales 5 phylotypes), Gammaproteobacteria (Oceanospirillales 3 phylotypes, Vibrionales 1 phylotype, Alteromonadales 1 phylotype), and Bacteroidetes (Flavobacteriales 2 phylotypes). In the coral mucus, phylotypes belonging to Oceanospirillales appeared only at the beginning of the incubation, whereas those belonging to Rhodobacterales appeared only in the late phase of the incubation. Thus, coral mucus can alter the AGB community structure and create an organic matter flux that is specific to a reef environment. (C) 2015 Elsevier B.V. All rights reserved.

Coral mucus influences the abundance and composition of microbes in the surrounding seawater. Here, the hypothesis that the mucus of the coral genera Acropora, Favia, Favites, and Goniastrea would influence bacterial growth (production) in seawater (measured using 3H-thymidine and 3H-leucine incorporation methods) was tested. The bacterial production in mucus-supplemented seawater was significantly greater than that in unsupplemented seawater: production in the mucus-supplemented seawater ranged from 108.2 to 4111 mu g C.L-1.d(-1) when measured by thymidine incorporation and from 72.9 to 115.9 mu g C.L-1.d(-1) when measured by leucine incorporation. This study showed that the enhanced production may be due to bacteria originating from seawater and not from coral mucus. Nonetheless, the effect of mucus on bacterial production was different among the coral genera: the mucus of Favia did not always enhance bacterial production. Additionally, even a very small amount of coral mucus, only similar to 300 mu L. of mucus per liter of seawater, was sufficient to enhance bacterial production by approximately 2-fold compared to unsupplemented seawater. These results suggest that coral mucus has a significant impact on the marine biogeochemical cycle around coral reefs. (C) 2014 Elsevier B.V. All rights reserved.

Roseobacter/Rhodobacter and SAR11, affiliated with Alphaproteobacteria, and the phylum Bacteroidetes constitute a large proportion of marine planktonic bacteria, but information about their growth and distribution patterns in the Southern Ocean is scarce. The aim of the present study is to determine patterns in the biomass and productivity of Roseobacter/Rhodobacter, SAR11, and Bacteroidetes groups along the steep temperature, salinity, and organic matter gradients in the Southern Ocean by using catalyzed reporter deposition-fluorescence in situ hybridization and bromodeoxyuridine (BrdU) immunocytochemistry FISH. We found that Roseobacter/Rhodobacter, SAR11, and Bacteroidetes are prominent contributors to total bacterial biomass and production. SAR11 bacteria were the predominant lineage, but their biomass was low in the coldest regions. In contrast, the biomasses of Roseobacter/Rhodobacter and Bacteroidetes lineages were positively correlated with organic matter concentrations. The Roseobacter/Rhodobacter had the highest proportion of BrdU-positive (i.e., actively growing) cells among the three phylotypes at all stations, despite their low abundance. The relative contribution of Bacteroidetes to the total bacterial productivity (number of active cells) was negatively correlated with temperature. These results suggest that the growth and distribution patterns of Roseobacter/Rhodobacter, SAR11, and Bacteroidetes were determined by different environmental gradients (e.g., organic matter concentrations or temperature) in the Southern Ocean.

Our objective was to track microbial processes associated with serial degradation of organic matter derived from algal blooms. To do this, we analyzed population fluctuations and growth responses of major phylogenetic groups of free-living marine bacteria. We used bromodeoxyuridine immunocytochemistry-fluorescence in situ hybridization methodology to examine marine bacterial community development during and after a diatom bloom in a mesocosm. We revealed that the Roseobacter/Rhodobacter, SAR11, Alteromonas, and Bacteroidetes groups were clearly major phylotypes responsible for most free-living bacterial biomass and production throughout the experiment. The clearest bacterial response was a proliferation of the Alteromonas group (cells with large volumes) during development of the bloom (up to 30 % of actively growing cells). Populations of these bacteria declined sharply thereafter, likely due to grazing. Alteromonas group responses suggest that these bacteria strongly influenced the flux of organic matter at an early bloom stage. The growth potential of Bacteroidetes was relatively large as the bloom peaked; this early development probably contributed to the initial stage of bloom decomposition. In contrast, the contribution of Roseobacter/Rhodobacter to total bacterial production increased at a late stage of decomposing of the bloom. The contributions of Betaproteobacteria, SAR11, and SAR86 groups to total bacterial abundance and production were relatively minor throughout the experiment. These results imply that the ability to utilize organic matter derived from diatoms varies among bacterial phylotypes, and, frequently, less abundant but ecological specialist taxa such as Alteromonas may play major roles in the flux of organic matter during diatom blooms.

Using bromodeoxyuridine (BrdU) magnetic beads immunocapture and a PCR-denaturing gradient gel electrophoresis (DGGE) technique (BUMP-DGGE), we determined seasonal variations in the community structures of actively growing bacteria in the neritic waters of Hiroshima Bay, western Japan. The community structures of actively growing bacteria were separated into two clusters, corresponding to the timing of phytoplankton blooms in the autumn-winter and spring-summer seasons. The trigger for changes in bacterial community structure was related to organic matter supply from phytoplankton blooms. We identified 23 phylotypes of actively growing bacteria, belonging to Alphaproteobacteria (Roseobacter group, 9 phylotypes), Gammaproteobacteria (2 phylotypes), Bacteroidetes (8 phylotypes), and Actinobacteria (4 phylotypes). The Roseobacter group and Bacteroidetes were dominant in actively growing bacterial communities every month, and together accounted for more than 70% of the total DGGE bands. We revealed that community structures of actively growing bacteria shifted markedly in the wake of phytoplankton blooms in the neritic waters of Hiroshima Bay.

We performed duplicate microcosm experiments to determine Vibrio species dynamics over 13 days in enriched natural seawater inoculated with Nannochloropsis. Chlorophyll a concentrations in Tanks 1 and 2 ranged from 8.71 to 235μg/l and 10.9 to 255μg/l, respectively; peak values were recorded on Day 6 in both tanks. Culturable Vibrio abundance on TCBS agar decreased from 180 CFU/ml on Day 2 to 50 CFU/ml on Day 5 in Tank 1, and from 120 CFU/ml on Day 1 to 20 CFU/ml on Day 6 in Tank 2. Total Vibrio abundance estimated by qPCR ranged from 3.2×102 to 2.6×104 cells/ml in Tank 1 and from 2.2×102 to 1.6×104 cells/ml in Tank 2. Interestingly, after the peak of chlorophyll a concentration in both tanks, no culturable Vibrio and only a few total Vibrio were detected. PCR-DGGE analysis showed that the Vibrio community was unexpectedly diverse at the onset, but the structure became simpler over time with the increasing chlorophyll a concentration. Hence, the addition of Nannochloropsis to larval-rearing water may control Vibrio species growth, a key factor responsible for the survival and growth of fish larvae.

We developed a quantitative PCR assay for detecting the parasitic ciliate Cryptocaryon irritans, which causes "white spot disease" in marine fishes, from the natural environment. A specific primer set for C. irritans was designed and its high specificity was confirmed in silico: almost all of the sequences deposited in the GenBank nucleotide database were covered, 22/23 for the forward primer and 7/7 for the reverse primer. We estimated that there were 3,415.9 rRNA gene copies per genome of C. irritans. In artificial mixture experiments to validate whether the qPCR assay is applicable to natural samples, the estimated copy numbers showed significantly positive correlations with the number of theronts added (p < 0.001). When we applied this qPCR assay to natural samples collected bimonthly from surface and bottom seawaters at an aquaculture site (water depth, 10 m) from May 2009 to March 2010, we only detected C. irritans (112.0 +/- A 6.3 cells/l) in the surface seawater sample in November. This qPCR assay is a useful tool for detecting C. irritans rapidly and quantitatively in natural environments; it could also help advance our understanding of the ecology of C. irritans, as well as facilitate the diagnosis of the disease.

Growth and productivity of phytoplankton substantially change organic matter characteristics, which affect bacterial abundance, productivity, and community structure in aquatic ecosystems. We analyzed bacterial community structures and measured activities inside and outside phytoplankton blooms in the western North Pacific Ocean by using bromodeoxyuridine immunocytochemistry and fluorescence in situ hybridization (BIC-FISH). Roseobacter/Rhodobacter, SAR11, Betaproteobacteria, Alteromonas, SAR86, and Bacteroidetes responded differently to changes in organic matter supply. Roseobacter/Rhodobacter bacteria remained widespread, active, and proliferating despite large fluctuations in organic matter and chlorophyll a (Chl-a) concentrations. The relative contribution of Bacteroidetes to total bacterial production was consistently high. Furthermore, we documented the unexpectedly large contribution of Alteromonas to total bacterial production in the bloom. Bacterial abundance, productivity, and growth potential (the proportion of growing cells in a population) were significantly correlated with Chl-a and particulate organic carbon concentrations. Canonical correspondence analysis showed that organic matter supply was critical for determining bacterial community structures. The growth potential of each bacterial group as a function of Chl-a concentration showed a bell-shaped distribution, indicating an optimal organic matter concentration to promote growth. The growth of Alteromonas and Betaproteobacteria was especially strongly correlated with organic matter supply. These data elucidate the distinctive ecological role of major bacterial taxa in organic matter cycling during open ocean phytoplankton blooms.

To explore the possibility of calculating phylotype-specific growth rates of marine bacteria, we optimized a protocol that combines bromodeoxyuridine (BrdU) immunocytochemistry with fluorescence in situ hybridization (FISH). The protocol worked well with 8 isolates tested, showing a significant positive correlation between average cellular fluorescence intensity and cell-specific BrdU contents. The BrdU immunocytochemistry-FISH (BIG-FISH) was then applied to neritic sea-water in Hiroshima Bay, western Japan. Rhodobacter/Roseobacter and Bacteroidetes groups were dominant in BrdU-positive cells (22 +/- 8 and 26 +/- 8%), suggesting a significant contribution to bacterial community productivity in the water. Analysis of single-cell immunofluorescence showed higher BrdU incorporation rates in the Gammaproteobacteria, SAR86, and Vibrio groups, suggesting that these less abundant groups were growing more rapidly than other phylotypes. Our study suggests that BIG-FISH has the potential to estimate phylotype-specific variability of bacterial productivity in aquatic systems.

We developed a method for the separate and simultaneous analysis of the community structure of heterotrophic nanopkankton (HNP) and autotrophic nanoplankton (ANP). This method consists of three steps. First, nanoplankton cells were concentrated using a cross-flow filtration system because cell densities in natural seawater are usually too low for genetic studies. Second, HNP and ANP were separated by flow cytometric sorting ("flow sorting") on the basis of the presence or absence of chlorophyll. Finally, the community structure was analyzed using denaturing gradient gel electrophoresis targeting 18S rRNA gene. The newly developed method was applied to the coastal surface water of Aburatsubo Inlet, Japan, in July 2008. The separation of nanoplankton into HNP and ANP was validated by phylogenetic analysis, and the trophic mode of uncultured nanoplankton was confirmed (e.g. Marine Alveolata group II [MALV II] and Marine Stramenopile clade-2 [MAST-2]). This new method involving cell concentration, flow sorting and phylogenetic analysis is a potentially powerful tool for evaluating the population dynamics and ecology of marine protozoa.

Among the fundamental questions in marine microbial ecology are which taxa or phylogenetic groups account for total bacterial productivity and what is the relative contribution of each. We combined bromodeoxyuridine (BrdU) immunocytochemistry and fluorescence in situ hybridization (BIC-FISH) to examine phylotype-specific contributions to total bacterial productivity in eutrophic seawater. We also examined year-round changes in phylotype-specific contributions and explored the factors controlling these changes. Monitoring by BIC-FISH throughout the year revealed the importance of the Roseobacter/Rhodobacter group as a constantly proliferating basic population (27% of all BrdU-positive cells), although their contribution was not significantly correlated with water temperature or with chlorophyll a or organic matter concentration. The Bacteroidetes were another important group, as they greatly increased in abundance after the end of phytoplankton blooms. Two other phylotypes tested, the SAR86 and Vibrio groups, changed their contributions to bacterial productivity with changes in water temperature. To our knowledge, this study was the first to estimate the yearly contribution of major subgroups of marine bacteria to total bacterial productivity in a seawater environment.

Bacterial community structures and their activities in the ocean are tightly coupled with organic matter fluxes and thus control ocean biogeochemical cycles. Bromodeoxyuridine (BrdU), halogenated nucleoside and thymidine analogue, has been recently used to monitor actively growing bacteria (AGB) in natural environments. We labelled DNA of proliferating cells in seawater bacterial assemblages with BrdU and determined community structures of the bacteria that were possible key species in mediating biochemical reactions in the ocean. Surface seawater samples were collected along a north-south transect in the North Pacific in October 2003 and subjected to BrdU magnetic beads immunocapture and PCR-DGGE (BUMP-DGGE) analysis. Change of BrdU-incorporated community structures reflected the change of water masses along a north-south transect from subarctic to subtropical gyres in the North Pacific. We identified 25 bands referred to AGB as BrdU-incorporated phylotypes, belonging to Alphaproteobacteria (5 bands), Betaproteobacteria (1 band), Gammaproteobacteria (4 bands), Cytophaga-Flavobacterium-Bacteroides (CFB) group bacteria (5 bands), Gram-positive bacteria (6 bands), and Cyanobacteria (4 bands). BrdU-incorporated phylotypes belonging to Vibrionales, Alteromonadales and Gram-positive bacteria appeared only at sampling stations in a subtropical gyre, while those belonging to Roseobacter-related bacteria and CFB group bacteria appeared at the stations in both subarctic and subtropical gyres. Our result revealed phylogenetic affiliation of AGB and their dynamic change along with north-south environmental gradients in open oceans. Different species of AGB utilize different amount and kinds of substrates, which can affect the change of organic matter fluxes along transect.

A fundamental question in microbial oceanography concerns the relationship between prokaryote diversity and biogeochemical function in an ecosystem context. We combined bromodeoxymidine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) to examine phylotype-specific growth in natural marine assemblages. We also examined a broad range of marine bacterial isolates to determine their abilities to incorporate BrdU in order to test the validity of the method for application to diverse marine assemblages. We found that 27 of 29 isolates belonging to different taxa could incorporate BrdU. BUMP-DGGE analysis revealed phylogenetic affiliations of DNA-synthesizing, presumably actively growing bacteria across a eutrophic to mesotrophic transect in the Inland Sea of Japan. We found that the BrdU-incorporating (growing) communities were substantially different from the total communities. The majority (34/56) of phylotypes incorporated BrdU and were presumably growing, and these phylotypes comprised 10 alphaproteobacteria, 1 betaproteobacterium, 11 gammaproteobacteria, 11 Cytophaga-Flavobacterium-Bateroides group bacteria, and 1 unclassified bacterium. All BrdU-responsive alphaproteobacteria were members of the Rhodobacterales, suggesting that such bacteria were dominant in the growing alphaproteobacterial populations in our samples. The BrdU-responsive gammaproteobacteria belonged to the Oceanospirillales, the SAR86 cluster, the Pseudomonadales, the Alteromonadales, and the Vibrionales. Thus, contemporaneous cooccurrence of diverse actively growing bacterial taxa was a consistent pattern in our biogeochemically varied study area.

養殖漁場の水質浄化は細菌が担っている。細菌が駆動する物質循環が乱れると水質が悪化するが、その鍵を握る細菌種は未だ分かっていない。本研究では、養殖漁場における有機物の分解・利用に直接寄与している細菌種を特定することを目的としている。令和3年度は、既得試料と新規採取試料の処理を実施するとともに、養殖漁場の特徴的な有機物である養殖魚の餌料に応答して増殖してくる細菌を分離培養し、細菌種を特定した。使用した餌料は、現場養殖漁場で使用されているドライペレット（DP）とモイストペレット（MP）で、細菌が直接利用できる有機物は溶存画分であるため、溶存画分のみを抽出した。養殖漁場の海水および非養殖漁場の海水を採取し、それぞれに約3 mg/LとなるようにDPならびにMP有機物を添加したマイクロコスム実験を実施した。培養3日後および培養8日後にサブサンプリングし用いた培地は、海洋細菌分離汎用培地1/2 ZoBell培地ならびにDPあるいはMP有機物を1/2 ZoBell培地と同じような有機物量となるように添加したDPあるいはMP寒天培地である。寒天培地に塗抹して2週間以降に単離を行い、最終的に91株を得て、グリセロールストックした。あわせて、これら分離株のうち57株のITS領域と16S rRNA遺伝子領域の塩基配列を読んだ。特定できた細菌種のなかに、Proteobacteria門Alphaproteobacteria綱Rhodobacterales目やBacteroidetes門Flavobacteriia綱Flavobacterialesに近縁な分離株が複数いた。これらの細菌群は、研究代表者の先行研究において沿岸域で活発に増殖して物質循環を司っている細菌鍵種であることが示唆されている。今後、既得データと照合することで、これら細菌群の本養殖漁場における季節的な動態を明らかにする予定である。

養殖漁場では、水域生態系に負荷される有機物量が生簀がない場合に比べて大きい。この有機物負荷が当該水域の持つ自浄能力を超えると、有機汚濁、富栄養化、底層の貧酸素化といった自家汚染を起こす。養殖漁場の持つ自浄能力を評価することは難しい。有効な評価指標の一つが、当該水域における高分子有機物（例えばタンパク質）を低分子有機物（例えばアミノ酸）に加水分解する酵素活性の測定である。この高分子有機物が細菌群が利用可能な低分子有機物に加水分解されるプロセスが水域における有機物フローの制限要因となり、これを"gate keeper"と呼ぶ。 この有機物の流れを主に担う海洋細菌群の加水分解活性は、水温、塩分、溶存酸素などの環境要因により変化する。今までの研究から、水域が成層し、海底が貧酸素化しやすい夏季には、この加水分解活性が特に底層で停滞しがちであり、強風により鉛直混合が起こる冬季に活性が上がることを確認している。本研究課題では、加水分解活性が停滞しやすい夏季底層に強いインパクトを与える台風の影響を評価しようとしている。 2021年の台風発生数は例年よりも少なく、2020年に引き続き台風が暴風域を伴ったまま上陸することは皆無であった。野外調査で台風の影響を評価することが出来なかったが、室内実験により興味深い成果を得ることができた。それは、雨水の影響である。降雨により表層海水の塩分が低下するとペプチドの末端からアミノ酸を一つずつ切り離していくエキソペプチダーゼの活性が有意に高くなるのである。タンパク質の内部のペプチド結合を切るエンドペプチダーゼや糖類の加水分解酵素ではそのような傾向は確認できなかった。雨水により加水分解活性が上昇する現象を室内実験で確認したことは新規な知見と言える。

近畿大学奄美実験場のクロマグロ養殖場では、サンゴが群棲している。環境に負荷を与える魚類養殖ときれいな海の象徴であるサンゴが共存しているこの事実は、至妙な物質循環が成立していることを示唆する。本研究では、サンゴが放出する粘液に注目して、この物質循環を支える細菌を解析した。具体的には、海水試料、ならびにサンゴ粘液をシリンジで直接採取した試料（粘液試料）および海水試料と粘液試料を混合した試料（混合試料）を用意した。平成25年度は、既得試料（平成21~24年5月および10月）と新規採取試料（平成25年5月および10月）の試料において、細菌群集構造を解析した。さらに、非養殖場海域における試料（既得試料）においても、細菌種の特定を行った。本研究では、増殖活性のある細菌群のみを標的にすることができるブロモデオキシウリジンを用いたDNAトレーサー法と、多様性解析手法の一つであるリボソーム遺伝子間転写領域自動解析あるいはCARD-FISH法を組み合わせて、サンゴ粘液によって増殖してくる細菌群の動態を明らかにした。現在のところ、粘液試料および混合試料においてのみ活発に増殖していた細菌を11種を特定した。これらのうち、6種はもともとの全体の細菌群では検出されなかった。一方で、粘液が加わると増殖活性がなくなる細菌種が9種存在した。活発に増殖している細菌の組成はAlphaproteobacteriaとBacteroidetesに属する細菌系統群で半分以上が占められていた。しかし、同じAlphaproteobacteriaに属していても、サンゴ粘液によって活発に増殖する系統群とそうでない系統群が存在していた。本研究で対象とする養殖場域では、AlphaproteobacteriaやBacteroidetesに属する系統群が重要な細菌群であることを示唆する。

近畿大学のクロマグロ養殖イケスは、奄美大島の花天湾にある。ここのイケスロープには、美しいサンゴが育っている。このクロマグロ養殖とサンゴ共存のメカニズムを物質循環の駆動力となる海洋細菌の動態とサンゴが大量に放出する粘液に注目して調べた。イケス周辺の「海水のみ」と「海水+粘液」を一定時間培養し、比較すると、「海水のみ」ではあまり細菌群集構造が変化しなかったが、「海水+粘液」ではその質も量も大きく変化した。

本年度は、海洋における有機物フラックスの変動機構を解明するために、細菌群集と各環境条件の相互関係について明らかにすることを目的とし、以下の試料についてさらなるデータを得た。 1.2004、2006、2007年に採取した北海道サロマ湖の海水・海氷試料についてBrdU/DGGE解析を行った。この結果、海水・海氷中の全体の細菌群と増殖速度の速い細菌群のDGGEバンドパターンは異なっており、0℃以下の低温環境に適応し増殖する細菌群の存在が示唆された。また、各年で海氷細菌群集構造が異なっていたことと、海氷植物プランクトン組成が年によって大きく異なるという報告から、海氷中における細菌群と植物プランクトン細胞あるいはその産生有機物との相互関係が示唆された。 2.広島県呉湾にて採取した試料について一年を通した経時的変動解析を行った。その結果、観測期間中RoseobacterやCFBグループといった細菌群が優占しており、その細菌群集構造は海洋の一大イベントである植物プランクトンブルームによって大きく変化していることが分かった。 これまで特定してきた細菌群を、有機物の分解・生成に直接的に大きく貢献している"key species"として捉え、その時空間変動や環境条件の変動から生態的ニッチを考察し、これを"key species eco-typing仮説"として提案した。本研究は、天然環境の細菌群の増殖に着目し、それに基づいた生態的ニッチを初めて提案した独創的な研究であり、有機物フラックスの変動機構解明の先駆的研究である。各生態的ニッチを代表する"key species"に焦点を当て、その現存量や増殖、活性などの定量的な解析を行うことで、細菌群集を介する有機物フラックスの変動を説明することが出来ると期待される。