Metagenomic analysis of the microbial communities and associated network of nitrogen metabolism genes in the Ryukyu limestone aquifer.

While microbial biogeochemical activities such as those involving denitrification and sulfate reduction have been considered to play important roles in material cycling in various aquatic ecosystems, our current understanding of the microbial community in groundwater ecosystems is remarkably insufficient. To assess the groundwater in the Ryukyu limestone aquifer of Okinawa Island, which is located in the southernmost region of Japan, we performed metagenomic analysis on the microbial communities at the three sites and screened for functional genes associated with nitrogen metabolism. 16S rRNA amplicon analysis showed that bacteria accounted for 94-98% of the microbial communities, which included archaea at all three sites. The bacterial communities associated with nitrogen metabolism shifted by month at each site, indicating that this metabolism was accomplished by the bacterial community as a whole. Interestingly, site 3 contained much higher levels of the denitrification genes such as narG and napA than the other two sites. This site was thought to have undergone denitrification that was driven by high quantities of dissolved organic carbon (DOC). In contrast, site 2 was characterized by a high nitrate-nitrogen (NO3-N) content and a low amount of DOC, and this site yielded a moderate amount of denitrification genes. Site 1 showed markedly low amounts of all nitrogen metabolism genes. Overall, nitrogen metabolism in the Ryukyu limestone aquifer was found to change based on environmental factors.

Mechanism of denitrification in subsurface-dammed Ryukyu limestone aquifer, southern Okinawa Island, Japan.

Denitrification crucially regulates the attenuation of groundwater nitrate and is unlikely to occur in a fast-flowing aquifer such as the Ryukyu limestone aquifer in southern Okinawa Island, Japan. However, evidences of denitrification have been observed in several wells within this region. This study analyzed environmental isotopes (δ15NNO3 and ẟ18ONO3) to derive the rationale for denitrification at this site. Additionally, the presence of two subsurface dams in the study area may influence the processes involved in nitrate attenuation. Herein, we analyzed 150 groundwater samples collected spatially and seasonally to characterize the variations in the groundwater chemistry and stable isotopes during denitrification. The values of δ15NNO3 and δ18ONO3 displayed a progressive trend up to +59.7 ‰ and + 21 ‰, respectively, whereas the concentrations of NO3--N decreased to 0.1 mg L-1. In several wells, the enrichment factors of δ15NNO3 ranged from -6.6 to -2.1, indicating rapid denitrification, and the δ15NNO3 to δ18ONO3 ratios varied from 1.3:1 to 2:1, confirming the occurrence of denitrification. Denitrification intensively proceeds under conditions of depleted dissolved oxygen concentrations (<2 mg L-1), sluggish groundwater flow with longer residence times, high concentrations of dissolved organic carbon (>1.2 mg L-1), and low groundwater levels during the dry season with precipitation rates of <100 mm per month (Jun-Sep). SF6 analysis indicated the exclusive occurrence of denitrification in specific wells with groundwater residence times exceeding 30 years. These wells are located in close proximity to the major NE-SW fault system in the Komesu area, where the hydraulic gradient was below 0.005. Detailed geological and lithological investigations based on borehole data revealed that subsurface dams did not cause denitrification while the major NE-SW fault system uplifted the impermeable basement rock of the Shimajiri Group, creating a lithological gap at an equivalent depth that ultimately formed a sluggish groundwater area, promoting denitrification.

Visualisation of Phosphate in Subcalicoblastic Extracellular Calcifying Medium and on a Skeleton of Coral by Using a Novel Probe, Fluorescein-4-Isothiocyanate-Labelled Alendronic Acid.

The overload of nutrients of anthropogenic origin, including phosphate, onto coastal waters has been reported to have detrimental effects on corals. However, to the best of our knowledge, the phosphate concentration threshold for inhibiting coral calcification is unclear owing to a lack of information on the molecular mechanisms involved in the inhibitory effect of phosphate. Therefore, in this study, we prepared a new phosphate analogue, fluorescein-4-isothiocyanate (FITC)-labelled alendronic acid (FITC-AA), from commercially available reagents and used it as a novel probe to demonstrate its transfer pathway from ambient seawater into Acropora digitifera. When the juveniles at 1 d post-settlement were treated with FITC-AA in a laboratory tank, this phosphate analogue was found in the subcalicoblastic extracellular calcifying medium (SCM) and was absorbed on the basal plate in the juveniles within a few minutes. When the juveniles bear zooxanthellae at 3 months post-settlement, FITC-AA was observed on the corallite walls within a few minutes after adding ambient seawater. We concluded that FITC-AA in ambient seawater was transferred via a paracellular pathway to SCM and then absorbed on the coral CaCO3 skeletons because FITC-AA with a high polarity group cannot permeate through cell membranes.

Identification and classification of Croceivirga thetidis sp. nov., a marine Flavobacteriaceae isolated from the hard coral Acropora.

A taxonomic investigation using a polyphasic method was conducted to identify a novel marine flavobacterium, designated as DJ-13T, isolated from the hard coral Acropora sp. collected at Okinawa, Japan. Bacterial cells were Gram-stain-negative, yellow-colored, strictly aerobic, rod-shaped, catalase- and oxidase-positive, non-motile, and chemoorganoheterotrophic. The novel isolate grew at NaCl concentrations of 0.5-7%, pH 6.5-9.0, and 15-37 °C. A phylogenetic study on the basis of the 16S rRNA gene sequence revealed that strain DJ-13T belongs to the family Flavobacteriaceae and that it shared the greatest sequence similarity (95.9%) with Croceivirga lutea CSW06T. Strain DJ-13T comprised iso-C17:0 3-OH, iso-C15:0, and iso-C15:1 G as the main (> 10%) cellular fatty acids. Menaquinone-6 (MK-6) was the only respiratory quinone. The assembled draft genome size of strain DJ-13T was 3.71 Mbp with G + C content of 38.7 mol%. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and average amino acid identity (AAI) values of DJ-13T and the species of the genus Croceivirga were found to be 74.9-75.5%, 13.4-14.7%, and 68.2-72.4%, respectively. Strain DJ-13T contained phosphatidylethanolamine, two unidentified aminolipids, and five unidentified lipids as polar lipids. From the polyphasic taxonomic results presented, the strain is considered to represent a novel species of the genus Croceivirga for which the name Croceivirga thetidis sp. nov. is proposed. The type strain of C. thetidis sp. nov. is DJ-13T (= KCTC 72790T = NBRC 114252T).

Phosphate bound to calcareous sediments hampers skeletal development of juvenile coral.

To test the hypothesis that terrestrial runoff affects the functions of calcareous sediments in coral reefs and hampers the development of corals, we analysed calcareous sediments with different levels of bound phosphate, collected from reef areas of Okinawajima, Japan. We confirmed that phosphate bound to calcareous sediments was readily released into ambient seawater, resulting in much higher concentrations of phosphorous in seawater from heavily polluted areas (4.3-19.0 µM as compared with less than 0.096 µM in natural ambient seawater). Additionally, we examined the effect of phosphate released from calcareous sediments on the development of Acropora digitifera coral juveniles. We found that high phosphate concentrations in seawater clearly inhibit the skeletal formation of coral juveniles. Our results demonstrate that calcareous sediments in reef areas play a crucial role in mediating the impact of terrestrial runoff on corals by storing and releasing phosphate in seawater.

First Report of Microcystis Strains Producing MC-FR and -WR Toxins in Japan.

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography-mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) inhibition assay. The representative MC, MC-LR, and related cyanobacterial toxins strongly inhibit PP2A activity and can therefore be assayed by measuring the extent of PP2A inhibition. However, these methods require reference toxin standards for the quantification and identification of known MCs. To obtain various MC-producing cyanobacterial strains, we surveyed and collected MC-producing cyanobacteria from environmental sources of water in Okinawa, Japan. Using a dual assay (LC-MS analysis and PP2A inhibition assay), we identified and isolated Microcystis strains producing five MC variants (MC-LR, -RR, -LA, -FR and -WR). Approximately 4 mg of MC-WR and -FR toxins were purified from the laboratory culture of the Microcystis isolate NIES-4344. Pure MC-WR and -FR variants were prepared for future use as toxin standards in LC-MS analysis. Phylogenetic analysis based on ftsZ revealed that the NIES-4344 strain belongs to the identified groups in Microcystis aeruginosa. This is the first report of Microcystis strains producing mainly MC-WR and -FR toxins in Japan.

Phosphate Enrichment Hampers Development of Juvenile Acropora digitifera Coral by Inhibiting Skeleton Formation.

Coral reef degradation due to various local stresses, such as nutrient enrichment and terrestrial run-off into coastal waters, is an increasing global concern. Inorganic phosphates have been considered to possibly inhibit skeleton formation in corals. Despite many studies available on the effects of nutrients on corals, a clear consensus on how nutrients exert deteriorative effects on corals has not been established satisfactorily. In this study, we examined the effects of phosphates and nitrates on in vitro aragonite CaCO3 formation by using biogenic polyamines and in vivo aragonite formation in the skeleton of juvenile Acropora digitifera corals. We showed that the phosphates at similar concentrations clearly inhibited both in vitro and in vivo CaCO3 formation. In contrast, nitrates inhibited neither in vitro aragonite CaCO3 formation nor in vivo aragonite formation in juvenile coral skeleton. Furthermore, our findings showed that inhibition of coral skeleton formation was due to absorption of phosphate on the skeleton, which inorganically inhibited normal development of juvenile coral skeleton.

Seonamhaeicola acroporae sp. nov., a marine species of the family Flavobacteriaceae isolated from the hard coral Acropora formosa.

A novel marine flavobacterial species, designated 3KA7-17T, was isolated from the hard coral Acropora formosa D. collected in Japan. The strain was pale-orange pigmented, Gram-stain negative, strictly aerobic, coccus shaped, and non-motile. Preliminary analysis based on the 16S rRNA gene sequence revealed an affiliation with the family Flavobacteriaceae of the phylum Bacteroidetes, and it had the greatest sequence similarity (96.0%) to Seonamhaeicola algicola Gy8T. The DNA G + C content was 34.3 mol%. MK-6 was the major menaquinone, with iso-C15:1 H and/or C13:0 3-OH (24.3%), iso-C15:0 (19.5%), iso-C15:0 3-OH (14.2%), and iso-C17:0 3-OH (15.9%) as the main (> 10%) cellular fatty acids. The major polar lipid profile consisted of phosphatidylethanolamine, two unidentified aminolipids, and two unidentified lipids. Based on distinct phylogenetic and phenotypic evidence, the strain represents a novel species of the genus Seonamhaeicola, for which the name Seonamhaeicola acroporae sp. nov. is proposed and the type strain is 3KA7-17T (= KCTC 62713T = NBRC 113410T).

Correction to: Coraliitalea coralii gen. nov., sp. nov., a Marine Bacterium of the Family Flavobacteriaceae Isolated from the Hard Coral Galaxea fascicularis.

In the original version of this paper, the Chemotaxonomic Characteristics in the Results and Discussion section and legend of Table 2 given in the above paper are incorrect. These errors are corrected with this erratum.

Atmospheric CO2 captured by biogenic polyamines is transferred as a possible substrate to Rubisco for the carboxylation reaction.

Biogenic polyamines are involved in a wide range of plant cellular processes, including cell division, morphogenesis and stress responses. However, the exact roles of biogenic polyamines are not well understood. We recently reported that biogenic polyamines that have multiple amino groups can react with CO2 and accelerate calcium carbonate formation in seawater. The ability of biogenic polyamines to capture atmospheric CO2 prompted us to examine their roles in photosynthesis. Here, we demonstrated that atmospheric CO2 captured by biogenic polyamines is a candidate substrate for the carboxylation reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is an enzyme involved in the first major step of carbon fixation during photosynthesis, and that biogenic polyamines can accelerate the carboxylation reaction of this enzyme because of their specific affinity for CO2. Moreover, the results of our nuclear magnetic resonance (NMR) analysis showed that putrescine, which is the most common biogenic polyamine, reacts with atmospheric CO2 and promotes the formation of carbamate derivatives and bicarbonate in aqueous environments. A sufficient amount of CO2 is well known to be produced by carbonic anhydrase from bicarbonate in vivo. The present study indicates that CO2 would be also produced by the equilibrium reaction from carbonate produced by biogenic polyamines and would be used as a substrate of Rubisco, too. Our results may suggest a new photosynthetic research strategy that involves CO2-concentrating mechanisms and also possibly constitutes a potential tool for reducing atmospheric CO2 levels and, consequently, global warming.

Spongiibacterium fuscum sp. nov., a marine Flavobacteriaceae isolated from the hard coral Galaxea fascicularis.

A novel marine bacterium, designated 04OKA-3-218T, was isolated from the hard coral Galaxea fascicularis L. collected in Japan. The strain was dark-brown-pigmented, Gram-negative, strictly aerobic, curved-rod-shaped and non-motile. Phylogenetic analysis based on the 16S rRNA gene sequence showed the affiliation of the isolate with members of the family Flavobacteriaceae of the phylum Bacteroidetes, with the highest sequence similarity (95.2%) to Spongiibacterium pacificum SW169T. The DNA G+C content was 42.9 mol%; MK-6 was the major menaquinone; with iso-C17:0 3-OH (28.8%), iso-C15:0 (26.8%) and iso-C15:1 H and/or C13:0 3-OH (21.2%) as the main (>  10%) cellular fatty acids. The major polar lipid profile consisted of phosphatidylethanolamine, two unidentified aminolipids, two unidentified phosphoaminolipids and three unidentified lipids. On the basis of distinct phylogenetic and phenotypic evidences, the strain represents a novel species of the genus Spongiibacterium, for which the name Spongiibacterium fuscum sp. nov. is proposed. The type strain of S. fuscum sp. nov. is 04OKA-3-218T (= KCTC 62504T = NBRC 113248T).

Coraliitalea coralii gen. nov., sp. nov., a Marine Bacterium of the Family Flavobacteriaceae Isolated from the Hard Coral Galaxea fascicularis.

A polyphasic taxonomic study was performed on a novel strain designated as 04OKA-3-121T, which was isolated from the hard coral Galaxea fascicularis L. collected at Akajima, Okinawa, Japan. These bacterial cells were observed to be pale-yellow, Gram-stain-negative, strictly aerobic, chemoheterotrophic, non-spore forming, non-motile, and rod-shaped. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the novel marine isolate is affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it shared the highest (93.6%) sequence similarity with Pseudozobellia thermophila KMM 3531T. The strain could be phenotypically differentiated from related members of the family Flavobacteriaceae. Major fatty acids of strain 04OKA-3-121T were iso-C15:0, iso-C15:1 G, and C16:1 ω7c and/or C16:1 ω6c. The DNA G + C content of the strain was determined to be 38.8 mol% and the major respiratory quinone was identified as menaquinone 6 (MK-6). Strain 04OKA-3-121T had phosphatidylethanolamine, two unidentified aminolipids, and eight unidentified lipids as polar lipids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel genus in the family Flavobacteriaceae, for which the name Coraliitalea coralii gen. nov., sp. nov. is proposed. The type strain of C. coralii gen. nov., sp. nov. is 04OKA-3-121T (= KCTC 52378T = NBRC 112329T).

Frondibacter mangrovi sp. nov., a member of the family Flavobacteriaceae isolated from seawater by in situ cultivation, and emended description of Frondibacter aureus.

A Gram-stain-negative, rod-shaped, non-motile, strictly aerobic, chemoheterotrophic, light-yellow-pigmented bacterium, designated strain 02OK1/10-76T, was isolated from a mangrove estuary in Japan by use of an in situ cultivation technique. Preliminary analysis based on the 16S rRNA gene sequence revealed that the novel isolate was affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (96.9 %) to Frondibacter aureus A5Q-67T. The DNA G+C content of strain 02OK1/10-76T was 35 mol%; MK-6 was the only menaquinone; and iso-C15 : 0, iso-C17 : 0 3-OH and iso-C16 : 0 3-OH were the major (>10 %) cellular fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, three unidentified aminolipids, an unidentified aminophospholipid and an unidentified lipid. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain represents a novel species of the genus Frondibacter, for which the name Frondibacter mangrovi sp. nov. is proposed. The type strain is 02OK1/10-76T (= KCTC 52666T = NBRC 112695T). An emended description of F. aureus is also provided.

Aureisphaera galaxeae gen. nov., sp. nov., a marine member of the family Flavobacteriaceae isolated from the hard coral Galaxea fascicularis.

A novel Gram-stain negative, spherical, non-motile, strictly aerobic, heterotrophic, yellow pigmented bacterium, designated strain 04OKA003-7(T) was isolated from the hard coral Galaxea fascicularis L. collected at Akajima, Okinawa, Japan. Phylogenetic analysis based on the 16S rRNA gene sequence revealed the novel isolate is affiliated with the family Flavobacteriaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (92.9 %) to Vitellibacter aestuarii JC2436(T) and Aureitalea marina S1-66(T). The strain could be differentiated phenotypically from recognized members of the family Flavobacteriaceae. The major fatty acids of strain 04OKA003-7(T) were identified as iso-C15:0 and iso-C17:0 3-OH as defined by the MIDI system. The DNA G+C content was determined to be 41 mol%, the major respiratory quinone was identified as menaquinone 6 (MK-6) and a polar lipid profile was present consisting of phosphatidylethanolamine, two unidentified aminolipids and an unidentified lipid. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel genus for which the name Aureisphaera galaxeae gen. nov., sp. nov. is proposed. The type strain of A. galaxeae is 04OKA003-7(T) (=KCTC 32993(T) = NBRC 110018(T)).

Biogenic polyamines capture CO2 and accelerate extracellular bacterial CaCO3 formation.

Bacteria, including cyanobacteria, as well as some fungi, are known to deposit calcium carbonate (CaCO(3)) extracellularly in calcium-containing artificial medium. Despite extensive investigation, the mechanisms involved in extracellular formation of CaCO(3) by bacteria have remained unclear. The ability of synthetic amines to remove carbon dioxide (CO(2)) from natural gas led us to examine the role of biogenic polyamines in CaCO(3) deposition by bacteria. Here, we demonstrated that biogenic polyamines such as putrescine, spermidine, and spermine were able to react with atmospheric CO(2) and the resultant carbamate anion was characterized by using nuclear magnetic resonance (NMR) analysis. Biogenic polyamines accelerated the formation of CaCO(3), and we artificially synthesized the dumbbell-shaped calcites, which had the same form as observed with bacterial CaCO3 precipitates, under nonbacterial conditions by using polyamines. The reaction rate of calcification increased with temperature with an optimum of around 40 °C. Our observation suggests a novel scheme for CO(2) dissipation that could be a potential tool in reducing atmospheric CO(2) levels and, therefore, global warming.

Okicamelliaside, an extraordinarily potent anti-degranulation glucoside isolated from leaves of Camellia japonica.

Guided by anti-degranulation assays, we isolated from leaves of Camellia japonica an ellagic acid glucoside named okicamelliaside. The structure was elucidated as 3,4-dioxoloellagic acid 4'-O-ß-D-glucopyranoside by spectroscopic and chemical methods. Okicamelliaside was 12,000 times more potent than the antihistaminic drug, ketotifen fumarate, in inhibiting the degranulation of RBL-2H3 cells.

Psychromonas agarivorans sp. nov., a novel agarolytic bacterium.

Two heterotrophic, agarolytic bacteria were isolated from marine environments. A 16S rRNA gene sequence analysis showed the isolates (designated strains J42-3A(T) and 04OZ-AS15-7A) to be positioned in a separate lineage within the genus Psychromonas. Members of the genus Psychromonas are recognized as being psychrophilic or psychrotolerant, whereas none of the currently established Psychromonas species is known to be agarolytic. The DNA G+C contents of the isolates were about 42 mol% and the predominant cellular fatty acids were 16 : 1omega7c and 16 : 0. Based on the results of the phylogenetic and phenotypic analyses and DNA-DNA hybridization data, the isolates represent a novel species, for which the name Psychromonas agarivorans sp. nov. is proposed. The type strain is J42-3A(T) (=NBRC 104585(T)=KCTC 22285(T)).

Leptobacterium flavescens gen. nov., sp. nov., a marine member of the family Flavobacteriaceae, isolated from marine sponge and seawater.

Six obligately aerobic, Gram-negative, non-motile, pale-yellow-pigmented, rod-shaped bacterial strains, designated YM3-301(T), HG868, 04PA2 Co4-8B, 04PA2 Co4-99A, 04PA2 018SW-3 and 04PA2 018SW-18, were isolated from a marine sponge and seawater and were subjected to a polyphasic taxonomic investigation. Phylogenetic analyses based on 16S rRNA gene sequences revealed that the novel isolates were affiliated with the family Flavobacteriaceae (phylum Bacteroidetes) and that they showed the highest sequence similarity (90.3-90.9 %) to members of the genus Leeuwenhoekiella and to Zhouia amylotica. The 16S rRNA gene sequence similarities with respect to members of other related genera were only 86.3-89.1 %. In contrast, the six isolates shared high levels of 16S rRNA gene sequence similarity (99.7-100 %) and DNA-DNA relatedness (72-94 %) with each other. The novel isolates were phenotypically and physiologically different from members of related genera. The G+C content of the DNA was 41.8-43.5 mol%, MK-6 was the major menaquinone and i15 : 0, i15 : 1 and i17 : 0 3-OH were the major fatty acids. On the basis of the data from the taxonomic studies, it was concluded that these six novel strains represent a novel genus and species of the family Flavobacteriaceae, for which the name Leptobacterium flavescens gen. nov., sp. nov. is proposed. The type strain of Leptobacterium flavescens is YM3-301(T) (=KCTC 22160(T)=MBIC 06275(T)=NBRC 104141(T)).

Psychromonas heitensis sp. nov., a psychrotolerant bacterium isolated from seawater in Japan.

Four bacterial strains, 04HE-4-40, A4I-9, A4I-21 and AK15-027(T), were isolated from Heita Bay off Kamaishi in Japan. Based on 16S rRNA gene sequence analysis, the isolates were placed as a new lineage within the genus Psychromonas. The 16S rRNA gene sequence similarity values between the isolates and type strains of recognized Psychromonas species were less than 97 %. The DNA G+C contents of the isolates were about 38 mol%. The major respiratory quinone was Q-8, and the predominant cellular fatty acids were 16 : 1omega7c and 16 : 0. The isolates were able to grow at 30 degrees C, unlike other Psychromonas species. Based on the results of the phylogenetic analysis and phenotypic characteristics and DNA-DNA hybridization data, the isolates represent a novel species, for which the name Psychromonas heitensis sp. nov. is proposed. The type strain is AK15-027(T) (=MBIC 06898(T)=NCIMB 14416(T)).

Janibacter corallicola sp. nov., isolated from coral in Palau.

A novel Janibacter species is described on the basis of phenotypic, chemotaxonomic and genotypic data. Two bacterial strains were isolated in Palau, which were both Gram-positive, catalase-positive bacteria with meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan. The major menaquinone was MK-8(H(4)). Mycolic acids were not detected. The G+C content of the DNA was 70-71 mol%. Comparative 16S rDNA studies of the two isolated strains revealed that they both belonged to the genus Janibacter. DNA-DNA relatedness data revealed that 04PA2-Co5-61(T) and 02PA-Ca-009 belong to the same species, a new species of the genus Janibacter. From these results, Janibacter corallicola sp. nov. is proposed, with the type strain 04PA2-Co5-61(T) (=MBIC 08265(T), DSM 18906(T)).