2000年04月, 日本水産学会, 日本水産学会奨励賞　　　　　　　　　　　　　　　
日本国

Identification of a novel monocyclic carotenoid and prediction of its biosynthetic genes in Algoriphagus sp. oki45.
Naoki Takatani, Takashi Maoka, Tomoo Sawabe, Fumiaki Beppu, Masashi Hosokawa
Applied microbiology and biotechnology, 108, 1, 102, 102, 2024年12月, ［国際誌］
英語, 研究論文（学術雑誌）, Bacteria belonging to the genus Algoriphagus have been isolated from various sources, such as Antarctic sea ice, seawater, and sediment, and some strains are known to produce orange to red pigments. However, the pigment composition and biosynthetic genes have not been fully elucidated. A new red-pigmented Algoriphagus sp. strain, oki45, was isolated from the surface of seaweed collected from Senaga-Jima Island, Okinawa, Japan. Genome comparison revealed oki45's average nucleotide identity of less than 95% to its closely related species, Algoriphagus confluentis NBRC 111222 T and Algoriphagus taiwanensis JCM 19755 T. Comprehensive chemical analyses of oki45's pigments, including 1H and 13C nuclear magnetic resonance and circular dichroism spectroscopy, revealed that the pigments were mixtures of monocyclic carotenoids, (3S)-flexixanthin ((3S)-3,1'-dihydroxy-3',4'-didehydro-1',2'-dihydro-β,ψ-caroten-4-one) and (2R,3S)-2-hydroxyflexixanthin ((2R,3S)-2,3,1'-trihydroxy-3',4'-didehydro-1',2'-dihydro-β,ψ-caroten-4-one); in particular, the latter compound was new and not previously reported. Both monocyclic carotenoids were also found in A. confluentis NBRC 111222 T and A. taiwanensis JCM 19755 T. Further genome comparisons of carotenoid biosynthetic genes revealed the presence of eight genes (crtE, crtB, crtI, cruF, crtD, crtYcd, crtW, and crtZ) for flexixanthin biosynthesis. In addition, a crtG homolog gene encoding 2,2'-β-hydroxylase was found in the genome of the strains oki45, A. confluentis NBRC 111222 T, and A. taiwanensis JCM 19755 T, suggesting that the gene is involved in 2-hydroxyflexixanthin synthesis via 2-hydroxylation of flexixanthin. These findings expand our knowledge of monocyclic carotenoid biosynthesis in Algoriphagus bacteria. KEY POINTS: • Algoriphagus sp. strain oki45 was isolated from seaweed collected in Okinawa, Japan. • A novel monocyclic carotenoid 2-hydroxyflexixanthin was identified from strain oki45. • Nine genes for 2-hydroxyflexixanthin biosynthesis were found in strain oki45 genome.

Hydrogenimonas leucolamina sp. nov., a hydrogen- and sulphur-oxidizing mesophilic chemolithoautotroph isolated from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Western Pacific Ocean
Shuya Hatakeyama, Sayaka Mino, Mana Mizobata, Mako Takada, Jiro Tsuchiya, Shogo Yamaki, Yasuhiro Ando, Tomoo Sawabe, Ken Takai
International Journal of Systematic and Evolutionary Microbiology, 74, 10, Microbiology Society, 2024年10月22日
研究論文（学術雑誌）, A novel mesophilic bacterium, strain SS33^T, was isolated from a deep-sea hydrothermal vent chimney at Suiyo Seamount, Izu-Bonin Arc, Western Pacific Ocean. The cells of strain SS33^T were motile short rods with a single polar flagellum. The growth of strain SS33^T was observed at the temperature range between 33 and 55 °C (optimum growth at 45 °C), at the pH range between 5.0 and 7.1 (optimum growth at pH 6.0) and in the presence of between 2.0 and 4.5% (w/v) NaCl [optimum growth at 3.5% (w/v)]. Strain SS33^T was a facultative anaerobic chemolithoautotroph using molecular hydrogen and elemental sulphur as the sole electron donor. Nitrate, nitrous oxide, sulphate, elemental sulphur and molecular oxygen were capable of serving as the sole electron acceptor. Phylogenetic analysis based on 16S rRNA gene sequences placed strain SS33^T in the genus Hydrogenimonas belonging to the class Epsilonproteobacteria. The closely related species of strain SS33^T were Hydrogenimonas urashimensis SSM-Sur55^T (95.96%), Hydrogenimonas thermophila EP1-55-1%^T (95.75%) and Hydrogenimonas cancrithermarum ISO32^T (95.24%). According to the taxonomic and physiological characteristics, it is proposed that strain SS33^T was classified into a novel species of genus Hydrogenimonas, Hydrogenimonas leucolamina sp. nov., with SS33^T (=JCM 39184^T =KCTC 25253^T) as the type strain. Furthermore, the genome comparison of Epsilonproteobacteria revealed that their [NiFe] hydrogenase genes belonging to Group 1b could be divided into two phylogenetic lineages and suggested that the reverse gyrase gene has been lost after division to the genus Hydrogenimonas.

Time-course transcriptomic profiling suggests Crp/Fnr transcriptional regulation of nosZ gene in a N2O-reducing thermophile
Jiro Tsuchiya, Sayaka Mino, Fuki Fujiwara, Nao Okuma, Yasunori Ichihashi, Robert M. Morris, Brook L. Nunn, Emma Timmins-Schiffman, Tomoo Sawabe
iScience, 111074, 111074, Elsevier BV, 2024年09月
研究論文（学術雑誌）

The Description of Pseudoalteromonas apostichopi sp. nov., Vibrio apostichopi sp. nov., and Marinobacter apostichopi sp. nov. from the Fertilized Eggs and Larvae of Apostichopus japonicus
Rika Kudo, Ryota Yamano, Juanwen Yu, Shuya Hatakeyama, Chunqi Jiang, Sayaka Mino, Shogo Yamaki, Yasuhiro Ando, Yuichi Sakai, Tomoo Sawabe
Current Microbiology, 81, 8, Springer Science and Business Media LLC, 2024年06月28日
研究論文（学術雑誌）

Hydrogenimonas cancrithermarum sp. nov., a hydrogen- and thiosulfate-oxidizing mesophilic chemolithoautotroph isolated from diffuse-flow fluids on the East Pacific Rise, and an emended description of the genus Hydrogenimonas
Sayaka Mino, So Fukazawa, Jiro Tsuchiya, Jesse C. McNichol, Stefan M. Sievert, Shogo Yamaki, Yasuhiro Ando, Tomoo Sawabe
International Journal of Systematic and Evolutionary Microbiology, 73, 11, Microbiology Society, 2023年11月03日
研究論文（学術雑誌）, A novel mesophilic, hydrogen- and thiosulfate-oxidizing bacterium, strain ISO32^T, was isolated from diffuse-flow hydrothermal fluids from the Crab Spa vent on the East Pacific Rise. Cells of ISO32^T were rods, being motile by means of a single polar flagellum. The isolate grew at a temperature range between 30 and 55 °C (optimum, 43 °C), at a pH range between 5.3 and 7.6 (optimum, pH 5.8) and in the presence of 2.0–4.0 % NaCl (optimum, 2.5 %). The isolate was able to grow chemolithoautotrophically with molecular hydrogen, thiosulfate or elemental sulfur as the sole electron donor. Thiosulfate, elemental sulfur, nitrate and molecular oxygen were each used as a sole electron acceptor. Phylogenetic analysis of 16S rRNA gene sequences placed ISO32^T in the genus Hydrogenimonas of the class Epsilonproteobacteria, with Hydrogenimonas thermophila EP1-55–1 %^T as its closest relative (95.95 % similarity). On the basis of the phylogenetic, physiological and genomic characteristics, it is proposed that the organism represents a novel species within the genus Hydrogenimonas, Hydrogenimonas cancrithermarum sp. nov. The type strain is ISO32^T (=JCM 39185^T =KCTC 25252^T). Furthermore, the genomic properties of members of the genus Hydrogenimonas are distinguished from those of members of other thermophilic genera in the orders Campylobacterales (Nitratiruptor and Nitrosophilus) and Nautiliales (Caminibacter, Nautilia and Lebetimonas), with larger genome sizes and lower 16S rRNA G+C content values. Comprehensive metabolic comparisons based on genomes revealed that genes responsible for the Pta–AckA pathway were observed exclusively in members of mesophilic genera in the order Campylobacterales and of the genus Hydrogenimonas. Our results indicate that the genus Hydrogenimonas contributes to elucidating the evolutionary history of Epsilonproteobacteria in terms of metabolism and transition from a thermophilic to a mesophilic lifestyle.

Unveiling the early life core microbiome of the sea cucumber Apostichopus japonicus and the unexpected abundance of the growth-promoting Sulfitobacter.
Juanwen Yu, Chunqi Jiang, Ryota Yamano, Shotaro Koike, Yuichi Sakai, Sayaka Mino, Tomoo Sawabe
Animal microbiome, 5, 1, 54, 54, 2023年10月24日, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: Microbiome in early life has long-term effects on the host's immunological and physiological development and its disturbance is known to trigger various diseases in host Deuterostome animals. The sea cucumber Apostichopus japonicus is one of the most valuable marine Deuterostome invertebrates in Asia and a model animal in regeneration studies. To understand factors that impact on host development and holobiont maintenance, host-microbiome association has been actively studied in the last decade. However, we currently lack knowledge of early life core microbiome during its ontogenesis and how it benefits the host's growth. RESULTS: We analyzed the microbial community in 28 sea cucumber samples from a laboratory breeding system, designed to replicate aquaculture environments, across six developmental stages (fertilized eggs to the juvenile stage) over a three years-period to examine the microbiomes' dynamics and stability. Microbiome shifts occurred during sea cucumber larval ontogenesis in every case. Application of the most sophisticated core microbiome extraction methodology, a hybrid approach with abundance-occupancy core microbiome analyses (top 75% of total reads and > 70% occupation) and core index calculation, first revealed early life core microbiome consisted of Alteromonadaceae and Rhodobacteraceae, as well as a stage core microbiome consisting of pioneer core microbe Pseudoalteromonadaceae in A. japonicus, suggesting a stepwise establishment of microbiome related to ontogenesis and feeding behavior in A. japonicus. More interestingly, four ASVs affiliated to Alteromonadaceae and Rhodobacteraceae were extracted as early life core microbiome. One of the ASV (ASV0007) was affiliated to the Sulfitobactor strain BL28 (Rhodobacteraceae), isolated from blastula larvae in the 2019 raring batch. Unexpectedly, a bioassay revealed the BL28 strain retains a host growth-promoting ability. Further meta-pangenomics approach revealed the BL28 genome reads were abundant in the metagenomic sequence pool, in particular, in that of post-gut development in early life stages of A. japonicus. CONCLUSION: Repeated rearing efforts of A. japonicus using laboratory aquaculture replicating aquaculture environments and hybrid core microbiome extraction approach first revealed particular ASVs affiliated to Alteromonadaceae and Rhodobacteraceae as the A. japonicus early life core microbiome. Further bioassay revealed the growth promoting ability to the host sea cucumber in one of the core microbes, the Sulfitobactor strain BL28 identified as ASV0007. Genome reads of the BL28 were abundant in post-gut development of A. japonicus, which makes us consider effective probiotic uses of those core microbiome for sea cucumber resource production and conservation. The study also emphasizes the importance of the core microbiome in influencing early life stages in marine invertebrates. Understanding these dynamics could offer pathways to improve growth, immunity, and disease resistance in marine invertebrates.

A lesson from polybutylene succinate plastisphere to the discovery of novel plastic degrading enzyme genes in marine vibrios.
Yutaro Kimura, Yutaka Fukuda, Rumi Otsu, Juwanen Yu, Sayaka Mino, Satoru Misawa, Satoshi Maruyama, Yuta Ikeda, Remi Miyamachi, Hiroshi Noguchi, Satoshi Kato, Yasuhito Yamamoto, Tomoo Sawabe
Environmental microbiology, 2023年09月29日, ［国際誌］
英語, 研究論文（学術雑誌）, Polybutylene succinate (PBS) is an eco-friendly green plastic. However, PBS was shown as being non-biodegradable in marine environments, and up until now, only a limited number of PBS-degrading marine microbes have been discovered. We first set up in vitro PBS- and PBSA (polybutylene succinate adipate)-plastispheres to characterize novel PBS-degrading marine microbes. Microbial growth and oxygen consumption were observed in both PBS- and PBSA-plastispheres enriched with natural seawater collected from Usujiri, Hokkaido, Japan, and Vibrionaceae and Pseudoalteromonadaceae were significantly enriched on these films. Further gene identification indicated that vibrios belonging to the Gazogenes clade possess genes related to a PBS degrading enzyme (PBSase). The PBS degradation assay for six Gazogenes clade vibrios identified Vibrio ruber, Vibrio rhizosphaerae, and Vibrio spartinae as being capable of degrading PBS. We further identified the gene responsible for PBSase from the type strain of V. ruber, and the purified recombinant vibrio PBSase was found to have low-temperature adaptation and was active under high NaCl concentrations. We also provided docking models between the vibrio PBSase and PBS and PBSA units to show how vibrio PBSase interacts with each substrate compared to the Acidovorax PBSase. These results could contribute to a more sustainable society through further utilization of PBS in marine environments and plastic recycling.

Genome taxonomy of the genus Thalassotalea and proposal of Thalassotalea hakodatensis sp. nov. isolated from sea cucumber larvae.
Ryota Yamano, Juanwen Yu, Alfabetian Harjuno Condro Haditomo, Chunqi Jiang, Sayaka Mino, Jesús L Romalde, Kyuhee Kang, Yuichi Sakai, Tomoo Sawabe
PloS one, 18, 6, e0286693, 2023年, ［国際誌］
英語, 研究論文（学術雑誌）, The genus Thalassotalea is ubiquitous in marine environments, and up to 20 species have been described so far. A Gram-staining-negative, aerobic bacterium, designated strain PTE2T was isolated from laboratory-reared larvae of the Japanese sea cucumber Apostichopus japonicus. Phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PTE2T was closely related to Thalassotalea sediminis N211T (= KCTC 42588T = MCCC 1H00116T) with 97.9% sequence similarity. ANI and in silico DDH values against Thalassotalea species were 68.5-77.0% and 19.7-24.6%, respectively, indicating the novelty of PTE2T. Based on genome-based taxonomic approaches, strain PTE2T (= JCM 34608T = KCTC 82592T) is proposed as a new species, Thalassotalea hakodatensis sp. nov.

Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae.
Rika Kudo, Ryota Yamano, Juanwen Yu, Shotaro Koike, Alfabetian Harjuno Condro Haditomo, Mayanne A M de Freitas, Jiro Tsuchiya, Sayaka Mino, Fabiano Thompson, Jesús L Romalde, Hisae Kasai, Yuichi Sakai, Tomoo Sawabe
PloS one, 18, 8, e0290060, 2023年, ［国際誌］
英語, 研究論文（学術雑誌）, A Gram-staining-negative, oxidase-positive, strictly aerobic rod-shaped bacterium, designated strain PT1T, was isolated from the laboratory-reared larvae of the sea cucumber Apostichopus japonicus. A phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PT1T was closely related to Neptuniibacter marinus ATR 1.1T (= CECT 8938T = DSM 100783T) and Neptuniibacter caesariensis MED92T (= CECT 7075T = CCUG 52065T) showing 98.2% and 98.1% sequence similarity, respectively. However, the average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values among these three strains were 72.0%-74.8% and 18.3%-19.5% among related Neptuniibacter species, which were below 95% and 70%, respectively, confirming the novel status of PT1T. The average amino acid identity (AAI) values of PT1T showing 74-77% among those strains indicated PT1T is a new species in the genus Neptuniibacter. Based on the genome-based taxonomic approach, Neptuniibacter victor sp. nov. is proposed for PT1T. The type strain is PT1T (JCM 35563T = LMG 32868T).

Nitrosophilus kaiyonis sp. nov., a hydrogen-, sulfur- and thiosulfate-oxidizing chemolithoautotroph within "Campylobacteria" isolated from a deep-sea hydrothermal vent in the Mid-Okinawa Trough.
So Fukazawa, Sayaka Mino, Jiro Tsuchiya, Satoshi Nakagawa, Ken Takai, Tomoo Sawabe
Archives of microbiology, 205, 1, 12, 12, 2022年12月03日, ［国際誌］
英語, 研究論文（学術雑誌）, A novel bacterium, strain MOT50T, was isolated from the chimney structure at the Iheya North field in the Mid-Okinawa Trough. The cells were motile short rods with a single polar flagellum. Growth was observed between 40 and 65 ℃ (optimum, 52 ℃), at pH values between 5.0 and 7.1 (optimum, pH 6.1) and in the presence of 2.0-4.0% NaCl (optimum, 2.5%). The isolates utilized molecular hydrogen, thiosulfate, or elemental sulfur as the sole electron donor. Thiosulfate, elemental sulfur, nitrate, and molecular oxygen are utilized as the sole electron acceptor. Ammonium is required as a nitrogen source. Thiosulfate, elemental sulfur, sulfate, or sulfite serves as a sulfur source for growth. The G + C content of the genomic DNA was 28.9%. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain MOT50T belonged to the genus Nitrosophilus of the class "Campylobacteria", and its closest relative was Nitrosophilus labii HRV44T (97.20%). On the basis of the phylogenetic, physiological, and molecular characteristics, it is proposed that the organism represents a novel species within the genus Nitrosophilus, Nitrosophilus kaiyonis sp. nov. The type strain is MOT50T (= JCM 39187T = KCTC 25251T).

Light response of Vibrio parahaemolyticus
Yunato Kuroyanagi, Jiro Tsuchiya, Chunqi Jiang, Sayaka Mino, Hisae Kasai, Daisuke Motooka, Tetsuya Iida, Masataka Satomi, Tomoo Sawabe
Frontiers in Marine Science, 9, Frontiers Media SA, 2022年11月10日, ［責任著者］
研究論文（学術雑誌）, Light is one of the most critical stimuli in the majority of living organisms. In the last two decades, blue light (BL) has become a major subject of attention because of developments in light-emitting diodes (LED). The effects of BL on eukaryotic organisms and phototrophic prokaryotes have been well studied, but the knowledge of its effects on non-phototrophic prokaryotes remains unclear. Since BL can penetrate seawater, it is expected that most prokaryotes living in the ocean possess molecular mechanisms which protect against BL. The aim of this study is to assess the molecular mechanisms of Vibrio parahaemolyticus cells against BL as a marine bacterial model compared to other wavelength light exposures. Physiological and transcriptomic analyses of BL-exposed cells compared to other light treated cells revealed the highest ROS fold change, the highest number of differentially expressed genes (DEGs), and up-regulation in the gene responsible to not only compatible solute such as glycine betaine and ectoine but also iron-sulfur biosynthesis related to ROS formation. Furthermore, red light (RL) up-regulated the expression of cryptochrome DASH, a protein known to be excited by BL, and orange light (OL) decreased the expression of thermostable direct hemolysin (TDH), suggesting that OL attenuates the virulence of V. parahaemolyticus. In addition, the expression of VtrA (V. parahaemolyticus type III secretion system 2 (T3SS2) regulator A) but not VtrB (V. parahaemolyticus T3SS2 regulator B) increased under both light treatments, indicating that light exposure is unlikely to be involved in T3SS2-mediated pathogenicity. These results expand our knowledge on unique light responses in non-phototrophic marine prokaryotes.

Comparative Physiology and Genomics of Hydrogen-Producing Vibrios
Yuta Matsumura, Kazumich Sato, Chunqi Jiang, Sayaka Mino, Tomoo Swabe
Current Microbiology, 79, 12, Springer Science and Business Media LLC, 2022年11月, ［責任著者］
研究論文（学術雑誌）

Draft genome sequence data of Vibrio harveyi VH1 isolated from a diseased tiger grouper, Epinephelus fuscoguttatus, cultured in Malaysia.
Md Ali Amatul-Samahah, Aslah Mohamad, Nurhidayu Al-Saari, Mohd Zamri-Saad, Mohamad Noor Amal Azmai, Mohd Termizi Yusof, Md Yasin Ina-Salwany, Mami Tanaka, Sayaka Mino, Tomoo Sawabe
Data in brief, 44, 108533, 108533, 2022年10月, ［国際誌］
英語, 研究論文（学術雑誌）, Vibriosis accounts for 66.7% of diseases reported in groupers' cultures and affects almost all stages of growth. The disease could lead up to mortality up to 50% mortality, and it was reported that high stocking density and poor fish handling were among the factors that contributed to the disease dissemination. V. harveyi has been reported to be among the causative agent and has caused acute mortality in cage groupers. In this study, we report the genome of V. harveyi VH1 isolated from a diseased tiger grouper Epinephelus fuscoguttatus, reared in a cage farm located in the coastal area of Langkawi.

The pan-genome of Splendidus clade species in the family Vibrionaceae: insights into evolution, adaptation, and pathogenicity.
Chunqi Jiang, Hisae Kasai, Sayaka Mino, Jesús L Romalde, Tomoo Sawabe
Environmental microbiology, 24, 10, 4587, 4606, 2022年09月15日, ［国際誌］
英語, 研究論文（学術雑誌）, The Splendidus clade is the largest clade in Vibrionaceae, and its members are often related to mortality of marine animals with huge economic losses. The molecular bases of their pathogenicity and virulence, however, remain largely unknown. In particular, the complete genome sequences of the Splendidus clade species are rarely registered, which is one of the obstacles to predict core and/or unique genes responsible to their adaptation and pathogenicity, and to perform a fine scale meta-transcriptome during bacterial infection to their hosts. In this study, we obtained the complete genomes of all type strains in the Splendidus clade and revealed that 1) different genome sizes (4.4-5.9 Mb) with V. lentus the biggest and most of them had several big plasmids, likely because of the different features on mobilome elements, 2) the Splendidus clade consists of 19 species except V. cortegadensis, and 3 sub-clades (SC) were defined with the 15 most closely related members as SC1; 3) different carbohydrate degradation preferences may be the result of environmental adaptation, 4) a broad prediction of virulence factors (VFs) revealed core and species unique VF genes. This article is protected by copyright. All rights reserved.

Genomic Analyses of Halioticoli Clade Species in Vibrionaceae Reveal Genome Expansion With More Carbohydrate Metabolism Genes During Symbiotic to Planktonic Lifestyle Transition
Chunqi Jiang, Sayaka Mino, Tomoo Sawabe
Frontiers in Marine Science, 9, Frontiers Media SA, 2022年03月23日, ［責任著者］
研究論文（学術雑誌）, Vibrionaceae is one of the most diverse bacterial families and is currently classified into over 50 clades, some members of which play an important role in the symbiotic relationships with humans and animals. Halioticoli clade, which currently consists of 10 species: 8 species associated with the gut of abalone (symbiotic), 1 species (V. breoganii) from bivalves, and 1 species (V. ishigakensis) from subtropical seawater (planktonic). To accelerate studies in the evolution, ecogenomics, and biotechnology of Halioticoli clade species, the genomic backbones and pangenome analyses based on complete genome sequences are needed. Genome sizes of Halioticoli clade species ranged from 3.5 Mb to 4.8 Mb, with V. ishigakensis the biggest. The evolutionary relationships using multilocus sequence analysis based on eight housekeeping genes and 125 single-copy core genes revealed a division of five sub-clades in this clade; 1) V. breoganii, V. comitans, V. inusitatus and V. superstes, 2) V. ezurae, V. neonatus, and V. halioticoli, 3) V. rarus, 4) V. gallicus, and 5) V. ishigakensis. The pan-genomic analysis combined with function and metabolism estimations showed that the planktonic group (sub-clade 5) contained the greatest number of specific genes, and more genes responsible for carbohydrate metabolisms, especially the genes encoding D-galactonate degradation. These results demonstrated that the genome expanded by acquiring more abilities for utilizing various carbohydrates during the evolution from symbiotic to a planktonic lifestyle. Moreover, according to Carbohydrate-Active enZYmes (CAZy) profiling, genes encoding alginate degrading enzymes (aly), classified into PL6, PL7, PL15, and PL17 were common in the ten genomes, but sub-clade 1 had the most. Meanwhile, sub-clade 1and 5 also possessed abundant genes related to macroalgae substrates degradation (GHs), which are also responsible for the genome expansion of sub-clade 1 and 5.

Taxonomic revision of the genus Amphritea supported by genomic and in silico chemotaxonomic analyses, and the proposal of Aliamphritea gen. nov.
Ryota Yamano, Juanwen Yu, Chunqi Jiang, Alfabetian Harjuno Condro Haditomo, Sayaka Mino, Yuichi Sakai, Tomoo Sawabe
PloS one, 17, 8, e0271174, 2022年, ［国際誌］
英語, 研究論文（学術雑誌）, A Gram-staining-negative, aerobic bacterium, designated strain PT3T was isolated from laboratory-reared larvae of the Japanese sea cucumber Apostichopus japonicus. Phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PT3T was closely related to Amphritea ceti RA1T (= KCTC 42154T = NBRC 110551T) and Amphritea spongicola MEBiC05461T (= KCCM 42943T = JCM 16668T) both with 98.3% sequence similarity, however, average nucleotide identity (ANI) and in silico DNA-DNA hybridization (in silico DDH) values among these three strains were below 95% and 70%, respectively, confirming the novelty of PT3T. Furthermore, the average amino acid identity (AAI) values of PT3T against other Amphritea species were on the reported genus delineation boundary (64-67%). Multilocus sequence analysis using four protein-coding genes (recA, mreB, rpoA, and topA) further demonstrated that PT3T, Amphritea ceti and Amphritea spongicola formed a monophyletic clade clearly separate from other members of the genus Amphritea. Three strains (PT3T, A. ceti KCTC 42154T and A. spongicola JCM 16668T) also showed higher similarities in their core genomes compared to those of the other Amphritea spp. Based on the genome-based taxonomic approach, Aliamphritea gen. nov. was proposed together with the reclassification of the genus Amphritea and Aliamphritea ceti comb. nov. (type strain RA1T = KCTC 42154T = NBRC 110551T), Aliamphritea spongicola comb. nov. (type strain MEBiC05461T = KCCM 42943T = JCM 16668T), and Aliamphritea hakodatensis sp. nov. (type strain PT3T = JCM 34607T = KCTC 82591T) were suggested.

Characterization of Microbiomes Associated With the Early Life Stages of Sea Cucumber Apostichopus japonicus Selenka
Juanwen Yu, Yuichi Sakai, Sayaka Mino, Tomoo Sawabe
FRONTIERS IN MARINE SCIENCE, 9, FRONTIERS MEDIA SA, 2022年01月
英語, 研究論文（学術雑誌）, There is a lot of evidence indicating pioneer microbes in early life having various effects on later host biology. Because of the influential phylogenetic position of sea cucumber, which is a deep branching clade in Deuterostomia, the attention on the microbiome in sea cucumber has been increasing. Although microbes in sea cucumber have been reported in several studies, there is a lack of knowledge regarding the pioneer microbiota in the early life stages of sea cucumber. In this study, microbiota changes during the larval development of sea cucumber were assessed using a laboratory rearing system. Microbial community structure was likely to be related to the developmental stage and significant alterations were detected in the late auricularia stage. The relative abundances of Oceanospirillales, Alteromonadales, and Rhodobacterales significantly varied after gut formation. A total of 257 strains were isolated from larval developmental stages of sea cucumber and affiliated to 124 ASVs in the metagenomic analysis. This data demonstrates for the first-time dynamic changes of sea cucumber microbiota in the developmental stages in early life.

The Structure and Function of Gut Microbiomes of Two Species of Sea Urchins, Mesocentrotus nudus and Strongylocentrotus intermedius, in Japan
Alfabetian Harjuno Condro Haditomo, Masanori Yonezawa, Juanwen Yu, Sayaka Mino, Yuichi Sakai, Tomoo Sawabe
Frontiers in Marine Science, 8, Frontiers Media SA, 2021年12月22日, ［査読有り］, ［責任著者］
研究論文（学術雑誌）, Sea urchin is an indicator of coastal environmental changes in the global warming era, and is also a model organism in developmental biology and evolution. Due to the depletion of wild resources, new aquaculture techniques for improving stocks have been well studied. The gut microbiome shapes various aspects of a host’s physiology. However, these microbiome structures and functions on sea urchins, particularly Mesocentrotus nudus and Strongylocentrotus intermedius which are important marine bioresources commonly found in Japan, have not been fully investigated yet. Using metagenomic approaches including meta16S and shotgun metagenome sequencings, the structures, functions, and dynamics of the gut microbiome of M. nudus and S. intermedius, related to both habitat environment and host growth, were studied. Firstly, a broad meta16S analysis revealed that at the family level, Psychromonadaceae and Flavobacteriaceae reads (38–71%) dominated in these sea urchins, which is a unique feature observed in species in Japan. Flavobacteriaceae reads were more abundant in individuals after rearing in an aquarium with circulating compared to one with running water. Campylobacteraceae and Vibrionaceae abundances increased in both kinds of laboratory-reared sea urchins in both types of experiments. 2-weeks feeding experiments of M. nudus and S. intermedius transplanted from the farm to laboratory revealed that these gut microbial structures were affected by diet rather than rearing environments and host species. Secondly, further meta16S analysis of microbial reads related to M. nudus growth revealed that at least four Amplicon Sequence Variant (ASV) affiliated to Saccharicrinis fermentans, which is known to be a nitrogen (N₂) fixing bacterium, showed a significant positive correlation to the body weight and test diameter. Interestingly, gut microbiome comparisons using shotgun metagenome sequencing of individuals showing higher and lower growth rates revealed a significant abundance of “Nitrate and nitrite ammonification” genes in the higher-grown individuals under the circulating water rearing. These findings provide new insights on the structure-function relationship of sea urchin gut microbiomes beyond previously reported nitrogen fixation function in sea urchin in 1950s; we discovered a nitrate reduction function into ammonium for the growth promotion of sea urchin.

Vibrio Clade 3.0: New Vibrionaceae Evolutionary Units Using Genome-Based Approach.
Chunqi Jiang, Mami Tanaka, Sayo Nishikawa, Sayaka Mino, Jesús L Romalde, Fabiano L Thompson, Bruno Gomez-Gil, Tomoo Sawabe
Current microbiology, 79, 1, 10, 10, 2021年12月14日, ［国際誌］
英語, 研究論文（学術雑誌）, Currently, over 190 species in family Vibrionaceae, including not-yet-cultured taxa, have been described and classified into over nine genera, in which the number of species has doubled compared to the previous vibrio evolutionary update (Vibrio Clade 2.0) (Sawabe et al. 2014). In this study, "Vibrio Clade 3.0," the second update of the molecular phylogenetic analysis was performed based on nucleotide sequences of eight housekeeping genes (8-HKGs) retrieved from genome sequences, including 22 newly determined genomes. A total of 51 distinct clades were observed, of which 21 clades are newly described. We further evaluated the delineation powers of the clade classification based on nucleotide sequences of 34 single-copy genes and 11 ribosomal protein genes (11-RPGs) retrieved from core-genome sequences; however, the delineation power of 8-HKGs is still high and that gene set can be reliably used for the classification and identification of Vibrionaceae. Furthermore, the 11-RPGs set proved to be useful in identifying uncultured species among metagenome-assembled genome (MAG) and/or single-cell genome-assembled genome (SAG) pools. This study expands the awareness of the diversity and evolutionary history of the family Vibrionaceae and accelerates the taxonomic applications in classifying as not-yet-cultured taxa among MAGs and SAGs.

Genetic Environment Surrounding blaOXA-55-like in Clinical Isolates of Shewanella algae Clade and Enhanced Expression of blaOXA-55-like in a Carbapenem-Resistant Isolate.
Yuki Ohama, Kotaro Aoki, Sohei Harada, Tatsuya Nagasawa, Tomoo Sawabe, Lisa Nonaka, Kyoji Moriya, Yoshikazu Ishii, Kazuhiro Tateda
mSphere, 6, 5, e0059321, 2021年10月27日, ［国際誌］
英語, 研究論文（学術雑誌）, Although Shewanella spp. are most frequently isolated from marine environments; more rarely, they have been implicated in human infections. Shewanella spp. are also recognized as the origin of genes for carbapenem-hydrolyzing class D β-lactamases. Due to the spread globally among Enterobacterales in recent years, risk assessments of both clinical and environmental Shewanella strains are urgently needed. In this study, we analyzed the whole-genome sequences of 10 clinical isolates and 13 environmental isolates of Shewanella spp. and compared them with those of Shewanella species strains registered in public databases. In addition, the levels of blaOXA-55-like transcription and β-lactamase activity of a carbapenem-resistant Shewanella algae isolate were compared with those of carbapenem-susceptible S. algae clade isolates. All clinical isolates were genetically identified as S. algae clade (S. algae, Shewanella chilikensis, and Shewanella carassii), whereas all but one of the environmental isolates were identified as various Shewanella spp. outside the S. algae clade. Although all isolates of the S. algae clade commonly possessed an approximately 12,500-bp genetic region harboring blaOXA-55-like, genetic structures outside this region were different among species. Among S. algae clade isolates, only one showed carbapenem resistance, and this isolate showed a high level of blaOXA-55-like transcription and β-lactamase activity. Although this study documented the importance of the S. algae clade in human infections and the relationship between enhanced production of OXA-55-like and resistance to carbapenems in S. algae, further studies are needed to elucidate the generalizability of these findings. IMPORTANCE Shewanella spp., which are known to carry chromosomally located blaOXA genes, have mainly been isolated from marine environments; however, they can also cause infections in humans. In this study, we compared the molecular characteristics of clinical isolates of Shewanella spp. with those originating from environmental sources. All 10 clinical isolates were genetically identified as members of the Shewanella algae clade (S. algae, S. chilikensis, and S. carassii); however, all but one of the 13 environmental isolates were identified as Shewanella species members outside the S. algae clade. Although all the S. algae clade isolates possessed an approximately 12,500-bp genetic region harboring blaOXA-55-like, only one isolate showed carbapenem resistance. The carbapenem-resistant isolate showed a high level of blaOXA-55-like transcription and β-lactamase activity compared with the carbapenem-susceptible isolates. To confirm the clinical significance and antimicrobial resistance mechanisms of the S. algae clade members, analysis involving more clinical isolates should be performed in the future.

Hydrogenimonas urashimensis sp. nov., a hydrogen-oxidizing chemolithoautotroph isolated from a deep-sea hydrothermal vent in the Southern Mariana Trough.
Sayaka Mino, Taiki Shiotani, Satoshi Nakagawa, Ken Takai, Tomoo Sawabe
Systematic and applied microbiology, 44, 1, 126170, 126170, 2021年01月, ［国際誌］
英語, 研究論文（学術雑誌）, A novel thermophilic bacterium, strain SSM-sur55T, was isolated from a chimney structure at the Urashima site on the Southern Mariana Trough in the Pacific Ocean. Growth was observed at temperatures between 25 and 60°C (optimum, 55°C; 180min doubling time), at pH values between 5.3 and 7.2 (optimum, pH 5.9) and in the presence of between 1.6 and 5.6% (w/v) NaCl (optimum, 3.2%). The isolate used molecular hydrogen as its sole energy source, carbon dioxide as its sole carbon source, ammonium as its sole nitrogen source, and elemental sulfur as its sole sulfur source. Thiosulfate, molecular oxygen (0.1%, v/v) or elemental sulfur was utilized as its sole electron acceptor. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SSM-sur55T belonged to the genus Hydrogenimonas of the class "Campylobacteria", and its closest relative was Hydrogenimonas thermophila EP1-55-1%T (94.9%). On the basis of the phylogenetic, physiological and molecular characteristics, strain SSM-sur55T represents a novel species within the genus Hydrogenimonas, for which the name Hydrogenimonas urashimensis sp. nov. is proposed, with the type strain SSM-sur55T (JCM 19825=KCTC 15926).

An annual faecal 16S amplicon sequencing of individual sea cucumber (Apostichopus japonicus) demonstrates the feeding behaviours against eukaryotes in natural environments
Yohei Yamazaki, Yuichi Sakai, Sayaka Mino, Tomoo Sawabe
AQUACULTURE RESEARCH, 51, 9, 3602, 3608, WILEY, 2020年09月
英語, 研究論文（学術雑誌）, To fill in the gaps in knowledge as to how individual sea cucumber Apostichopus japonicus behave consuming eukaryotic food sources in natural environments, eukaryotic communities in the faeces of sea cucumbers and sediments were analysed through one whole year based on 16S rRNA gene sequencing of the organelle genomes. A total of 390 eukaryotic features were obtained, and 99.7% of the features were assigned to chloroplasts. The eukaryotic communities in faeces and sediments showed seasonal fluctuations through one whole year based on Bray-Curtis distance and community composition. Comparison of eukaryotic communities between faeces and sediments showed that 12 families including Chaetocerotaceae and Laminariaceae were more abundant in faeces than in sediments, suggesting that sea cucumbers may choose sediment containing these algal taxa more often compared with others in natural environments. All features of Laminariaceae were assigned to Saccharina japonica, which is consistent with the fact that this alga is one of the most suitable diets in the aquaculture of A. japonicus. Assessments of individual 16S amplicon sequences of both faecal and sediment samples could be an alternative tool to help us understand dynamic feeding behaviours of sea cucumber populations in contributing to bioresource conservation and development of a superior approach to aquaculture.

Biogeochemical Implications of N2O-Reducing Thermophilic Campylobacteria in Deep-Sea Vent Fields, and the Description of Nitratiruptor labii sp. nov.
Muneyuki Fukushi, Sayaka Mino, Hirohisa Tanaka, Satoshi Nakagawa, Ken Takai, Tomoo Sawabe
iScience, 23, 9, 101462, 101462, 2020年08月15日, ［国際誌］
英語, 研究論文（学術雑誌）, Nitrous oxide (N2O) is a potent greenhouse gas and has significantly increased in the atmosphere. Deep-sea hydrothermal fields are representative environments dominated by mesophilic to thermophilic members of the class Campylobacteria that possess clade II nosZ encoding nitrous oxide reductase. Here, we report a strain HRV44T representing the first thermophilic campylobacterium capable of growth by H2 oxidation coupled to N2O reduction. On the basis of physiological and genomic properties, it is proposed that strain HRV44T (=JCM 34002 = DSM 111345) represents a novel species of the genus Nitratiruptor, Nitratiruptor labii sp. nov. The comparison of the N2O consumption ability of strain HRV44T with those of additional Nitratiruptor and other campylobacterial strains revealed the highest level in strain HRV44T and suggests the N2O-respiring metabolism might be the common physiological trait for the genus Nitratiruptor. Our findings provide insights into contributions of thermophilic Campylobacteria to the N2O sink in deep-sea hydrothermal environments.

Ecogenomics of the Marine Benthic Filamentous Cyanobacterium Adonisia
Juline M. Walter, Felipe H. Coutinho, Luciana Leomil, Paulo I. Hargreaves, Mariana E. Campeao, Veronica V. Vieira, Beatriz S. Silva, Giovana O. Fistarol, Paulo S. Salomon, Tomoo Sawabe, Sayaka Mino, Masashi Hosokawa, Hideaki Miyashita, Fumito Maruyama, Marcel C. van Verk, Bas E. Dutilh, Cristiane C. Thompson, Fabiano L. Thompson
MICROBIAL ECOLOGY, 80, 2, 249, 265, SPRINGER, 2020年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Turfs are among the major benthic components of reef systems worldwide. The nearly complete genome sequences, basic physiological characteristics, and phylogenomic reconstruction of two phycobiliprotein-rich filamentous cyanobacteria strains isolated from turf assemblages from the Abrolhos Bank (Brazil) are investigated. Both Adonisia turfae CCMR0081(T) (= CBAS 745(T)) and CCMR0082 contain approximately 8 Mbp in genome size and experiments identified that both strains exhibit chromatic acclimation. Whereas CCMR0081(T) exhibits chromatic acclimation type 3 (CA3) regulating both phycocyanin (PC) and phycoerythrin (PE), CCMR0082 strain exhibits chromatic acclimation type 2 (CA2), in correspondence with genes encoding specific photosensors and regulators for PC and PE. Furthermore, a high number and diversity of secondary metabolite synthesis gene clusters were identified in both genomes, and they were able to grow at high temperatures (28 degrees C, with scant growth at 30 degrees C). These characteristics provide insights into their widespread distribution in reef systems.

Genomic characterization of closely related species in the Rumoiensis clade infers ecogenomic signatures to non-marine environments
Mami Tanaka, Daiki Kumakura, Sayaka Mino, Hidetaka Doi, Yoshitoshi Ogura, Tetsuya Hayashi, Iseo Yumoto, Man Cai, Yu-Guang Zhou, Bruno Gomez-Gil, Toshiyoshi Araki, Tomoo Sawabe
ENVIRONMENTAL MICROBIOLOGY, 22, 8, 3205, 3217, WILEY, 2020年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Members of the family Vibrionaceae are generally found in marine and brackish environments, playing important roles in nutrient cycling. The Rumoiensis clade is an unconventional group in the genus Vibrio, currently comprising six species from different origins including two species isolated from non-marine environments. In this study, we performed comparative genome analysis of all six species in the clade using their complete genome sequences. We found that two non-marine species, Vibrio casei and Vibrio gangliei, lacked the genes responsible for algal polysaccharide degradation, while a number of glycoside hydrolase genes were enriched in these two species. Expansion of insertion sequences was observed in V. casei and Vibrio rumoiensis, which suggests ongoing genomic changes associated with niche adaptations. The genes responsible for the metabolism of glucosylglycerate, a compound known to play a role as compatible solutes under nitrogen limitation, were conserved across the clade. These characteristics, along with genes encoding species-specific functions, may reflect the habit expansion which has led to the current distribution of Rumoiensis clade species. Genome analysis of all species in a single clade give us valuable insights into the genomic background of the Rumoiensis clade species and emphasize the genomic diversity and versatility of Vibrionaceae.

Nitrosophilus alvini gen. nov., sp. nov., a hydrogen-oxidizing chemolithoautotroph isolated from a deep-sea hydrothermal vent in the East Pacific Rise, inferred by a genome-based taxonomy of the phylum "Campylobacterota".
Taiki Shiotani, Sayaka Mino, Wakana Sato, Sayo Nishikawa, Masanori Yonezawa, Stefan M Sievert, Tomoo Sawabe
PloS one, 15, 12, e0241366, PUBLIC LIBRARY SCIENCE, 2020年, ［国際誌］
英語, 研究論文（学術雑誌）, A novel bacterium, strain EPR55-1T, was isolated from a deep-sea hydrothermal vent on the East Pacific Rise. The cells were motile rods. Growth was observed at temperatures between 50 and 60°C (optimum, 60°C), at pH values between 5.4 and 8.6 (optimum, pH 6.6) and in the presence of 2.4-3.2% (w/v) NaCl (optimum, 2.4%). The isolate used molecular hydrogen as its sole electron donor, carbon dioxide as its sole carbon source, ammonium as its sole nitrogen source, and thiosulfate, sulfite (0.01 to 0.001%, w/v) or elemental sulfur as its sole sulfur source. Nitrate, nitrous oxide (33%, v/v), thiosulfate, molecular oxygen (0.1%, v/v) or elemental sulfur could serve as the sole electron acceptor to support growth. Phylogenetic analyses based on both 16S rRNA gene sequences and whole genome sequences indicated that strain EPR55-1T belonged to the family Nitratiruptoraceae of the class "Campylobacteria", but it had the distinct phylogenetic relationship with the genus Nitratiruptor. On the basis of the physiological and molecular characteristics of the isolate, the name Nitrosophilus alvini gen. nov. sp. nov. is proposed, with EPR55-1T as the type strain (= JCM 32893T = KCTC 15925T). In addition, it is shown that "Nitratiruptor labii" should be transferred to the genus Nitrtosophilus; the name Nitrosophilus labii comb. nov. (JCM 34002T = DSM 111345T) is proposed for this organism. Furthermore, 16S rRNA gene-based and genome-based analyses showed that Cetia pacifica is phylogenetically associated with Caminibacter species. We therefore propose the reclassification of Cetia pacifica as Caminibacter pacificus comb. nov. (DSM 27783T = JCM 19563T). Additionally, AAI thresholds for genus classification and the reclassification of subordinate taxa within "Campylobacteria" are also evaluated, based on the analyses using publicly available genomes of all the campylobacterial species.

Tracking the dynamics of individual gut microbiome of sea cucumber Apostichopus japonicus during gut regeneration.
Yohei Yamazaki, Yuichi Sakai, Juanwen Yu, Sayaka Mino, Tomoo Sawabe
PeerJ, 8, e10260, PEERJ INC, 2020年, ［国際誌］
英語, 研究論文（学術雑誌）, Sea cucumbers possess the remarkable capacity to regenerate their body parts or organs. Regeneration of host organs and/or body parts involves reconstruction of the host associated microbiota, however, the dynamics and contribution of microbiota to the regeneration process are largely unknown due to a lack of experimental models. To track the dynamics of individual gut microbiomes during gut regeneration, both caged mariculture and laboratory isolator systems of sea cucumbers (Apostichopus japonicus) were developed and longitudinal meta16S analyses were performed. Under natural environmental conditions in the caged mariculture system, both bacterial and eukaryotic communities in sea cucumbers' guts appeared to be reconstructed within 4 months after evisceration. Using the laboratory isolator, which can trace daily dynamics, we found that fecal microbiota collected before evisceration were clearly different from those collected after evisceration. We also identified eight key bacteria, belonging to Alteromonadaceae, Rhodobacteraceae, Oceanospirillaceae and family-unassigned Gammaproteobacteria, suggesting that these bacteria might interact with the host during the gut regeneration process. Six of the eight key bacteria were isolated for further bioassay using the isolator developed in this study to test whether these isolates affect gut regeneration.

Vibrio taketomensis sp. nov. by genome taxonomy
Mami Tanaka, Bi Hongyu, Chunqi Jiang, Sayaka Mino, Pedro Milet Meirelles, Fabiano Thompson, Bruno Gomez-Gil, Tomoo Sawabe
SYSTEMATIC AND APPLIED MICROBIOLOGY, 43, 1, 126048, 126048, ELSEVIER GMBH, 2020年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Two novel strains C4III282(T) and C4III291 were isolated from seawater collected a site off the Taketomi coral reef. Phylogenetic analysis based on the I 6 rRNA sequences revealed that the two strains belong to the genus Vibrio. MLSA using eight protein-coding genes (ftsZ, gapA, gyrB, mreB, pyres, recA, rpoA, and topA) showed that C4III282(T) and C4III291 are closely related to the members of the Ponticus Glade, namely Vibrio panuliri JCM I9500(T), Vibrio ponticus DSM 16217(T), and "Vibrio rhodolitus" G98. ANI and in silico DDH values with members of the Ponticus Glade were 77.6-78.7% and 22.2-23.1, respectively. The name Vibrio taketomensis sp. nov. is proposed with C4III282(T) (CAIM 1928(T)=DSM 106943(T)=JCM 33434(T)) as the type strain. (C) 2019 Elsevier GmbH. All rights reserved.

Repeated selective enrichment process of sediment microbiota occurred in sea cucumber guts
Yohei Yamazaki, Yuichi Sakai, Sayaka Mino, Wataru Suda, Masahira Hattori, Pedro Milet Meirelles, Fabiano Thompson, Tomoo Sawabe
ENVIRONMENTAL MICROBIOLOGY REPORTS, 11, 6, 797, 807, WILEY, 2019年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Deposit-feeding sea cucumbers repeat ingestion of sediments and excretion of faeces daily and consequently increase bacterial abundance in sediments and promote organic matter mineralization. Such ecological roles are expected to be collaborative activities of sea cucumbers and the gut microbiota. Here, we performed a spatiotemporally broad 16S rRNA gene analysis using 109 samples from sea cucumber faeces and habitat sediments to explore potential contribution of their gut microbiota to the ecological roles. Most operational taxonomic units (OTUs) observed in the faecal samples were shared with the sediment samples, nevertheless faecal and sediment microbiota differed from each other in UniFrac analysis. Lower bacterial diversity and increased relative abundance of specific OTUs in the faecal microbiota strongly suggest selective enrichment of ingested sediment microbiota in their guts. Interestingly, representative faecal OTUs were more abundant in sea cucumber-populated sediments than in un-inhabited sediments, indicating bacteria selectively enriched in the guts were spread on ambient sediments via faeces. Moreover, the predicted microbial community metabolic potential showed a higher abundance of genes related to carbohydrate and xenobiotics metabolisms in faeces than in sediments. Our study suggests the repeated selective enrichment transforms ambient sediment microbial communities and maintains the host's ecological roles by promoting organic matter mineralization.

Recent update on the prevalence of Vibrio species among cultured grouper in Peninsular Malaysia
Nor Zulkiply Amalina, Zulperi Dzarifah, Mohammad Noor Azmai Amal, Mohd Termizi Yusof, Mohd Zamri-Saad, Nurhidayu Al-saari, Mami Tanaka, Sayaka Mino, Tomoo Sawabe, Md Yasin Ina-Salwany
Aquaculture Research, 50, 11, 3202, 3210, Blackwell Publishing Ltd, 2019年11月01日
英語, 研究論文（学術雑誌）, Vibrio infections are common among marine fish and lead to serious problems in the aquaculture sector. This study reports a recent occurrence of Vibrio species (spp.) isolated from cultured groupers in Peninsular Malaysia using the gyrB and pyrH genes. A total of 147 Vibrio strains were successfully isolated from 77 (64%) groupers using culture method and subjected to gyrB and pyrH sequencing for species identification and confirmation. Results showed that 89% of Vibrio strains were identified and clustered to six groups of Vibrio spp., while 11% were not clustered to any Vibrio spp. using the gyrB sequences. Meanwhile, by analysis of the pyrH sequences all the 147 Vibrio strains (100%) were successfully identified and clustered into 11 groups of Vibrio spp., including the gyrB non-identified strains. The pyrH gene provides a better resolution for identification of Vibrio spp. compared with the gyrB gene. Thus, the pyrH gene was more suitable for a rapid determination of Vibrio spp. distribution in Peninsular Malaysia. Using the pyrH gene, our study found higher prevalence of Vibrio vulnificus (33%), V. alginolyticus (24%) and V. parahaemolyticus (22%), followed by V. rotiferianus (5%), V. harveyi (3%), V. tubiashii (2%), V. campbellii (2%), V. ponticus (1%), V. diabolicus (1%), V. owensii (1%) and others Vibrio sp. (7%). Thus, the results of this study revealed that the occurrence of pathogenic vibrios among grouper fish is still high in Malaysian aquaculture. In addition, the pyrH gene was proved as a suitable marker for rapid identification of Vibrio species compared with the gyrB gene.

Vibriosis in Fish: A Review on Disease Development and Prevention.
M Y Ina-Salwany, Nurhidayu Al-Saari, Aslah Mohamad, Fathin-Amirah Mursidi, Aslizah Mohd-Aris, M N A Amal, Hisae Kasai, Sayaka Mino, Tomoo Sawabe, M Zamri-Saad
Journal of aquatic animal health, 31, 1, 3, 22, 2019年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Current growth in aquaculture production is parallel with the increasing number of disease outbreaks, which negatively affect the production, profitability, and sustainability of the global aquaculture industry. Vibriosis is among the most common diseases leading to massive mortality of cultured shrimp, fish, and shellfish in Asia. High incidence of vibriosis can occur in hatchery and grow-out facilities, but juveniles are more susceptible to the disease. Various factors, particularly the source of fish, environmental factors (including water quality and farm management), and the virulence factors of Vibrio, influence the occurrence of the disease. Affected fish show weariness, with necrosis of skin and appendages, leading to body malformation, slow growth, internal organ liquefaction, blindness, muscle opacity, and mortality. A combination of control measures, particularly a disease-free source of fish, biosecurity of the farm, improved water quality, and other preventive measures (e.g., vaccination) might be able to control the infection. Although some control measures are expensive and less practical, vaccination is effective, relatively cheap, and easily implemented. In this review, the latest knowledge on the pathogenesis and control of vibriosis, including vaccination, is discussed.

Understanding the NaCl-dependent behavior of hydrogen production of a marine bacterium, Vibrio tritonius.
Nurhidayu Al-Saari, Eri Amada, Yuta Matsumura, Mami Tanaka, Sayaka Mino, Tomoo Sawabe
PeerJ, 7, e6769, 2019年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Biohydrogen is one of the most suitable clean energy sources for sustaining a fossil fuel independent society. The use of both land and ocean bioresources as feedstocks show great potential in maximizing biohydrogen production, but sodium ion is one of the main obstacles in efficient bacterial biohydrogen production. Vibrio tritonius strain AM2 can perform efficient hydrogen production with a molar yield of 1.7 mol H2/mol mannitol, which corresponds to 85% theoretical molar yield of H2 production, under saline conditions. With a view to maximizing the hydrogen production using marine biomass, it is important to accumulate knowledge on the effects of salts on the hydrogen production kinetics. Here, we show the kinetics in batch hydrogen production of V. tritonius strain AM2 to investigate the response to various NaCl concentrations. The modified Han-Levenspiel model reveals that salt inhibition in hydrogen production using V. tritonius starts precisely at the point where 10.2 g/L of NaCl is added, and is critically inhibited at 46 g/L. NaCl concentration greatly affects the substrate consumption which in turn affects both growth and hydrogen production. The NaCl-dependent behavior of fermentative hydrogen production of V. tritonius compared to that of Escherichia coli JCM 1649 reveals the marine-adapted fermentative hydrogen production system in V. tritonius. V. tritonius AM2 is capable of producing hydrogen from seaweed carbohydrate under a wide range of NaCl concentrations (5 to 46 g/L). The optimal salt concentration producing the highest levels of hydrogen, optimal substrate consumption and highest molar hydrogen yield is at 10 g/L NaCl (1.0% (w/v)).

Availability of Nanopore sequences in the genome taxonomy for Vibrionaceae systematics: Rumoiensis Glade species as a test case
Mami Tanaka, Sayaka Mino, Yoshitoshi Ogura, Tetsuya Hayashi, Tomoo Sawabe
PEERJ, 6, e5018, PEERJ INC, 2018年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Whole genome sequence comparisons have become essential for establishing a robust scheme in bacterial taxonomy. To generalize this genome-based taxonomy, fast, reliable, and cost-effective gnome sequencing methodologies are required. MinION, the palm -sized sequencer from Oxford Nanopore Technologies, enables rapid sequencing of bacterial genomes using minimal laboratory resources. Here we tested the ability of Nanopore sequences for the genome-based taxonomy of Vibrionaceae and compared Nanopore- only assemblies to complete gnomes of fiw Rumoiensis clade species: Vibrio aphrogenes, V. algivorus, V. casei, V. litoralis, and V. rumoiensis. Comparison of overall gnome relatedness indices (OGRI) and multilocus sequence analysis (MLSA) based on Nanopore-only assembly and Illumina or hybrid assemblies revealed that errors in Nanopore-only assembly do not Influence average nucleotide identity (ANI), in silico DNA -DNA hybridization (DDH), G+C content, or MLSA tree topology in Vibrionaceae. Our results show that the genome sequences frorn Nanopore-based approach can be used for rapid species identification based on the OGRI and MLSA.

Corrigendum to "Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. within the family Vibrionaceae, isolated from coral reef seawater off Ishigaki Island, Japan" [Syst. Appl. Microbiol. 40 (5) (2017) 290-296].
A K M Rohul Amin, Mami Tanaka, Nurhidayu Al-Saari, Gao Feng, Sayaka Mino, Yoshitoshi Ogura, Tetsuya Hayashi, Pedro M Meirelles, Fabiano L Thompson, Bruno Gomez-Gil, Toko Sawabe, Tomoo Sawabe
Systematic and applied microbiology, 41, 1, 62, 63, 2018年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Enrichment and Genomic Characterization of a N2O-Reducing Chemolithoautotroph From a Deep-Sea Hydrothermal Vent.
Sayaka Mino, Naoki Yoneyama, Satoshi Nakagawa, Ken Takai, Tomoo Sawabe
Frontiers in bioengineering and biotechnology, 6, 184, 184, 2018年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Nitrous oxide (N2O) is a greenhouse gas and also leads to stratospheric ozone depletion. In natural environments, only a single N2O sink process is the microbial reduction of N2O to N2, which is mediated by nitrous oxide reductase (NosZ) encoded by nosZ gene. The nosZ phylogeny has two distinct clades, clade I and formerly overlooked clade II. In deep-sea hydrothermal environments, several members of the class Campylobacteria are shown to harbor clade II nosZ gene and perform the complete denitrification of nitrate to N2; however, little is known about their ability to grow on exogenous N2O as the sole electron acceptor. Here, we obtained an enrichment culture from a deep-sea hydrothermal vent in the Southern Mariana Trough, which showed a respiratory N2O reduction with H2 as an electron donor. The single amplicon sequence variant (ASV) presenting 90% similarity to Hydrogenimonas species within the class Campylobacteria was predominant throughout the cultivation period. Metagenomic analyses using a combination of short-read and long-read sequence data succeeded in reconstructing a complete genome of the dominant ASV, which encoded clade II nosZ gene. This study represents the first cultivation analysis that shows the occurrence of N2O-respiring microorganisms in a deep-sea hydrothermal vent and provides the opportunity to assess their capability to reduce N2O emission from the environments.

Metagenomics of Coral Reefs Under Phase Shift and High Hydrodynamics.
Pedro Milet Meirelles, Ana Carolina Soares, Louisi Oliveira, Luciana Leomil, Luciana Reis Appolinario, Ronaldo Bastos Francini-Filho, Rodrigo Leão de Moura, Renato Tenan de Barros Almeida, Paulo S Salomon, Gilberto Menezes Amado-Filho, Ricardo Kruger, Eduardo Siegle, Diogo A Tschoeke, Isao Kudo, Sayaka Mino, Tomoo Sawabe, Cristiane C Thompson, Fabiano L Thompson
Frontiers in microbiology, 9, 2203, 2203, 2018年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Local and global stressors have affected coral reef ecosystems worldwide. Switches from coral to algal dominance states and microbialization are the major processes underlying the global decline of coral reefs. However, most of the knowledge concerning microbialization has not considered physical disturbances (e.g., typhoons, waves, and currents). Southern Japan reef systems have developed under extreme physical disturbances. Here, we present analyses of a three-year investigation on the coral reefs of Ishigaki Island that comprised benthic and fish surveys, water quality analyses, metagenomics and microbial abundance data. At the four studied sites, inorganic nutrient concentrations were high and exceeded eutrophication thresholds. The dissolved organic carbon (DOC) concentration (up to 233.3 μM) and microbial abundance (up to 2.5 × 105 cell/mL) values were relatively high. The highest vibrio counts coincided with the highest turf cover (∼55-85%) and the lowest coral cover (∼4.4-10.2%) and fish biomass (0.06 individuals/m2). Microbiome compositions were similar among all sites and were dominated by heterotrophs. Our data suggest that a synergic effect among several regional stressors are driving coral decline. In a high hydrodynamics reef environment, high algal/turf cover, stimulated by eutrophication and low fish abundance due to overfishing, promote microbialization. Together with crown-of-thorns starfish (COTS) outbreaks and possible of climate changes impacts, theses coral reefs are likely to collapse.

Corrigendum to "Vibrio ishigakensis sp. nov., in Halioticoli clade isolated from seawater in Okinawa coral reef area, Japan" [Syst. Appl. Microbiol. 39 (5) (2016) 330-335].
Feng Gao, Nurhidayu Al-Saari, A K M Rohul Amin, Kazumichi Sato, Sayaka Mino, Wataru Suda, Kenshiro Oshima, Masahira Hattori, Moriya Ohkuma, Paulo Iiboshi Hargreaves, Pedro Milet Meirelles, Fabiano L Thompson, Cristiane Thompson, Bruno Gomez-Gil, Toko Sawabe, Tomoo Sawabe
Systematic and applied microbiology, 40, 8, 516, 516, 2017年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed (vol 12, e0180053, 2017)
Mami Tanaka, Shoko Endo, Fumihito Kotake, Nurhidayu Al-Saari, A. K. M. Rohul Amin, Gao Feng, Sayaka Mino, Hidetaka Doi, Yoshitoshi Ogura, Tetsuya Hayashi, Wataru Suda, Masahira Hattori, Isao Yumoto, Toko Sawabe, Tomoo Sawabe, Toshiyoshi Araki
PLOS ONE, 12, 12, e0189555, PUBLIC LIBRARY SCIENCE, 2017年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, [This corrects the article DOI: 10.1371/journal.pone.0180053.].

Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. within the family Vibrionaceae, isolated from coral reef seawater off Ishigaki Island, Japan.
A K M Rohul Amin, Mami Tanaka, Nurhidayu Al-Saari, Gao Feng, Sayaka Mino, Yoshitoshi Ogura, Tetsuya Hayashi, Pedro M Meirelles, Fabiano L Thompson, Bruno Gomez-Gil, Toko Sawabe, Tomoo Sawabe
Systematic and applied microbiology, 40, 5, 290, 296, ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2017年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Two phylogenetically distinct Vibrionaceae strains C4II189T and C4V358T isolated from reef seawater off Ishigaki Island, Japan, in 2014 were studied with advanced genome-based taxonomy approaches. All aspects of phylogenetic (16S rRNA phylogeny, MLSA), phenotypic and genetic (ANI, DDH, AAI, and the number of core genes) cohesions between the two identified species were high enough to propose them as members of a new genus within the family Vibrionaceae. Consequently, an eighth genus Thaumasiovibrio gen. nov. is proposed that contains two new species Thaumasiovibrio occultus sp. nov. strain C4II189T (=DSM 101554T=JCM 31629T) (type species) and Thaumasiovibrio subtropicus sp. nov. strain C4V358T (=DSM 101555T=JCM 31630T). Thaumasiovibrio species were phylogenetically distinct from the other Vibrionaceae species based on pyrH gene sequences. The combination of catalase negative, sensitivity to vibriostatic agent O/129, and green colony formation on TCBS for the phylogenetically affiliated strains was the diagnostic features for the current tentative identification of this genus.

Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed
Mami Tanaka, Shoko Endo, Fumihito Kotake, Nurhidayu Al-Saari, A. K. M. Rohul Amin, Gao Feng, Sayaka Mino, Hidetaka Doi, Yoshitoshi Ogura, Tetsuya Hayashi, Wataru Suda, Masahira Hattori, Isao Yumoto, Toko Sawabe, Tomoo Sawabe, Toshiyoshi Araki
PLOS ONE, 12, 6, e0180053, PUBLIC LIBRARY SCIENCE, 2017年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A novel strain Vibrio aphrogenes sp. nov. strain CA-1004(T) isolated from the surface of seaweed collected on the coast of Mie Prefecture in 1994 [1] was characterized using poly phasic taxonomy including multilocus sequence analysis (MLSA) and a genome based comparison. Both phylogenetic analyses on the basis of 16S rRNA gene sequences and MLSA based on eight protein-coding genes (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA) showed the strain could be placed in the Rumoiensis Glade in the genus Vibrio. Sequence similarities of the 16S rRNA gene and the multilocus genes against the Rumoiensis Glade members, V. rumoiensis, V. algivorus, V. casei, and V. litoralis, were low enough to propose V. aphrogenes sp. nov. strain CA-1004(T) as a separate species. The experimental DNA-DNA hybridization data also revealed that the strain CA-1004(T) was separate from four known Rumoiensis Glade species. The G+C content of the V. aphrogenes strain was determined as 42.1% based on the genome sequence. Major traits of the strain were non motile, halophilic, fermentative, alginolytic, and gas production. A total of 27 traits (motility, growth temperature range, amylase, alginase and lipase productions, and assimilation of 19 carbon compounds) distinguished the strain from the other species in the Rumoiensis Glade. The name V. aphrogenes sp. nov. is proposed for this species in the Rumoiensis Glade, with CA-1004(T) as the type strain (JCM 31643(T) = DSM 103759(T)).

International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Aeromonadaceae, Vibrionaceae and related organisms  Minutes of the meetings, 31 March 2016, Roscoff, France.
Tomoo Sawabe, James D Oliver
International journal of systematic and evolutionary microbiology, 67, 3, 759, 760, 2017年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Endemicity of the cosmopolitan mesophilic chemolithoautotroph Sulfurimonas at deep-sea hydrothermal vents.
Sayaka Mino, Satoshi Nakagawa, Hiroko Makita, Tomohiro Toki, Junichi Miyazaki, Stefan M Sievert, Martin F Polz, Fumio Inagaki, Anne Godfroy, Shingo Kato, Hiromi Watanabe, Takuro Nunoura, Koichi Nakamura, Hiroyuki Imachi, Tomo-O Watsuji, Shigeaki Kojima, Ken Takai, Tomoo Sawabe
The ISME journal, 11, 4, 909, 919, NATURE PUBLISHING GROUP, 2017年04月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Rich animal and microbial communities have been found at deep-sea hydrothermal vents. Although the biogeography of vent macrofauna is well understood, the corresponding knowledge about vent microbial biogeography is lacking. Here, we apply the multilocus sequence analysis (MLSA) to assess the genetic variation of 109 Sulfurimonas strains with ⩾98% 16S rRNA gene sequence similarity, which were isolated from four different geographical regions (Okinawa Trough (OT), Mariana Volcanic Arc and Trough (MVAT), Central Indian Ridge (CIR) and Mid-Atlantic Ridge (MAR)). Sequence typing based on 11 protein-coding genes revealed high genetic variation, including some allele types that are widespread within regions, resulting in 102 nucleotide sequence types (STs). This genetic variation was predominantly due to mutation rather than recombination. Phylogenetic analysis of the 11 concatenated genes showed a clear geographical isolation corresponding to the hydrothermal regions they originated from, suggesting limited dispersal. Genetic differentiation among Sulfurimonas populations was primarily influenced by geographical distance rather than gas composition of vent fluid or habitat, although in situ environmental conditions of each microhabitat could not be examined. Nevertheless, Sulfurimonas may possess a higher dispersal capability compared with deep-sea hydrothermal vent thermophiles. This is the first report on MLSA of deep-sea hydrothermal vent Epsilonproteobacteria, which is indicative of allopatric speciation.

Correction: Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed.
Mami Tanaka, Shoko Endo, Fumihito Kotake, Nurhidayu Al-Saari, A K M Rohul Amin, Gao Feng, Sayaka Mino, Hidetaka Doi, Yoshitoshi Ogura, Tetsuya Hayashi, Wataru Suda, Masahira Hattori, Isao Yumoto, Toko Sawabe, Tomoo Sawabe, Toshiyoshi Araki
PloS one, 12, 12, e0189555, PUBLIC LIBRARY SCIENCE, 2017年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, [This corrects the article DOI: 10.1371/journal.pone.0180053.].

Microbial biofuels: Potential and challenges
Nurhidayu Al-saari, Tomoo Sawabe, Yuta Matsumura, Kazumichi Sato, Sayaka Mino, Toko Sawabe
Molecular Diversity of Environmental Prokaryotes, 285, 294, CRC Press, 2016年08月19日
英語, 論文集(書籍)内論文

Vibrio ishigakensis sp. nov., in Halioticoli clade isolated from seawater in Okinawa coral reef area, Japan.
Feng Gao, Nurhidayu Al-Saari, A K M Rohul Amin, Kazumichi Sato, Sayaka Mino, Wataru Suda, Kenshiro Oshima, Masahira Hattori, Moriya Ohkuma, Paulo Iiboshi Hargreaves, Pedro Milet Meirelles, Fabiano L Thompson, Cristiane Thompson, Bruno Gomez-Gil, Toko Sawabe, Tomoo Sawabe
Systematic and applied microbiology, 39, 5, 330, 5, ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2016年07月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Five novel strains showing non-motile, alginolytic, halophilic and fermentative features were isolated from seawater samples off Okinawa in coral reef areas. These strains were characterized by an advanced polyphasic taxonomy including genome based taxonomy using multilocus sequence analysis (MLSA) and in silico DNA-DNA similarity (in silico DDH). Phylogenetic analyses on the basis of 16S rRNA gene sequences revealed that the isolates could be assigned to the genus Vibrio, however they were not allocated into any distinct cluster with known Vibrionaceae species. MLSA based on eight protein-coding genes (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA) showed the vibrios formed an outskirt branch of Halioticoli clade. The experimental DNA-DNA hybridization data revealed that the five strains were in the range of being defined as conspecific but separate from nine Halioticoli clade species. The G+C contents of the Vibrio ishigakensis strains were 47.3-49.1mol%. Both Amino Acid Identity and Average Nucleotide Identity of the strain C1(T) against Vibrio ezurae HDS1-1(T), Vibrio gallicus HT2-1(T), Vibrio halioticoli IAM 14596(T), Vibrio neonatus HDD3-1(T) and Vibrio superstes G3-29(T) showed less than 95% similarity. The genome-based taxonomic approach by means of in silico DDH values also supports the V. ishigakensis strains being distinct from the other known Halioticoli clade species. Sixteen traits (growth temperature range, DNase and lipase production, indole production, and assimilation of 10 carbon compounds) distinguished these strains from Halioticoli clade species. The names V. ishigakensis sp. nov. is proposed for the species of Halioticoli clade, with C1(T) as the type strain (JCM 19231(T)=LMG 28703(T)).

First microbiota assessments of children's paddling pool waters evaluated using 16S rRNA gene-based metagenome analysis
Toko Sawabe, Wataru Suda, Kenshiro Ohshima, Masahira Hattori, Tomoo Sawabe
JOURNAL OF INFECTION AND PUBLIC HEALTH, 9, 3, 362, 365, ELSEVIER SCIENCE LONDON, 2016年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Insufficient chloric sterilization of children's paddling pool waters increases the risk of diarrheal illness. Therefore, we investigated the micro biota changes after children use pools. First, we applied 16S rRNA gene-based metagenome analysis to understand the dynamics of microbiota in pool water, especially with respect to the bio-contamination by potential pathogens. Proteobacteria were major taxa detected in every pool water sample after children spent time in the pool. In more detail, Gammaproteobacteria comprised the dominant class, which was followed by Betaproteobacteria. Five phyla, Bacteroidetes, Firmicutes, Actinobacteria and Deinococcus-Thermus phyla were minor groups. The pool water microbiota are likely to be a consortium of intestinal and skin microbiota from humans. Interestingly, the ratio of Gammaproteobacteria and Betaproteobacteria differed according to the age of the children who used the pool, which means the pool water was additionally contaminated by soil microbiota as a result of the children's behavior. Furthermore, potential pathogens, such as Campylobacter spp., Comamonas testosteroni and Burkholderia pseudomallei, were also found. Considering the standard plate counts, the abundances of these human pathogens are unlikely to be a sufficiently infectious dose. We suggest the importance of sanitary measures in paddling pool waters to reduce bio-contamination from both humans and the environment. (C) 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Limited. All rights reserved.

Individual Apostichopus japonicus fecal microbiome reveals a link with polyhydroxybutyrate producers in host growth gaps
Yohei Yamazaki, Pedro Milet Meirelles, Sayaka Mino, Wataru Suda, Kenshiro Oshima, Masahira Hattori, Fabiano L. Thompson, Yuichi Sakai, Toko Sawabe, Tomoo Sawabe
SCIENTIFIC REPORTS, 6, 21631, 21631, NATURE PUBLISHING GROUP, 2016年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Gut microbiome shapes various aspects of a host's physiology, but these functions in aquatic animal hosts have yet to be fully investigated. The sea cucumber Apostichopus japonicus Selenka is one such example. The large growth gap in their body size has delayed the development of intensive aquaculture, nevertheless the species is in urgent need of conservation. To understand possible contributions of the gut microbiome to its host's growth, individual fecal microbiome comparisons were performed. High-throughput 16S rRNA sequencing revealed significantly different microbiota in larger and smaller individuals; Rhodobacterales in particular was the most significantly abundant bacterial group in the larger specimens. Further shotgun metagenome of representative samples revealed a significant abundance of microbiome retaining polyhydroxybutyrate (PHB) metabolism genes in the largest individual. The PHB metabolism reads were potentially derived from Rhodobacterales. These results imply a possible link between microbial PHB producers and potential growth promotion in Deuterostomia marine invertebrates.

The First Temporal and Spatial Assessment of Vibrio Diversity of the Surrounding Seawater of Coral Reefs in Ishigaki, Japan.
A K M R Amin, Gao Feng, Nurhidayu Al-Saari, Pedro M Meirelles, Yohei Yamazaki, Sayaka Mino, Fabiano L Thompson, Toko Sawabe, Tomoo Sawabe
Frontiers in microbiology, 7, 1185, 1185, 2016年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Coral reefs perform a major role in regulating marine biodiversity and serve as hotspot for highly dynamic and diverse microbiomes as holobionts. Corals around Ishigaki, however, are at risk due to tremendous stressors including elevation of seawater temperature, eutrophication and so on. However, no information is currently available on how Vibrio diversity fluctuates spatially and temporally due to environmental determinants in Ishigaki coral reef ecosystems. The aim of this study is to elucidate spatiotemporal Vibrio diversity dynamic at both community and population levels and to assess the environmental drivers correlated to Vibrio abundance and diversity. The Vibrio community identified based on pyrH gene phylogeny of 685 isolates from seawater directly connecting to Ishigaki coral holobionts consisted of 22 known and 12 potential novel Vibrionaceae species. The most prominent species were V. hyugaensis, V. owensii and V. harveyi followed by V. maritimus/V. variabillis, V. campbellii, V. coralliilyticus, and Photobacterium rosenbergii. The Vibrio community fluctuations, assessed by PCoA with UniFrac distance and clustering with Euclidiean distance were varied less not only by year but also by site. Interestingly, significant positive correlation was observed between rising seawater temperature and the abundance of V. campbellii (r = 0.62; P < 0.05) whereas the opposite was observed for V. owensii (r = -0.58; P < 0.05) and the C6 group of V. hyugaensis (r = -0.62; P < 0.05). AdaptML-based microhabitat differentiation revealed that V. harveyi, V. campbellii, P. rosenbergii, and V. coralliilyticus populations were less-ecologically distinctive whereas V. astriarenae and V. ishigakensis were ecologically diverse. This knowledge could be important clue for the future actions of coral conservation.

Effects of Hemagglutination Activity in the Serum of a Deep-Sea Vent Endemic Crab, Shinkaia Crosnieri, on Non-Symbiotic and Symbiotic Bacteria
So Fujiyoshi, Hiroaki Tateno, Tomoo Watsuji, Hideyuki Yamaguchi, Daisuke Fukushima, Sayaka Mino, Makoto Sugimura, Tomoo Sawabe, Ken Takai, Shigeki Sawayama, Satoshi Nakagawa
MICROBES AND ENVIRONMENTS, 30, 3, 228, 234, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2015年09月, ［査読有り］
英語, 研究論文（学術雑誌）, In deep-sea hydrothermal environments, most invertebrates associate with dense populations of symbiotic microorganisms in order to obtain nutrition. The molecular interactions between deep-sea animals and environmental microbes, including their symbionts, have not yet been elucidated in detail. Hemagglutinins/lectins, which are carbohydrate-binding proteins, have recently been reported to play important roles in a wide array of biological processes, including the recognition and control of non-self materials. We herein assessed hemagglutination activity in the serum of a deep-sea vent endemic crab, Shinkaia crosnieri, which harbors chemosynthetic epibionts on its plumose setae. Horse and rabbit erythrocytes were agglutinated using this serum (opt. pH 7.5 and opt. temperature 15 degrees C). Agglutinating activity was inhibited by eight kinds of sugars and several divalent cations, did not require any divalent metal ions, and remained detectable even after heating the serum at 100 degrees C for 30 min. By using fluorescently labeled serum, we demonstrated that deep-sea crab serum components bound to the epibionts even in the presence of sugars. This study represents the first immunological assessment of a deep-sea vent endemic crab and demonstrated the possibility of a non-lectin-mediated symbiont-host interaction.

Identification of a gene cluster responsible for hydrogen evolution in Vibrio tritonius strain AM2 with transcriptional analyses
Yuta Matsumura, Hidayu Al-saari, Sayaka Mino, Satoshi Nakagawa, Fumito Maruyama, Yoshitoshi Ogura, Tetsuya Hayashi, Ken Kurokawa, Toko Sawabe, Tomoo Sawabe
INTERNATIONAL JOURNAL OF HYDROGEN ENERGY, 40, 30, 9137, 9146, PERGAMON-ELSEVIER SCIENCE LTD, 2015年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Vibrio tritonius strain AM2 shows high-yield hydrogen production even under saline conditions (1.7 mol hydrogen/mol mannitol). However, the molecular mechanism of efficient hydrogen production has never been studied in the genus Vibrio. The aim of this study is to identify the genes responsible for hydrogen evolution in V. tritonius and the gene expression pattern. Complete genome analysis revealed an existence of a single 24-kb gene cluster containing 21 genes, which are essential for the formation of an energy-conserving formate hydrogen lyase (FHL) complex, to be more specific the vibrio FHL was structurally rather similar to the hyf (hydrogenase four) gene cluster found in Escherichia coli. Moreover, genes responsible to the formate dehydrogenase (FDH-H), fh1A-type transcriptional activator and hydrogenase maturation proteins (hyp) were also located downstream of the vibrio hyf gene cluster to form a "super-gene-set" of the FHL complex gene cluster. The vibrio gene for the large subunit of the FHL complex hyfG possessed typical motifs coordinating the [NiFe] center at the active site, which indicates the V. tritonius hydrogenase was able to be classified as a [NiFe]-hydrogenase. Furthermore, transcriptional analysis revealed that the expression level of the hyfG gene slightly increased upon pH decrease, which correlates to the pH-dependent hydrogen production of V. tritonius. Therefore, we can conclude that the FHL complex of V. tritonius is key enzyme in the hydrogen production under acidic conditions. Moreover hyfABCDEFGHIJ-hycI-hydN-fdhF and hyp genes could be co-transcribed respectively during the efficient hydrogen production state. Details of the gene cluster are discussed here. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Advanced Microbial Taxonomy Combined with Genome-Based-Approaches Reveals that Vibrio astriarenae sp nov., an Agarolytic Marine Bacterium, Forms a New Clade in Vibrionaceae
Nurhidayu Al-Saari, Feng Gao, Amin A. K. M. Rohul, Kazumichi Sato, Keisuke Sato, Sayaka Mino, Wataru Suda, Kenshiro Oshima, Masahira Hattori, Moriya Ohkuma, Pedro M. Meirelles, Fabiano L. Thompson, Cristiane Thompson, Gilberto M. A. Filho, Bruno Gomez-Gil, Toko Sawabe, Tomoo Sawabe
PLOS ONE, 10, 8, PUBLIC LIBRARY SCIENCE, 2015年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Advances in genomic microbial taxonomy have opened the way to create a more universal and transparent concept of species but is still in a transitional stage towards becoming a defining robust criteria for describing new microbial species with minimum features obtained using both genome and classical polyphasic taxonomies. Here we performed advanced microbial taxonomies combined with both genome-based and classical approaches for new agarolytic vibrio isolates to describe not only a novel Vibrio species but also a member of a new Vibrio clade. Two novel vibrio strains (Vibrio astriarenae sp. nov. C7(T) and C20) showing agarolytic, halophilic and fermentative metabolic activity were isolated from a seawater sample collected in a coral reef in Okinawa. Intraspecific similarities of the isolates were identical in both sequences on the 16S rRNA and pyrH genes, but the closest relatives on the molecular phylogenetic trees on the basis of 16S rRNA and pyrH gene sequences were V. hangzhouensis JCM 15146(T) (97.8% similarity) and V. agarivorans CECT 5085(T) (97.3% similarity), respectively. Further multilocus sequence analysis (MLSA) on the basis of 8 protein coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) obtained by the genome sequences clearly showed the V. astriarenae strain C7(T) and C20 formed a distinct new clade protruded next to V. agarivorans CECT 5085(T). The singleton V. agarivorans has never been included in previous MLSA of Vibrionaceae due to the lack of some gene sequences. Now the gene sequences are completed and analysis of 100 taxa in total provided a clear picture describing the association of V. agarivorans into pre-existing concatenated network tree and concluded its relationship to our vibrio strains. Experimental DNA-DNA hybridization (DDH) data showed that the strains C7(T) and C20 were conspecific but were separated from all of the other Vibrio species related on the basis of both 16S rRNA and pyrH gene phylogenies (e.g., V. agarivorans CECT 5085(T), V. hangzhouensis JCM 15146(T) V. maritimus LMG 25439(T), and V. variabilis LMG 25438(T)). In silico DDH data also supported the genomic relationship. The strains C7(T) also had less than 95% average amino acid identity (AAI) and average nucleotide identity (ANI) towards V. maritimus C210, V. variabilis C206, and V. mediterranei AK1(T), V. brasiliensis LMG 20546(T), V. orientalis ATCC 33934(T), and V. sinaloensis DSM 21326. The name Vibrio astriarenae sp. nov. is proposed with C7 as the type strains. Both V. agarivorans CECT 5058(T) and V. astriarenae C7(T) are members of the newest clade of Vibrionaceae named Agarivorans.

Baseline Assessment of Mesophotic Reefs of the Vitoria-Trindade Seamount Chain Based on Water Quality, Microbial Diversity, Benthic Cover and Fish Biomass Data
Pedro M. Meirelles, Gilberto M. Amado-Filho, Guilherme H. Pereira-Filho, Hudson T. Pinheiro, Rodrigo L. de Moura, Jean-Christophe Joyeux, Eric F. Mazzei, Alex C. Bastos, Robert A. Edwards, Elizabeth Dinsdale, Rodolfo Paranhos, Eidy O. Santos, Tetsuya Iida, Kazuyoshi Gotoh, Shota Nakamura, Tomoo Sawabe, Carlos E. Rezende, Luiz M. R. Gadelha, Ronaldo B. Francini-Filho, Cristiane Thompson, Fabiano L. Thompson
PLOS ONE, 10, 6, PUBLIC LIBRARY SCIENCE, 2015年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Seamounts are considered important sources of biodiversity and minerals. However, their biodiversity and health status are not well understood; therefore, potential conservation problems are unknown. The mesophotic reefs of the Vitoria-Trindade Seamount Chain (VTC) were investigated via benthic community and fish surveys, metagenomic and water chemistry analyses, and water microbial abundance estimations. The VTC is a mosaic of reef systems and includes fleshy algae dominated rhodolith beds, crustose coralline algae (CCA) reefs, and turf algae dominated rocky reefs of varying health levels. Macro-carnivores and larger fish presented higher biomass at the CCA reefs (4.4 kg per frame) than in the rhodolith beds and rocky reefs (0.0 to 0.1 kg per frame). A larger number of metagenomic sequences identified as primary producers (e.g., Chlorophyta and Streptophyta) were found at the CCA reefs. However, the rocky reefs contained more diseased corals (>90%) than the CCA reefs (similar to 40%) and rhodolith beds (similar to 10%). Metagenomic analyses indicated a heterotrophic and fast-growing microbiome in rocky reef corals that may possibly lead to unhealthy conditions possibly enhanced by environmental features (e.g. light stress and high loads of labile dissolved organic carbon). VTC mounts represent important hotspots of biodiversity that deserve further conservation actions.

Microbial taxonomy in the post-genomic era: Rebuilding from scratch?
Cristiane C. Thompson, Gilda R. Amaral, Mariana Campeao, Robert A. Edwards, Martin F. Polz, Bas E. Dutilh, David W. Ussery, Tomoo Sawabe, Jean Swings, Fabiano L. Thompson
ARCHIVES OF MICROBIOLOGY, 197, 3, 359, 370, SPRINGER, 2015年04月, ［査読有り］
英語, Microbial taxonomy should provide adequate descriptions of bacterial, archaeal, and eukaryotic microbial diversity in ecological, clinical, and industrial environments. Its cornerstone, the prokaryote species has been re-evaluated twice. It is time to revisit polyphasic taxonomy, its principles, and its practice, including its underlying pragmatic species concept. Ultimately, we will be able to realize an old dream of our predecessor taxonomists and build a genomic-based microbial taxonomy, using standardized and automated curation of high-quality complete genome sequences as the new gold standard.

Structure of a novel monocyclic carotenoid, 3 ''-hydroxy-2 '-isopentenylsaproxanthin ((3R,2 ' S)-2 -(3-hydroxy-3 -methylbuty1)-3 ', 4 '-didehydro-1 ', 2 '-dihydro-beta, Psi-carotene-3, 1 '-diol), from a flavobacterium Gillisia limnaea strain DSM 15749
Naoki Takatani, Tomoo Sawabe, Takashi Maoka, Kazuo Miyashita, Masashi Hosokawa
BIOCATALYSIS AND AGRICULTURAL BIOTECHNOLOGY, 4, 2, 174, 179, ELSEVIER SCIENCE BV, 2015年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Gillisia lirtinaea strain DSM 15749 (=R-8282(T), =LMG21470,=CIP108418) is a Gram-negative, strictly aerobic, psychrophilic, and non-photosynthetic bacterium that belongs to the family Flavobacteriaceae, which was isolated from a microbial mat in Lake Fryxell, Antarctica (Van. Trapp en eI. al., 2004). The bacteriurn shows yellow colored colony, but the cat otenoid composition has never been determined yet. In this study, we elucidated the structure of carotenoids produced by the strain DSM 15749. G. lirtinaea strain DSM 15749 synthesized a novel monocyclic-type carotenoid, 3 ''-hydroxy-2'-isopentenylsaproxanthin ((3R, 2'S')-2 '-(3-hydroxy-3-methylbutyl)-3 '', 4'-didehydro-1', 2'-dihydro-beta, Psi-carotene-3, 1'-diol), as well as (3R, TR)-zeaxanthin. Furthermore, another monocyclic carotenoid, 2'-isopentenylsaproxanthin, was also detected in the strain DSM 15749. 3 ''-Hydroxy-2'-isopentenylsaproxanthin is "Chimera'like unique structures, in that one end group forms the same structure of zeaxanthin, and the other end group is the same as bacterioruberin which is produced by several extremophilic bacteria and archaea. This structure involved the hydroxy derivative of 2'-isopentenylsaproxanthin. The present study is the First report about the carotenoids produced by the genus Gillisto, of the family Flavobacteriaceae. (C) 2015 Elsevier Ltd. All rights reserved.

Microbial community diversity and physical-chemical features of the Southwestern Atlantic Ocean
Nelson Alves Junior, Pedro Milet Meirelles, Eidy de Oliveira Santos, Bas Dutilh, Genivaldo G. Z. Silva, Rodolfo Paranhos, Anderson S. Cabral, Carlos Rezende, Tetsuya Iida, Rodrigo L. de Moura, Ricardo Henrique Kruger, Renato C. Pereira, Rogerio Valle, Tomoo Sawabe, Cristiane Thompson, Fabiano Thompson
ARCHIVES OF MICROBIOLOGY, 197, 2, 165, 179, SPRINGER, 2015年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Microbial oceanography studies have demonstrated the central role of microbes in functioning and nutrient cycling of the global ocean. Most of these former studies including at Southwestern Atlantic Ocean (SAO) focused on surface seawater and benthic organisms (e.g., coral reefs and sponges). This is the first metagenomic study of the SAO. The SAO harbors a great microbial diversity and marine life (e.g., coral reefs and rhodolith beds). The aim of this study was to characterize the microbial community diversity of the SAO along the depth continuum and different water masses by means of metagenomic, physical-chemical and biological analyses. The microbial community abundance and diversity appear to be strongly influenced by the temperature, dissolved organic carbon, and depth, and three groups were defined [1. surface waters; 2. sub-superficial chlorophyll maximum (SCM) (48-82 m) and 3. deep waters (236-1,200 m)] according to the microbial composition. The microbial communities of deep water masses [South Atlantic Central water, Antarctic Intermediate water and Upper Circumpolar Deep water] are highly similar. Of the 421,418 predicted genes for SAO metagenomes, 36.7 % had no homologous hits against 17,451,486 sequences from the North Atlantic, South Atlantic, North Pacific, South Pacific and Indian Oceans. From these unique genes from the SAO, only 6.64 % had hits against the NCBI non-redundant protein database. SAO microbial communities share genes with the global ocean in at least 70 cellular functions; however, more than a third of predicted SAO genes represent a unique gene pool in global ocean. This study was the first attempt to characterize the taxonomic and functional community diversity of different water masses at SAO and compare it with the microbial community diversity of the global ocean, and SAO had a significant portion of endemic gene diversity. Microbial communities of deep water masses (236-1,200 m) are highly similar, suggesting that these water masses have very similar microbiological attributes, despite the common knowledge that water masses determine prokaryotic community and are barriers to microbial dispersal. The present study also shows that SCM is a clearly differentiated layer within Tropical waters with higher abundance of phototrophic microbes and microbial diversity.

Biofuel innovation by microbial diversity
Thiago Bruce, Astria D. Ferrão-Gonzales, Yutaka Nakashimada, Yuta Matsumura, Fabiano Thompson, Tomoo Sawabe
Springer Handbook of Marine Biotechnology, 1163, 1180, Springer Berlin Heidelberg, 2015年01月01日, ［査読有り］
英語, 論文集(書籍)内論文, Biofuels represent a viable alternative to the use of fossil fuels. They contribute to the reduction of greenhouse gas emission and do not compete with agricultural land. However, biofuels are not yet capable of replacing the current energy matrix based on fossil fuels because they cannot compete with standard fuels such as diesel and gasoline. Therefore, innovation is necessary to promote technical, economical, and environmental viability of biofuels. For this purpose, the marine realm is a promising source of bioresources to promote innovation in biofuel production. The marine biomass can be converted into biofuels such as biodiesel, bioethanol, and biogases (i. e, methane and hydrogen) through microbial activity. Microbial diversity plays a fundamental role in marine ecosystems by recycling of organic matter, which is a reflection of the vast genetic diversity associated to the marine microbial species available for biotechnological exploitation. The present study provides an overview of the importance of microbial fuels and presents innovative findings which can be applied to the production of biofuels in the near future.

Corrigendum: Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp nov.
Tomoo Sawabe, Yoshitoshi Ogura, Yuta Matsumura, Gao Feng, A. K. M. Rohul Amin, Sayaka Mino, Satoshi Nakagawa, Toko Sawabe, Ramesh Kumar, Yohei Fukui, Masataka Satomi, Ryoji Matsushima, Fabiano L. Thompson, Bruno Gomez Gil, Richard Christen, Fumito Maruyama, Ken Kurokawa, Tetsuya Hayashi
FRONTIERS IN MICROBIOLOGY, 5, 583, FRONTIERS RESEARCH FOUNDATION, 2014年11月, ［査読有り］
英語

ミニシンポジウム記録 水産物の生産・加工・流通段階を保障するリスク管理研究の最新動向
澤辺 智雄, 里見 正隆, 山崎 浩司
日本水産学会誌, 80, 6, 1002, 1008, 日本水産学会, 2014年11月, ［査読有り］
日本語

Marinobacter salarius sp nov and Marinobacter similis sp nov., Isolated from Sea Water
Hooi Jun Ng, Mario Lopez-Perez, Hayden K. Webb, Daniela Gomez, Tomoo Sawabe, Jason Ryan, Mikhail Vyssotski, Chantal Bizet, Francois Malherbe, Valery V. Mikhailov, Russell J. Crawford, Elena P. Ivanova
PLOS ONE, 9, 9, PUBLIC LIBRARY SCIENCE, 2014年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Two non-pigmented, motile, Gram-negative marine bacteria designated R9SW1(T) and A3d10(T) were isolated from sea water samples collected from Chazhma Bay, Gulf of Peter the Great, Sea of Japan, Pacific Ocean, Russia and St. Kilda Beach, Port Phillip Bay, the Tasman Sea, Pacific Ocean, respectively. Both organisms were found to grow between 4 degrees C and 40 degrees C, between pH 6 to 9, and are moderately halophilic, tolerating up to 20% (w/v) NaCl. Both strains were found to be able to degrade Tween 40 and 80, but only strain R9SW1(T) was found to be able to degrade starch. The major fatty acids were characteristic for the genus Marinobacter including C-16:0, C-16:1 omega 7c, C-18:1 omega 9c and C-18:1 omega 7c. The G+C content of the DNA for strains R9SW1(T) and A3d10(T) were determined to be 57.1 mol% and 57.6 mol%, respectively. The two new strains share 97.6% of their 16S rRNA gene sequences, with 82.3% similarity in the average nucleotide identity (ANI), 19.8% similarity in the in silico genome-to-genome distance (GGD), 68.1% similarity in the average amino acid identity (AAI) of all conserved protein-coding genes, and 31 of the Karlin's genomic signature dissimilarity. A phylogenetic analysis showed that R9SW1(T) clusters with M. algicola DG893(T) sharing 99.40%, and A3d10(T) clusters with M. sediminum R65(T) sharing 99.53% of 16S rRNA gene sequence similarities. The results of the genomic and polyphasic taxonomic study, including genomic, genetic, phenotypic, chemotaxonomic and phylogenetic analyses based on the 16S rRNA, gyrB and rpoD gene sequence similarities, the analysis of the protein profiles generated using MALDI-TOF mass spectrometry, and DNA-DNA relatedness data, indicated that strains R9SW1(T) and A3d10(T) represent two novel species of the genus Marinobacter. The names Marinobacter salarius sp. nov., with the type strain R9SW1(T) (= LMG 27497(T) = JCM 19399(T) = CIP 110588(T) = KMM 7502(T)) and Marinobacter similis sp. nov., with the type strain A3d10(T) (= JCM 19398(T) = CIP 110589(T) = KMM 7501(T)), are proposed.

Enhanced hydrogen production by a newly described heterotrophic marine bacterium, Vibrio tritonius strain AM2, using seaweed as the feedstock
Yuta Matsumura, Kazumichi Sato, Nurhidayu Al-Saari, Satoshi Nakagawa, Tomoo Sawabe
International Journal of Hydrogen Energy, 39, 14, 7270, 7277, Elsevier Ltd, 2014年05月05日, ［査読有り］
英語, 研究論文（学術雑誌）, To achieve more stable bio-hydrogen (bioH2) production from non-food feedstocks, stable feedstock preparations of marine biomass and an efficient bioH2 system using marine bacteria under saline conditions are two important key technologies that needed to be developed. Vibrio tritonius strain AM2, which was isolated from the gut of a marine invertebrate, was cultured under various conditions in marine broth (at initial 2.25% (w/v) NaCl) supplemented with mannitol, a seaweed carbohydrate, to evaluate its hydrogen production. The maximum molar yield of bioH2 was recorded as 1.7 mol H2/mol mannitol at pH 6 and 37 °C. The mannitol-grown cells had higher yields of bioH2 than the glucose-grown cells in the pH range 5.5-7.5. Compared to glucose, mannitol might be a better substrate for bioH 2 production using strain AM2. Fermentation product profiling revealed that strain AM2 might be utilising the formate-hydrogen pathway for bioH2 production. Furthermore, strain AM2 was able to produce hydrogen from powdered brown macroalgae containing 31.1% dry weight of mannitol. The molar yield of hydrogen reached 1.6 mol H2/mol mannitol contained in the seaweed feedstock. In conclusion, strain AM2 has the ability to produce hydrogen from mannitol with high yields even under saline conditions. Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Zunongwangia mangrovi sp nov., isolated from mangrove (Avicennia marina) rhizosphere, and emended description of the genus Zunongwangia
N. Rameshkumar, Ramya Krishnan, Elke Lang, Yuta Matsumura, Toko Sawabe, Tomoo Sawabe
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 64, 545, 550, SOC GENERAL MICROBIOLOGY, 2014年02月, ［査読有り］
英語, 研究論文（学術雑誌）, A Gram-stain-negative, strictly aerobic, slightly halophilic, non-motile and rod-shaped bacterial strain, designated P2E16(T), was isolated from mangrove (Avicennia marina) rhizosphere, collected at Devipattinam mangroves, Tamil Nadu, India. Strain P2E16(T) grew optimally at pH 7.0-8.0, at 25-28 degrees C and in the presence of 2-3% (w/v) NaCl. 16S rRNA gene analysis showed that strain P2E16(T) was phylogenetically closely related to the genus Zunongwangia, with Zunongwangia pro funda SM-A87(T) as the closest related type strain (98.2 % 16S rRNA gene sequence similarity) and less than 93% 16S rRNA gene sequence similarity to all other members of the family Flavobacteriaceae. Strain P2E16(T) contained MK-6 as the major respiratory quinone,phosphatidylethanolamine as the predominant polar lipid and iso-C-15:0 (17.8%), iso-C-17:0 3-OH (15.1 %), C-15:0 (12.8%), iso-C-17:1 omega 9c (9.8%), iso-C-15:1 G (9.0%), and summed feature 3 (comprising C-16:1 omega 7c and/or iso-C-15:0 2-OH; 7.1 %) as the major fatty acids. The DNA G+C content was 34.3 mol%. Differential phenotypic properties, together with the phylogenetic distinctiveness and low DNA-DNA relatedness demonstrated that strain P2E16(T) was distinct from the type strain of Zunongwangia pro funda. On the basis of these presented data, strain P2E16(T) is considered to represent a novel species of the genus Zunongwangia, for which the name Zunongwangia mangrovi sp. nov. is proposed. The type strain is P2E16(T) (=DSM 24499(T)=LMG 26237(T)=KCTC 23496(T)). An emended description of the genus Zunongwangia is also provided.

Allying with armored snails: the complete genome of gammaproteobacterial endosymbiont
Satoshi Nakagawa, Shigeru Shimamura, Yoshihiro Takaki, Yohey Suzuki, Shun-ichi Murakami, Tamaki Watanabe, So Fujiyoshi, Sayaka Mino, Tomoo Sawabe, Takahiro Maeda, Hiroko Makita, Suguru Nemoto, Shin-Ichiro Nishimura, Hiromi Watanabe, Tomo-O Watsuji, Ken Takai
ISME JOURNAL, 8, 1, 40, 51, NATURE PUBLISHING GROUP, 2014年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Deep-sea vents harbor dense populations of various animals that have their specific symbiotic bacteria. Scaly-foot gastropods, which are snails with mineralized scales covering the sides of its foot, have a gammaproteobacterial endosymbiont in their enlarged esophageal glands and diverse epibionts on the surface of their scales. In this study, we report the complete genome sequencing of gammaproteobacterial endosymbiont. The endosymbiont genome displays features consistent with ongoing genome reduction such as large proportions of pseudogenes and insertion elements. The genome encodes functions commonly found in deep-sea vent chemoautotrophs such as sulfur oxidation and carbon fixation. Stable carbon isotope (C-13)-labeling experiments confirmed the endosymbiont chemoautotrophy. The genome also includes an intact hydrogenase gene cluster that potentially has been horizontally transferred from phylogenetically distant bacteria. Notable findings include the presence and transcription of genes for flagellar assembly, through which proteins are potentially exported from bacterium to the host. Symbionts of snail individuals exhibited extreme genetic homogeneity, showing only two synonymous changes in 19 different genes (13 810 positions in total) determined for 32 individual gastropods collected from a single colony at one time. The extremely low genetic individuality in endosymbionts probably reflects that the stringent symbiont selection by host prevents the random genetic drift in the small population of horizontally transmitted symbiont. This study is the first complete genome analysis of gastropod endosymbiont and offers an opportunity to study genome evolution in a recently evolved endosymbiont.

Physiologic and metagenomic attributes of the rhodoliths forming the largest CaCO3 bed in the South Atlantic Ocean
Giselle S. Cavalcanti, Gustavo B. Gregoracci, Eidy O. dos Santos, Cynthia B. Silveira, Pedro M. Meirelles, Leila Longo, Kazuyoshi Gotoh, Shota Nakamura, Tetsuya Iida, Tomoo Sawabe, Carlos E. Rezende, Ronaldo B. Francini-Filho, Rodrigo L. Moura, Gilberto M. Amado-Filho, Fabiano L. Thompson
ISME JOURNAL, 8, 1, 52, 62, NATURE PUBLISHING GROUP, 2014年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Rhodoliths are free-living coralline algae (Rhodophyta, Corallinales) that are ecologically important for the functioning of marine environments. They form extensive beds distributed worldwide, providing a habitat and nursery for benthic organisms and space for fisheries, and are an important source of calcium carbonate. The Abrolhos Bank, off eastern Brazil, harbors the world's largest continuous rhodolith bed (of similar to 21 000 km(2)) and has one of the largest marine CaCO3 deposits (producing 25 megatons of CaCO3 per year). Nevertheless, there is a lack of information about the microbial diversity, photosynthetic potential and ecological interactions within the rhodolith holobiont. Herein, we performed an ecophysiologic and metagenomic analysis of the Abrolhos rhodoliths to understand their microbial composition and functional components. Rhodoliths contained a specific microbiome that displayed a significant enrichment in aerobic ammonia-oxidizing betaproteobacteria and dissimilative sulfate-reducing deltaproteobacteria. We also observed a significant contribution of bacterial guilds (that is, photolithoautotrophs, anaerobic heterotrophs, sulfide oxidizers, anoxygenic phototrophs and methanogens) in the rhodolith metagenome, suggested to have important roles in biomineralization. The increased hits in aromatic compounds, fatty acid and secondary metabolism subsystems hint at an important chemically mediated interaction in which a functional job partition among eukaryal, archaeal and bacterial groups allows the rhodolith holobiont to thrive in the global ocean. High rates of photosynthesis were measured for Abrolhos rhodoliths (52.16 mu mol carbon m(-2)s(-1)), allowing the entire Abrolhos rhodolith bed to produce 5.65 x 10(5) tons C per day. This estimate illustrates the great importance of the Abrolhos rhodolith beds for dissolved carbon production in the South Atlantic Ocean.

はじめに
澤辺 智雄, 里見 正隆, 山崎 浩司
日本水産学会誌, 80, 6, 1002, 1002, The Japanese Society of Fisheries Science, 2014年
日本語

Draft genome sequence of marine flavobacterium Jejuia pallidilutea strain 11shimoA1 and pigmentation mutants
Naoki Takatani, Masato Nakanishi, Pedro Meirelles, Sayaka Mino, Wataru Suda, Kenshiro Oshima, Masahira Hattori, Moriya Ohkuma, Masashi Hosokawa, Kazuo Miyashita, Fabiano L. Thompson, Ako Niwa, Toko Sawabe, Tomoo Sawabe
Genome Announcements, 2, 6, American Society for Microbiology, 2014年, ［査読有り］
英語, 研究論文（学術雑誌）, Here, we present the draft genome sequence of a novel carotenoid 2'-isopentenylsaproxanthin producer, Jejuia pallidilutea strain 11shimoA1, isolated from the surface of seaweed in Japan, and the ethyl methanesulfonate-induced pigmentation mutants. This genomic information will help to not only elucidate the 2'-isopentenylsaproxanthin biosynthetic pathway but also understand the evolution of flavobacteria.

Draft genome sequences of marine flavobacterium Nonlabens strains NR17, NR24, NR27, NR32, NR33, and Ara13
Masato Nakanishi, Pedro Meirelles, Ryohei Suzuki, Naoki Takatani, Sayaka Mino, Wataru Suda, Kenshiro Oshima, Masahira Hattori, Moriya Ohkuma, Masashi Hosokawa, Kazuo Miyashita, Fabiano L. Thompson, Ako Niwa, Toko Sawabe, Tomoo Sawabe
Genome Announcements, 2, 6, American Society for Microbiology, 2014年, ［査読有り］
英語, 研究論文（学術雑誌）, Here, we present the draft genome sequences of six carotenoid producers affiliated with Nonlabens spp. isolated from marine environments in both the northern and southern parts of Japan. The genomic information will help to elucidate the function and evolution of carotenoid synthetic gene clusters not only in the genus Nonlabens but also in the family Flavobacteriaceae.

Draft genome sequences of two Vibrionaceae species, Vibrio ponticus C121 and Photobacterium aphoticum C119, isolated as coral reef microbiota
Nurhidayu Al-saari, Pedro Milet Meirelles, Sayaka Mino, Wataru Suda, Kenshiro Oshima, Masahira Hattori, Moriya Ohkuma, Fabiano L. Thompson, Bruno Gomez-Gil, Toko Sawabe, Tomoo Sawabe
Genome Announcements, 2, 5, American Society for Microbiology, 2014年, ［査読有り］
英語, 研究論文（学術雑誌）, Here, the draft genome sequences of two Vibrionaceae, Vibrio ponticus C121 and Photobacterium aphoticum C119, which were isolated from the coral reef vicinity in Okinawa, Japan, are reported. The genome provides further insight into the genomic plasticity, biocomplexity, and ecophysiology, including pathogenicity and evolution, of these genera.

Draft genome sequences of marine flavobacterium Algibacter lectus strains SS8 and NR4
Naoki Takatani, Masato Nakanishi, Pedro Meirelles, Sayaka Mino, Wataru Suda, Kenshiro Oshima, Masahira Hattori, Moriya Ohkuma, Masashi Hosokawa, Kazuo Miyashita, Fabiano L. Thompson, Ako Niwa, Toko Sawabe, Tomoo Sawabe
Genome Announcements, 2, 6, American Society for Microbiology, 2014年, ［査読有り］
英語, 研究論文（学術雑誌）, Here, we present the draft genome sequences of a zeaxanthin-producing flavobacterium, Algibacter lectus strains SS8 and NR4, isolated from coastal sediment and rock surfaces in Hakodate, Japan, respectively. This genomic information represents the first Algibacter genome sequences, which will help us to elucidate the biology and evolution of Flavobacteriaceae bacteria.

Draft genome sequences of Marinobacter similis A3d10T and Marinobacter salarius R9SW1T
Elena P. Ivanova, Hooi Jun Ng, Hayden K. Webb, Gao Feng, Kenshiro Oshima, Masahira Hattori, Moriya Ohkuma, Alexander F. Sergeev, Valery V. Mikhailov, Russell J. Crawford, Tomoo Sawabe
Genome Announcements, 2, 3, American Society for Microbiology, 2014年, ［査読有り］
英語, 研究論文（学術雑誌）, Here, we present the draft genomes of Marinobacter similis A3d10T, a potential plastic biodegrader, and Marinobacter salarius R9SW1T, isolated from radioactive waters. This genomic information will contribute information on the genetic basis of the metabolic pathways for the degradation of both plastic and radionuclides.

Identification of a novel carotenoid, 2′-isopentenylsaproxanthin, by Jejuia pallidilutea strain 11shimoA1 and its increased production under alkaline condition
N. Takatani, K. Nishida, T. Sawabe, T. Maoka, K. Miyashita, M. Hosokawa
Applied Microbiology and Biotechnology, 98, 15, 6633, 6640, Springer Verlag, 2014年, ［査読有り］
英語, 研究論文（学術雑誌）, Carotenoids are a class of naturally occurring pigment, carrying out important biological functions in photosynthesis and involved in environmental responses including nutrition in organisms. Saproxanthin and myxol, which have monocyclic carotenoids with a γ-carotene skeleton, have been reported to show a stronger antioxidant activity than those with β-carotene and zeaxanthin. In this research, a yellow-orange bacterium of strain 11shimoA1 (JCM19538) was isolated from a seaweed collected at Nabeta Bay (Shizuoka, Japan). The 16S rRNA gene sequence of strain 11shimoA1 revealed more than 99.99 % similarity with those of Jejuia pallidilutea strains in the family Flavobacteriaceae. Strain 11shimoA1 synthesized two types of carotenoids. One of them was (3R, 3′R)-zeaxanthin with dicyclic structure and another was identified as (3R, 2′S)-2′-isopentenylsaproxanthin, a novel monocyclic carotenoid with pentenyl residue at C-2′ position of saproxanthin, using FAB-MS, 1H NMR, and CD analyses. Culturing strain 11shimoA1 in an alkaline medium at pH 9.2 resulted in a markedly increased in production of 2′-isopentenylsaproxanthin per dry cell weight, but a decreased in zeaxanthin production as compared to their respective production levels in medium with pH 7.0. These carotenoids are likely to play some roles in the adaptation of the bacterium to the environmental conditions. © 2014 Springer-Verlag.

Metagenomic Analysis of Healthy and White Plague-Affected Mussismilia braziliensis Corals
Gizele D. Garcia, Gustavo B. Gregoracci, Eidy de O. Santos, Pedro M. Meirelles, Genivaldo G. Z. Silva, Rob Edwards, Tomoo Sawabe, Kazuyoshi Gotoh, Shota Nakamura, Tetsuya Iida, Rodrigo L. de Moura, Fabiano L. Thompson
MICROBIAL ECOLOGY, 65, 4, 1076, 1086, SPRINGER, 2013年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Coral health is under threat throughout the world due to regional and global stressors. White plague disease (WP) is one of the most important threats affecting the major reef builder of the Abrolhos Bank in Brazil, the endemic coral Mussismilia braziliensis. We performed a metagenomic analysis of healthy and WP-affected M. braziliensis in order to determine the types of microbes associated with this coral species. We also optimized a protocol for DNA extraction from coral tissues. Our taxonomic analysis revealed Proteobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, and Actinomycetes as the main groups in all healthy and WP-affected corals. Vibrionales, members of the Cytophaga-Flavobacterium-Bacteroides complex, Rickettsiales, and Neisseriales were more abundant in the WP-affected corals. Diseased corals also had more eukaryotic metagenomic sequences identified as Alveolata and Apicomplexa. Our results suggest that WP disease in M. braziliensis is caused by a polymicrobial consortium.

Biogeography of Persephonella in deep-sea hydrothermal vents of the Western Pacific
Sayaka Mino, Hiroko Makita, Tomohiro Toki, Junichi Miyazaki, Shingo Kato, Hiromi Watanabe, Hiroyuki Imachi, Tomo-o Watsuji, Takuro Nunoura, Shigeaki Kojima, Tomoo Sawabe, Ken Takai, Satoshi Nakagawa
FRONTIERS IN MICROBIOLOGY, 4, 107, FRONTIERS MEDIA SA, 2013年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Deep-sea hydrothermal vent fields are areas on the seafloor with high biological productivity fueled by microbial chemosynthesis. Members of the Aquificales genus Persephone//a are obligately chemosynthetic bacteria, and appear to be key players in carbon, sulfur, and nitrogen cycles in high temperature habitats at deep-sea vents. Although this group of bacteria has cosmopolitan distribution in deep-sea hydrothermal ecosystem around the world, little is known about their population structure such as intraspecific genomic diversity, distribution pattern, and phenotypic diversity. We developed the multi-locus sequence analysis (MLSA) scheme for their genomic characterization. Sequence variation was determined in five housekeeping genes and one functional gene of 36 Persephone//a hydrogeraphrla strains originated from the Okinawa Trough and the South Mariana Trough (SNT). Although the strains share >98.7% similarities in 16S rRNA gene sequences, MLSA revealed 35 different sequence types (ST), indicating their extensive genomic diversity. A phylogenetic tree inferred from all concatenated gene sequences revealed the clustering of isolates according to the geographic origin. In addition, the phenotypic clustering pattern inferred from whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TO F/MS) analysis can be correlated to their MLSA clustering pattern. This study represents the first MLSA combined with phenotypic analysis indicative of allopatric speciation of deep-sea hydrothermal vent bacteria.

Microbial Communities Associated with Holothurians: Presence of Unique Bacteria in the Coelomic Fluid
Masaki Enomoto, Satoshi Nakagawa, Tomoo Sawabe
MICROBES AND ENVIRONMENTS, 27, 3, 300, 305, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2012年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Marine invertebrates interact with various microorganisms ranging from pathogens to symbionts. One-to-one symbiosis between a single microbial species and a single host animal has served as a model for the study of host-microbe interactions. In addition, increasing attention has recently been focused on the complex symbiotic associations, e. g., associations between sponges and their symbionts, due to their biotechnological potential; however, relatively little is known about the microbial diversity associated with members of the phylum Echinodermata. Here, for the first time, we investigated microbial communities associated with a commercially important holothurian species, Apostichopus japonicus, using culture-dependent and -independent methods. Diverse and abundant heterotrophs, mostly Gammaproteobacteria members, were cultured semi-quantitatively. Using the cloning and sequencing technique, different microbial communities were found in different holothurian tissues. In the holothurian coelomic fluid, potentially metabolically active and phylogenetically unique members of Epsilonproteobacteria and Rickettsiales were discovered. This study suggests that coelomic fluids of marine invertebrates, at least those inhabiting intertidal areas where physical and chemical conditions fluctuate, provide microbes with unique and stable habitats.

A simple method to collect digestive fluid for monitoring digestive enzyme activities and gut microbes of the abalone Haliotis discus discus
Kentaro Niwa, Hideaki Aono, Tomoo Sawabe
NIPPON SUISAN GAKKAISHI, 78, 5, 951, 957, JAPANESE SOC FISHERIES SCIENCE, 2012年09月, ［査読有り］
日本語, 研究論文（学術雑誌）, A simple method using a syringe to collect digestive fluid from the gut of the disc abalone Haliotis discus discus is described. The activities of polysaccharide degrading enzymes and bacterial microflora were compared between the digestive fluid (DF) collected by using a syringe and the digestive diverticulum homogenate (DH) collected by the conventional dissection method. Specific activities of alginate lyase, cellulase and beta-1,4-mannanase of DF were higher than those of DH. Viable bacterial count of DF was lower (1.0 x 10(7) CFU/mL) than that of DH (2.1 x 10(8) CFU/g). Microflora analysis of isolates using the 16S rRNA gene indicated that DF and DH showed a similar microflora composition, in which vibrios including Vibrio halioticoli and V. ezurae were predominant. The present method may be useful to determine changes in the digestive activity and the gut microflora of abalone without sacrificing individuals.

Vibrio jasicida sp nov., a member of the Harveyi clade, isolated from marine animals (packhorse lobster, abalone and Atlantic salmon)
Susumu Yoshizawa, Yasuhiro Tsuruya, Youhei Fukui, Tomoo Sawabe, Akira Yokota, Kazuhiro Kogure, Melissa Higgins, Jeremy Carson, Fabiano L. Thompson
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 62, 1864, 1870, MICROBIOLOGY SOC, 2012年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Six isolates of a facultatively anaerobic bacterium were recovered in culture from marine invertebrates and vertebrates, including packhorse lobster (Jasus verreauxi, abalone (Haliotis sp.) and Atlantic salmon (Salmo salar), between 1 994 and 2002. The bacteria were Gramnegative, rod-shaped and motile by means of more than one polar flagellum, oxidase-positive, catalase-positive and able to grow in the presence of 0.5-8.0% NaCl (optimum 3.0-6.0 %) and at 10-37 degrees C (optimum 25-30 degrees C). On the basis of 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA) using five loci (2443 bp; gyrB, pyrH, ftsZ, mreB and gapA), the closest phylogenetic neighbours of strain TCFB 0772(T) were the type strains of Vibrio communis (99.8 and 94.6% similarity, respectively), Vibrio owensii (99.8 and 94.1 %), Vibrio natriegens (99.4 and 88.8 %), Vibrio parahaemolyticus (99.4 and 90.3 %), Vibrio rotiferianus (99.2 and 94.4%), Vibrio alginolyticus (99.1 and 89.3%) and Vibrio campbellii (99.1 and 92.3%). DNA DNA hybridization confirmed that the six isolates constitute a unique taxon that is distinct from other known species of Vibrio. In addition, this taxon can be readily differentiated phenotypically from other Vibrio species. The six isolates therefore represent a novel species, for which the name Vibrio jasicida sp. nov. is proposed; the novel species is represented by the type strain TCFB 0772(T) (=JCM 16453(T) =LMG 25398(T)) (DNA G+C content 45.9 mol%) and reference strains TCFB 1977 (=JCM 16454) and TCFB 1000 (=JCM 16455).

Thermotomaculum hydrothermale gen. nov., sp nov., a novel heterotrophic thermophile within the phylum Acidobacteria from a deep-sea hydrothermal vent chimney in the Southern Okinawa Trough
Hiroshi Izumi, Takuro Nunoura, Masayuki Miyazaki, Sayaka Mino, Tomohiro Toki, Ken Takai, Yoshihiko Sako, Tomoo Sawabe, Satoshi Nakagawa
EXTREMOPHILES, 16, 2, 245, 253, SPRINGER JAPAN KK, 2012年03月, ［査読有り］
英語, 研究論文（学術雑誌）, A novel heterotrophic, thermophilic bacterium, designated strain AC55(T), was isolated from a deep-sea hydrothermal vent chimney at the Hatoma Knoll in the Okinawa Trough, Japan. Cells of strain AC55(T) were non-motile, long rods (2.0- to 6.8-mu m long and 0.3- to 0.6-mu m wide). The strain was an obligatory anaerobic heterotroph capable of fermentative growth on complex proteinaceous substances. Elemental sulfur was reduced to hydrogen sulfide but did not stimulate growth. Growth was observed between 37 and 60A degrees C (optimum 55A degrees C), pH 5.5 and 8.5 (optimum pH 6.6), and in the presence of 1.5-4.5% (w/v) NaCl (optimum 2.5%, w/v). Menaquinone-7 and -8 were the major respiratory quinones. The G + C content of the genomic DNA from strain AC55(T) was 51.6 mol%. The 16S rRNA gene sequence analysis revealed that strain AC55(T) was the first cultivated representative of Acidobacteria subdivision 10. Based on the physiological and phylogenetic features of the novel isolate, the genus name Thermotomaculum gen. nov. is proposed, with Thermotomaculum hydrothermale sp. nov. as the type species. The type strain is AC55(T) (=JCM 17643(T) = DSM 24660(T) = NBRC 107904(T)).

クロアワビの消化酵素活性と消化管内容細菌叢モニタリングのための消化管内容液採取法
丹羽健太郎, 青野英明, 澤辺智雄
日水誌, 78, 5, 951, 957, 日本水産學會, 2012年, ［査読有り］
日本語, アワビ消化液の非致死的な消化管内容液（DF）の採取方法を開発した。この方法により採取した DF と解剖により採取した消化盲嚢ホモジネート（DH）との間で多糖類分解酵素活性と細菌叢を比較した結果，DF のアルギン酸リアーゼ，セルラーゼおよび β-1,4-マンナナーゼの比活性は DH のそれらに比べていずれも高いこと，また，DF の生菌数は DH よりも低い値を示すが，細菌種の組成は類似していることが明らかになった。本法は，アワビの消化生理の動態解析に有用と考えられる。

水産「プロバイオティクス」の創成
杉田 治男, 澤辺 智雄, 吉水 守
日本水産學會誌 = Bulletin of the Japanese Society of Scientific Fisheries, 78, 4, 779, 779, 公益社団法人 日本水産学会, 2012年, ［査読有り］
日本語

蛍光標識テクノロジーを利用した食中毒細菌エコロジーの理解
澤辺桃子, 澤辺智雄
New Food Industry, 53, 8, 57, 66, 食品資材研究会, 2011年, ［査読有り］
日本語

培養併用蛍光in situ ハイブリダイゼーション法を用いた黄色ブドウ球菌の迅速定量検出
清水 茂雅, 久保沢 洋介, 松浦 美里, 川合 祐史, 山崎 浩司
日本食品微生物学会雑誌, 28, 1, 29, 36, 日本食品微生物学会, 2011年, ［査読有り］
日本語, 研究論文（学術雑誌）, Conventional plating methods for enumerating Staphylococcus aureus are time-consuming and labor-intensive. Rapid methods for specific quantification are desirable. Fluorescence in situ hybridization with filter-cultivation (FISHFC) method has been already developed to enumerate viable specific microorganisms such as Listeria monocytogenes, Clostridium perfringens and Vibrio parahaemolyticus. The purpose of this study was to develop and estimate FISHFC method for S. aureus quantitative detection in food samples. Alexa Fluor^® 546-labeled oligonucleotide probe, STA68, was newly designed from the 16S rRNA gene sequences of S. aureus. STA68-conferred fluorescence was observed for S. aureus but not for any other organisms, suggesting that STA68 probe is highly specific for S. aureus. Results were achievable within 12 hours by FISHFC method, containing with 10 hours cultivation, as compared to more than 3 days required for confirmation of S. aureus by conventional plating methods. When inoculated to BPW and food samples, the numbers of viable counts determined by FISHFC method were not significantly different to those obtained by the conventional plating method (p>0.05). This result suggests that FISHFC method was preferable for the specific, rapid and accurate quantification of viable S. aureus in food.

培養併用蛍光 in situ ハイブリダイゼーション法を用いた汚染指標大腸菌の迅速定量検出
青井良平, 清水茂雅, 山崎浩司, 澤辺智雄, 川合祐史
日本食品科学工学会誌, 58, 10, 483, 489, 日本食品科学工学会, 2011年, ［査読有り］
日本語, ECO636プローブは E.coli と Shigella 属に対して特異性を示した．汚染指標として E. coli を検出するためのFISHFC法におけるマイクロコロニー形成のための培養時間はSEL液体培地で7時間が最適であった．
E. coli 新鮮培養菌を用いた FISHFC 法と平板塗抹法での生菌数には有意差は認められず（p >0.05），さらに，E. coli を接種した食品サンプル（8種類）からの検出でも，FISHFC 法と平板塗抹法での生菌数値に有意差は認められなかった．
したがって，本研究で設計した ECO636 プローブを用いた FISHFC 法による E. coli の定量検出法は，培養時間7時間およびFISH操作2時間の合計9時間で，E. coli を平板塗抹法と同等の精度かつ迅速に検出·定量できる方法であり，汚染指標としての E. coli 定量検出に有用な方法であることが明らかとなった．

[Biodiversity and evolution of vibrios].
Sawabe T
Nihon saikingaku zasshi. Japanese journal of bacteriology, 65, 2, 333, 342, 2-4, 2010年12月, ［査読有り］
日本語, ビブリオは，コレラ菌，腸炎ビブリオ及びビブリオ・バルニフィカスというヒト病原性種を含むことから，病原微生物学から環境微生物学までにおける幅広い学術分野でモデル微生物として活発に研究されている細菌群である。コレラ菌の発見から156年を経過した今でも，新種のビブリオが発見され続けており，自然界におけるビブリオの種多様性は驚くほど高い。1965年にM. Véonによって作られたビブリオ科（Vibrionaceae）は，6属，103種が包含される巨大な分類群となっている。ビブリオで新種が記載され続ける背景には，株の遺伝的多様性を精密に検出する指紋鑑定法や個体識別法が取り入れられたことにある。また，個体識別の過程で得られる多座位の遺伝子配列を解析することにより，ビブリオの進化の系譜や種分化機構の推定も試みられるようになってきた。さらに，ビブリオをモデルとしたゲノム情報に基づく分類規範の構築も始まっている。本稿ではビブリオの分類の歴史を紐解きながら，ビブリオの多様性と進化に関する最新の成果をまとめて紹介する。

腸炎ビブリオの走性行動における遺伝子発現解析－L-セリンとアジ鰓抽出物に対する応答－
澤辺桃子, 澤辺智雄
函館短期大学紀要, 38, 38, 13, 18, 函館短期大学, 2010年, ［査読有り］
日本語

Environmental determinants correlated to Vibrio harveyi-mediated death of marine gastropods
Youhei Fukui, Sei-Ichi Saitoh, Tomoo Sawabe
ENVIRONMENTAL MICROBIOLOGY, 12, 1, 124, 133, WILEY-BLACKWELL PUBLISHING, INC, 2010年01月, ［査読有り］
英語, 研究論文（学術雑誌）, P>Vibrio harveyi is an emerging pathogen that causes mass mortality in a wide variety of marine animal species; however, it is still unclear which environmental determinants correlate V. harveyi dynamics and the bacterium-mediated death of marine animal life. We conducted a correlation analysis over a 5-year period (2003-2007) analysing the following data: V. harveyi abundance, marine animal mortality and environmental variables (seawater temperature, salinity, pH, chlorophyll a, rainfall and total viable bacterial counts). The samples were collected from a coastal area in northern Japan, where deaths of a marine gastropod species (Haliotis discus hannai) have been reported. Our analysis revealed significant positive correlations between average seawater temperature and average V. harveyi abundance (R = 0.955; P < 0.05), and between average seawater temperature and V. harveyi-mediated abalone death (R = 0.931; P < 0.05). Based on the regression model, n degrees C rise in seawater temperature gave rise to a 21n-fold increase in the risk of mortality caused by V. harveyi infection. This is the first report providing evidence of the strong positive correlation between seawater temperature and V. harveyi-mediated death of marine species.

細菌の進化の系譜をたどる新手法：多遺伝子座配列解析（MLSA）－見えてきた地球史とダイナミックにリンクするビブリオの進化
澤辺 智雄
化学と生物, 47, 1, 4, 6, 公益社団法人 日本農芸化学会, 2009年, ［査読有り］
日本語

培養併用蛍光in situ ハイブリダイゼーション（FISHFC）法による緑膿菌の定量
澤辺桃子, 大坪雅史, 澤辺智雄
函館短期大学紀要, 35, 35, 51, 56, 函館短期大学, 2009年, ［査読有り］
日本語

クロアワビHaliotis discus discusにおける消化酵素の多様性と主要多糖分解酵素の酵素化学的特徴
丹羽健太郎, 青野英明, 澤辺智雄
水産増殖, 57, 4, 557, 565, 水産増殖談話会, 2009年, ［査読有り］
日本語, 本研究ではクロアワビの消化酵素の多様性を定量的に測定し，活性の高い多糖分解酵素の単離精製と主要性状を明らかにした。クロアワビの消化管内容液および消化盲嚢ホモジネートから脂質，タンパク質，糖質代謝酵素が検出され，その中でアルギン酸リアーゼ，セルラーゼおよびマンナナーゼの活性が高かった。これら3種の酵素を単離精製し，酵素化学的特性を調べたところ，精製酵素の分子量はそれぞれ27.5，64.6，38.1 kDaで，至適pHは7.0，5.0，5.0，至適温度は40，40，55°Cであった。

Poly(ethylene terephthalate) Polymer Surfaces as a Substrate for Bacterial Attachment and Biofilm Formation
Hayden K. Webb, Russell J. Crawford, Tom Sawabe, Elena P. Ivanova
MICROBES AND ENVIRONMENTS, 24, 1, 39, 42, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2009年, ［査読有り］
英語, 研究論文（学術雑誌）, Plastic debris causes extensive damage to the marine environment, largely due to its ability to resist degradation. Attachment on plastic surfaces is a key initiation process for their degradation. The tendency of environmental marine bacteria to adhere to poly(ethylene terephthalate) (PET) plastic surfaces as a model material was investigated. It was found that the overall number of heterotrophic bacteria in a sample of sea water taken from St. Kilda Beach, Melbourne, Australia, was significantly reduced after six months from 4.2-4.7 x 10(3) cfu mL(-1) to below detectable levels on both full-strength and oligotrophic marine agar plates. The extinction of oligotrophs after six months was detected in all samples. In contrast, the overall bacterial number recovered on full strength marine agar from the sample flasks with PET did not dramatically reduce. Heterotrophic bacteria recovered on full-strength marine agar plates six months after the commencement of the experiment were found to have suitable metabolic activity to survive in sea water while attaching to the PET plastic surface followed by the commencement of biofilm formation.

Multi-Probe-Fluorescence in situ Hybridization for the Rapid Enumeration of Viable Vibrio parahaemolyticus
Tomoo Sawabe, Ai Yoshizawa, Yuko Kawanishi, Eriko Komatsu-Takeda, Satoshi Nakagawa, Toko Sawabe, Masashi Ootubo, Masataka Satomi, Yutaka Yano, Koji Yamazaki
MICROBES AND ENVIRONMENTS, 24, 3, 259, 264, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2009年, ［査読有り］
英語, 研究論文（学術雑誌）, A one-step multi-probe FISH method of detecting viable Vibrio parahaemolyticus was developed. Three candidate regions, corresponding to Helix 440+441, Helix 588, and Helix 1241 in 16S rRNA, were selected for detection, the thermodynamic parameters (Delta G(overall)) of the probes were optimized, and VP437, VP612 and VP1253, whose fluorescence were 1.7 to 11.3 times that of Delta G(overall)-unadjusted sequences, were designed. The addition of competitive oligo-nucleotides to reactions with VP612 and VP1253 strengthened the specificity of the probes. The three probes were labeled with FITC, TAMRA, and Cy5, respectively, and using a mixture of the probes and six competitive oligo-nucleotides, one-step FISH was applied to the species-specific detection of V. parahaemolyticus including epidemic strains of O3:K6 and O4:K68 serotypes. V. alginolyticus, V. rotiferianus, and V. campbellii were not detected in the reaction. Microcolonies (30-80 mu m in diameter) of V. parahaemolyticus were observed within 6 hours at 37 degrees C on seawater agar plates in both fresh and heat-damaged V. parahaemolyticus. Viable bacterial counts based on the proposed method were significantly different from those measured with typical vibrio selective media (CHROMagar Vibrio and TCBS).

Mutation is the Main Driving Force in the Diversification of the Vibrio splendidus Clade
Tomoo Sawabe, Shinichi Koizumi, Youhei Fukui, Satoshi Nakagawa, Elena P. Ivanova, Kumiko Kita-Tsukamoto, Kazuhiro Kogure, Fabiano L. Thompson
MICROBES AND ENVIRONMENTS, 24, 4, 281, 285, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2009年, ［査読有り］
英語, 研究論文（学術雑誌）, The Vibrio splendidus clade is the biggest in Vibrionales composed of 11 described species (25). Diversification of these species may have occurred 260 million years ago. The main driving forces of speciation in this clade have never been studied. Population biological parameters (population base recombination rate (rho), population base mutation rate (theta), and index of association (Ia)) were determined among 16 strains of 9 defined species in the Splendidus cluster. A comparison of individual gene phylogeny indicated significant incongruence in tree topology, which suggests the occurrence of recombination between species. Homologous recombination between species was detected at four loci. However, the mutation rate theta was higher than the recombination rate rho, suggesting that mutation is the main driving force in the diversification of V. splendidus-related species.

Vibrio porteresiae sp nov., a diazotrophic bacterium isolated from a mangrove-associated wild rice (Porteresia coarctata Tateoka)
N. Rameshkumar, Youhei Fukui, Tomoo Sawabe, Sudha Nair
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 58, 1608, 1615, SOC GENERAL MICROBIOLOGY, 2008年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Two facultatively anaerobic, nitrogen-fixing bacteria (strains MSSRF30(T) and MSSRF31) were isolated from a mangrove-associated wild rice (Porteresia coarctata Tateolka). These strains were determined to be nitrogen-fixers using the acetylene reduction assay and by PCR detection of a nifH gene amplicon. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the novel strains were most closely related to Vibrio fluvialis LMG 7894(T) (96.8 % gene sequence similarity), Vibrio furnissii LMG 7910(T) (96.8 % sequence similarity) and Vibrio tubiashii CIP 102760(T) (96.7% sequence similarity). Further multilocus sequence analysis using recA, pyrH, rpoA and nifH genes also showed low levels of sequence similarities (83-93 %) with all species of the genus Vibrio with validly published names. A multigene phylogenetic tree using concatenated sequences of the four genes (16S rRNA, rpoA, recA and pyrH) showed that strains MSSRF30T and MSSRF31 occupied a distinct phylogenetic position, forming a long branching that was not clustered with-any other recognized Vibrio species. The fatty acid profile also suggested that the novel strains belonged to the genus Vibrio. The results of physiological and biochemical tests, genomic fingerprinting and DNA-DNA hybridization analyses clearly differentiated both novel strains from their closest phylogenetic neighbours, Vibrio cholerae IID6019, Vibric, mimicus LMG 7896(T), V. fluvialis LMG 7894(T) and V. furnissii LMG 7910(T). Several phenotypic traits enabled the differentiation of strain MSSRF30T from other species of the genus Vibrio. The DNA G + C content of strain MSSIRF30(T) was 44.4 +/- 3.1 mol%. Based on genotypic, phenotypic, chemotaxonomic, phylogenetic and DNA-DNA hybridization analyses, the name Vibrio porteresiae sp. nov. (type strain rVISSRF30(T) =LMG 24061(T) =DSM 19223(T)) is proposed for this novel taxon.

自然界におけるビブリオ－その多様性、進化、生態　　　　　　　　　　　　　　　
澤辺 智雄
化学療法の領域, 24, 6, 24, 32, (株)医薬ジャーナル社, 2008年, ［査読有り］
日本語, コレラ菌の発見から154年を経過した今でも新種のビブリオが相次いで発見され、自然界におけるビブリオの多様性は驚くほど高い。1965年にM.Veronによって作られたビブリオ科は、現在、6属、96種が包含される巨大な分類群に成長している。ここまで種数が増えた背景には、ビブリオの分類に染色体上の遺伝子変異を精密に検出する指紋鑑定や個体識別の方法が取り入れられたことにある。また個体識別で得られた遺伝子配列を基に、種分化や進化の系譜もたどれるようになってきた。最新の分子系統解析で、ビブリオには少なくとも14の単系統群があり、その共通祖先は6億年前に、そしてそのいくつかは魚類の爆発的進化が生じた3億5千万年以降に大きく種分化した可能性が示されている。(著者抄録)

集団遺伝学的方法論を取り入れたビブリオ科細菌の再分類
澤辺 智雄
IFO Res. Commun., 22, 17, 26, 2008年, ［査読有り］
日本語, 研究論文（学術雑誌）

Rapid detection of Vibrio harveyi in seawater by real-time PCR
Youhei Fukui, Tomoo Sawabe
MICROBES AND ENVIRONMENTS, 23, 2, 172, 176, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2008年, ［査読有り］
英語, 研究論文（学術雑誌）, Vibrio harveyi cause mass mortalities of cultured marine fish. To address the ecology of V. harveyi in aquaculture, intensive monitoring is needed. We first optimized a quantitative real-time PCR method to determine V. harveyi abundance. The designed TaqMan probe and primers based on the 16S rRNA gene were specific at 68 degrees C of annealing and extension. Furthermore, the method using a chelating resin method was able to enumerate 1.7 x 10(2) CFU/ml in breeding seawater at an abalone farm. This method represents a good tool for monitoring the ecology of V. harveyi in marine environments within 5 h.

Inferring the evolutionary history of vibrios by means of multilocus sequence analysis
Tomoo Sawabe, Kumiko Kita-Tsukamoto, Fabiano L. Thompson
JOURNAL OF BACTERIOLOGY, 189, 21, 7932, 7936, AMER SOC MICROBIOLOGY, 2007年11月, ［査読有り］
英語, 研究論文（学術雑誌）, We performed the first broad study aiming at the reconstruction of the evolutionary history of vibrios by means of multilocus sequence analysis of nine genes. Overall, 14 distinct clades were recognized using the SplitsTree decomposition method. Some of these clades may correspond to families, e.g., the clades Salinivibrio and Photobacteria, while other clades, e.g., Splendidus and Harveyi, correspond to genera. The common ancestor of all vibrios was estimated to have been present 600 million years ago. We can define species of vibrios as groups of strains that share >95% gene sequence similarity and >99.4% amino acid identity based on the eight protein-coding housekeeping genes. The gene sequence data were used to refine the standard online electronic taxonomic scheme for vibrios (http://www.taxvibrio.incc.br).

Multilocus sequence analysis reveals that Vibrio harveyi and V-campbellii are distinct species
Fabiano L. Thompson, Bruno Gomez-Gil, Ana Teresa Ribeiro Vasconcelos, Tomoo Sawabe
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 73, 13, 4279, 4285, AMER SOC MICROBIOLOGY, 2007年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Identification and classification of Vibrio species have relied upon band pattern methods (e.g., amplified fragment length polymorphism) and DNA-DNA hybridization. However, data generated by these methods cannot be used to build an online electronic taxonomy. In order to overcome these limitations, we developed the first standard multilocus sequence scheme focused on the ubiquitous and pathogenic Vibrio harveyi species group (i.e., V. harveyi, V. campbellii, V. rotiferianus, and a new as yet unnamed species). We examined a collection of 104 isolates from different geographical regions and hosts using segments of seven housekeeping genes. These two species formed separated clusters on the basis of topA, pyrH, ftsZ, and mreB gene sequences. The phylogenetic picture obtained by the other three loci, i.e., gyrB, recA, and gapA, was more complex though. V. campbellii appeared nested within V. harveyi in the recA trees, whereas V. harveyi formed a tight nested cluster within V. campbellii by gapA. The gyrB gene had no taxonomic resolution and grouped the two species together. The fuzziness observed in these three genes seems not be related to recombination but to low divergence due to the accumulation of only a few substitutions. In spite of this, the concatenated sequences provided evidence that the two species form two separated clusters. These clusters did not arise by recombination but by accumulation of point mutations. V. harveyi and V. campbellii isolates can be readily identified through the open database resource developed in this study (http://www.taxvibrio.Incc.br/). We argue that the species should be defined by evolutionary criteria. Strains of the same species will share at least 95% concatenated sequence similarity using the seven loci, and, most importantly, cospecific strains will form cohesive readily recognizable phylogenetic clades.

Vibrio comitans sp nov., Vibrio rarus sp nov and Vibrio inusitatus sp nov., from the gut of the abalones Haliotis discus discus, H-gigantea, H-madaka and H-rufescens
Tomoo Sawabe, Yusuke Fujimura, Kentaro Niwa, Hideaki Aono
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 57, 916, 922, SOC GENERAL MICROBIOLOGY, 2007年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Nine alginolytic, facultatively anaerobic, non-motile bacteria were isolated from the guts of the abalones Haliotis discus discus, H. gigantea, H. madaka and H. rufescens. Phylogenetic analyses based on 16S rRNA gene sequences indicated that these bacteria were closely related to Vibrio superstes G3-29(T) (98.6-99.3% sequence similarity). DNA-DNA hybridization and phylogenetic analysis based on the gapA gene demonstrated that six strains constituted one bacterial species, two strains represented a second species and one strain represented a third species. The three novel bacterial species were different from all currently known vibrios. The names Vibrio comitans sp. nov. (type strain GHG2-1(T) =LMG 23416(T) = NBRC 102076(T); DNA G+C content 45.0-48.0 mol%), Vibrio inusitatus sp. nov. (type strain RW14(T) =LMG 23434(T) = NBRC 102082(T); DNA G + C content 43.1-43.7 mol%) and Vibrio rarus sp. nov. (type strain RW22(T) =LMG 23674(T) =NBRC 102084(T); DNA G + C content 43.8 mol%) are proposed to encompass these new taxa. Several phenotypic features were revealed that discriminate V. comitans, V. rarus and V. inusitatus from other Vibrio species.

Monitoring of fish-pathogenic vibrios
Tomoo Sawabe, Youhei Fukui
NIPPON SUISAN GAKKAISHI, 73, 2, 310, 312, JAPANESE SOC FISHERIES SCIENCE, 2007年03月, ［査読有り］
日本語, 研究論文（学術雑誌）

V. haryeyi感染によるエゾアワビの大量死
Tomoo Sawabe, Sahoko Inoue, Youhei Fukui, Kaoru Yoshie, Yutaka Nishihara, Hiroki Miura
MICROBES AND ENVIRONMENTS, 22, 3, 300, 308, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2007年, ［査読有り］
英語, 研究論文（学術雑誌）, Sixty thousand of deaths among cultured Ezo abalone Haliotis discus hannai occurred within a few days at an abalone farm in Japan in the middle of August, 2002. Dead animals were characterized by a hemolymphatic edema around the major circulatory system. Vibrios showing swarming motility dominated in the edema. The pathogenic vibrios were identified as Vibrio harveyi based on a phylogenetic analysis and a phenotypic characterization. In both immersion and injection experiments, the swarming vibrios fulfilled Koch's postulates as a pathogen for Ezo abalone. Using a GFP-tagged V. harveyi S20, a clump of bacterium was detected on the gills of the abalone within 48 hours after contact with the bacterium. This is the first report of V. harveyi infection in Ezo abalone Haliotis discus hannai.

Improved one-step colony PCR detection of Vibrio harveyi
Youhei Fukui, Tomoo Sawabe
MICROBES AND ENVIRONMENTS, 22, 1, 1, 10, JAPANESE SOC MICROBIAL ECOLOGY, DEPT BIORESOURCE SCIENCE, 2007年, ［査読有り］
英語, 研究論文（学術雑誌）, Vibrio harveyi strains are pathogenic to a wide range of marine fish and shellfish, having a significant negative economic impact on aquaculture worldwide. However, a reliable and rapid method of detecting V. harveyi has yet to be established. We aimed to construct an improved one-step detection method for V. harveyi. Reanalysis of 16S rRNA gene sequences of type strains of V. harveyi revealed a unique consensus region compared to related Vibrio species. Using a VHARF-VHARR primer set from this region, a V. harveyi-specific PCR-based detection method was established and could differentiate V. harveyi from related species such as V. campbellii, V. rotiferianus, and V. alginolyticus. Furthermore, the new method (optimal amplification with 20 core cycles of (94 degrees C-30 s, 60 degrees C-30 s, 72 degrees C-30 s)) could be applied to the identification of V. harveyi strains of colonies. This PCR was able to detect V. harveyi grown on plates in the environment within 3 days without bacterial isolation, DNA extraction, or the assistance of biochemical tests. The specificity and rapidity of the detection is reliable enough to understand the ecology of V. harveyi in environments, and to predict outbreaks of mass mortality caused by V. harveyi in aquaculture.

Simple conjugation and outgrowth procedures for tagging vibrios with GFP, and factors affecting the stable expression of the gfp tag
T. Sawabe, Y. Fukui, E. V. Stabb
LETTERS IN APPLIED MICROBIOLOGY, 43, 5, 514, 522, BLACKWELL PUBLISHING, 2006年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Aim: Our goal was to develop a simple system for tagging wild-type marine bacteria with gfp.
Methods and Results: Escherichia coli strain CC118 lambda pir carrying the conjugative helper plasmid pEVS104 and the gfp-containing plasmid pKV111 was used to transfer gfp to Vibrio recipients. Four different media were tested for their ability to support the growth of recipients, but not the E. coli donor, to allow powerful enrichment of gfp-tagged wild-type vibrios from mating mixes. Forty-three vibrio strains, representing 39 different species, were successfully tagged with gfp using the conjugative transfer from E. coli followed by selective outgrowth at 15 degrees C on ZoBell 2216E agar containing 0.5% sodium alginate. Using this outgrowth medium, colonies of GFP-expressing vibrio clones were detectable within 4 days. The percentage of visibly fluorescent cells in three representative GFP-tagged vibrios was higher at 15 degrees C than at 20 or 25 degrees C (c. 50% vs. 45% or 40%, respectively), and was also higher during the aerobic rather than the anaerobic culturing (c. 50% vs. 35%, respectively).
Conclusions: We found a simple selective outgrowth technique that enabled us to isolate a wide variety of GFP-tagged marine vibrios following the conjugative transfer of gfp from E. coli.
Significance and Impact of the Study: Tagging cells with GFP and related fluorescent proteins is a powerful approach for investigating the bacteria in situ, particularly during the colonization of hosts. The simple and cost-effective outgrowth condition described in this study could be applied to construct a wide variety gfp-tagged marine bacteria.

Vibrio2005: The First International Conference on the Biology of Vibrios
Fabiano L. Thompson, Karl E. Klose, Brian Austin, Yan Boucher, Peter Dawyndt, Dirk Gevers, Shah M. Faruque, Bruno Gomez-Gil, John F. Heidelberg, Tetsuya Iida, Didier Mazel, Diane McDougald, G. Balakrish Nair, Frans Ollevier, John H. Paul III, Martin Polz, Tomoo Sawabe, Ana C. P. Vicente, Rita R. Colwell, Jean Swings
Journal of Bacteriology, 188, 13, 4592, 4596, 2006年07月, ［査読有り］
英語, 研究論文（国際会議プロシーディングス）

Effect of iron on resuscitation of Tenacibaculum sp., the causative bacterium of spotting disease of short-spined sea urchin Strongylocentrotus intermedius, from the viable but non-culturable (VBNC) state
T Yamase, T Sawabe, K Kuma, K Tajima
FISH PATHOLOGY, 41, 1, 1, 6, JAPAN SOC FISH PATHOL DEPT FISHERIES-FAC AGR, 2006年03月, ［査読有り］
日本語, 研究論文（学術雑誌）, Tenacibaculum sp., the causative bacterium of spotting disease of short-spined sea urchin Storongylocentrotus intermedius, has been known to enter into the viable but non-culturable (VBNC) state in 75% Herbst's artificial seawater at 5 degrees C. Previous studies indicated that elevation of water temperature or addition of iron induced the resuscitation of the bacterium from the VBNC state. The present study revealed that the resuscitation period from the VBNC state was further prolonged when Tenacibaculum sp. in the VBNC state was resuscitated under the presence of iron, catalase and the homogenate of the sea urchin. Environmental waters around a sea urchin hatchery in Hokkaido, Japan often contained iron at concentrations over 0.34 mg/L in summer. The infection experiment showed that the VBNC cells activated with iron produced the spotting disease to healthy sea urchins. These results suggest that the VBNC cells of Tenacibaculum sp. resuscitated during summer under iron-rich environment are associated with outbreaks of the disease.

Adhesion of Tenacibaculum sp to short-spined sea urchin Strongylocentroutus intermedius and control of spotting disease by inhibiting adhesion using carbohydrate
R Taniguchi, T Sawabe, K Tajima
FISH PATHOLOGY, 41, 1, 13, 17, JAPAN SOC FISH PATHOL DEPT FISHERIES-FAC AGR, 2006年03月, ［査読有り］
英語, 研究論文（学術雑誌）, We examined the adhesion of Tenacibaculum sp., the causative bacterium of spotting disease of short-spined sea urchin Stronglycentroutus intermedius, to the host. The number of adhesive cells of Tenacibaculum sp. strain F-2 isolated from diseased sea urchin was about 30 times more than those of two stains of non-pathogenic marine Cytophaga. sp. isolated from healthy sea urchins. The adhesion of Tenacibaculum sp. F-2 to the sea urchins was inhibited by about 90% when the sea urchins were pre-treated with 0.1% D-galactose or D-xylose for 1h. With this treatment, all sea urchins remained asymptomatic and were still alive at 7 1h day after being immersed with 10(6) or 10(7) CFU/mL Tenacibaculum sp. F-2 for 1 h at 23 degrees C, while mortality of control reached 100%. These results indicate that carbohydrate treatment of the sea urchin is useful to control the disease.

エゾバフンウニ斑点病原因菌Tenacibaculum sp.のVBNC状態からの蘇生に及ぼす鉄の影響
山瀬智久, 澤辺智雄, 久万健志, 田島研一
魚病研究, 41, 1, 1, 6, 2006年03月, ［査読有り］
日本語, 研究論文（学術雑誌）, Tenacibaculum sp., the causative bacterium of spotting disease of short-spined sea urchin Storongylocentrotus intermedius, has been known to enter into the viable but non-culturable (VBNC) state in 75% Herbst's artificial seawater at 5°C. Previous studies indicated that elevation of water temperature or addition of iron induced the resuscitation of the bacterium from the VBNC state. The present study revealed that the resuscitation period from the VBNC state was further prolonged when Tenacibaculum sp. in the VBNC state was resuscitated under the presence of iron, catalase and the homogenate of the sea urchin. Environmental waters around a sea urchin hatchery in Hokkaido, Japan often contained iron at concentrations over 0.34 mg/L in summer. The infection experiment showed that the VBNC cells activated with iron produced the spotting disease to healthy sea urchins. These results suggest that the VBNC cells of Tenacibaculum sp. resuscitated during summer under iron-rich environment are associated with outbreaks of the disease. © 2006 The Japanese Society of Fish Pathology.

Thalassomonas loyana sp nov, a causative agent of the white plague-like disease of corals on the Eilat coral reef
FL Thompson, Y Barash, T Sawabe, G Sharon, J Swings, E Rosenberg
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 56, 365, 368, SOC GENERAL MICROBIOLOGY, 2006年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The taxonomic position of the coral pathogen strain CBMAI 722(T) was determined on the basis of molecular and phenotypic data. We clearly show that the novel isolate CBMAI 722 T is a member of the family Colwelliaceae, with Thalassomonas ganghwensis as the nearest neighbour (95% 16S rRNA gene sequence similarity). CBMAI 722(T) can be differentiated from its nearest neighbour on the basis of phenotypic and chemotaxonomic features, including the utilization of cellobiose and L-arginine, the production of alginase and amylase, but not oxidase, and the presence of the fatty acids 12:0 3-OH and 14:0, but not 10:0 or 15:0. The DNA G + C content of CBMAI 722(T) is 39.3 mol%. We conclude that this strain represents a novel species for which we propose the name Thalassomonas loyana sp. nov., with the type strain CBMAI 722(T) (=LMG 22536(T)). This is the first report of the involvement of a member of the family Colwelliaceae in coral white plague-like disease.

Enhanced expression of active recombinant alginate lyase AlyPEEC cloned from a marine bacteriu Pseudoalteromonas elyakovii in Escherichia coli by calcium compounds　　　　　　　　　　　　　　　
Sawabe, T, Takahasi, H. Saeki, H, Niwa, K, Aono, H
Enzyme and Microbial Technology, 40, 285, 291, 2006年, ［査読有り］
英語, 研究論文（学術雑誌）

PCR-RFLP analysis for identification of Aeromonas isolates collected from diseased fish and aquatic animals
MM Rahman, T Somsiri, R Tanaka, T Sawabe, K Tajima
FISH PATHOLOGY, 40, 4, 151, 159, JAPAN SOC FISH PATHOL DEPT FISHERIES-FAC AGR, 2005年12月, ［査読有り］
英語, 研究論文（学術雑誌）, The study was conducted to develop a PCR and an improved PCR-RFLP analysis method for rapid species-identification of Aeromonas genospecies. The forward and reverse primers for PCR were designed from the complementary sequences of the 16S rDNA of all 15-recognized Aeromonas genospecies to amplify a 1206-bp PCR product. The PCR amplified the expected product from the DNA template of type or reference strains of all recognized Aeromonas genospecies as well as 106 Aeromonas strains, while no PCR product was obtained from any of the non-Aeromonas strains tested. The PCR-RFLP analysis using the restriction enzymes (Alul, Mbol, Pvull, Pstl and Narl) provided identification of almost all Aeromonas species. However, the Aeromonas sp. T8 group and A. caviae exhibited a similar RFLP pattern. Some selected biochemical tests were found to be helpful for differentiation of the two species.

細菌叢を制御したシオミズツボワムシを投与したヒラメの腸内細菌叢
清水 智子, 篠崎 大祐, 笠井 久会, 澤辺 智雄, 渡辺 研一, 吉水 守
水産増殖, 53, 3, 275, 278, 水産増殖談話会, 2005年09月20日
日本語, 消毒前のワムシ卵および未処理区の孵化ワムシの細菌叢は、共にFlavobacterium属が主体を成していたことから、孵化前後でワムシの菌叢は変化しないことが示された。また、消毒して孵化させた後に V. splendidus V-15株を添加した区のワムシの細菌叢はVibrio属が主体を成していた。これらの結果から、ワムシの細菌叢は制御可能であることが示唆された。次いで、ショ糖分解能のない抗ウイルス活性を有する V. splendidus V-15株優勢ワムシをヒラメに給餌した。ヒラメ腸管内における Vibrio属細菌のうち、ショ糖非分解菌の割合は未処理区で62％、消毒区で59％、菌添加区で100％となり、ヒラメの腸内細菌叢も制御可能であることが示唆された。

Presence of Ecophysiologically Diverse Populations within Cobetia marina Strains Isolated from Marine Invertebrate, Algae and the Environments
Elena P. Ivanova, Richard Christen, Tomoo Sawabe, Yulia V. Alexeeva, Anatoly M. Lysenko, Victor P. Chelomin, Valery V. Mikhailov
Microbes and Environments, 20, 4, 200, 207, 2005年, ［査読有り］
英語, 研究論文（学術雑誌）, Over the last decade, taxonomic surveys have recovered sixteen strains of Halomonas-like marine het-erotrophic bacteria from different ecological habitats. The sixteen strains were isolated from three N.W. Pacific Ocean habitats: seawater, the mussel Crenomytilus grayanus and the degraded thallus of brown alga Fucus evanescens. These strains were subjected to a taxonomic investigation of their phenotypic/physiological, genetic, and phylogenetic features. Analysis indicates these bacteria belong to Cobetia marina. The study found all strains tolerated CdCl2 concentrations up to 875 mM. Taxonomically, the sixteen strains belong to the same species, nevertheless, their physiological features revealed distinguishing characteristics. For instance, strain KMM 296, recovered from the mussel Crenomytilus grayanus, was distinct from other C. Marina strains by its ability to produce highly active alkaline phosphatase. The majority of C. Marina strains that were isolated from degraded alga thallus appeared to have a particular metabolic specialisation by utilizing a range of easily assimilable monosaccharides. Notably, despite a high level of genetic similarity (80% of DNA relatedness), the phenotypic features of the strains isolated from degraded alga thallus differed with the type strain C. Marina LMG 2217T. These differences suggest an ecologically adapted population of C. Marina at a subspecies level. © 2005, Japanese Society of Microbial Ecology &
The Japanese Society of Soil Microbiology. All rights reserved.

Characterization of fish myofibrillar protein by conjugation with alginate oligosaccharide prepared using genetic recombinant alginate lyase
R Sato, T Sawabe, H Saeki
JOURNAL OF FOOD SCIENCE, 70, 1, C58, C62, INST FOOD TECHNOLOGISTS, 2005年01月, ［査読有り］
英語, 研究論文（学術雑誌）, The production of alginate lyase using genetically modified Escherichia coli was superior to the purification of alginate lyase from a culture medium of Pseudoalteromonas elyakovii regarding production efficiency. When alginate oligosaccharide (AO) prepared using genetic recombinant alginate lyase was introduced to fish myofibrillar proteins, the protein obtained high water solubility and improved thermal stability, similarly to AO prepared using wild-type lyase. Therefore, the use of genetic recombinant technology for the production of alginate lyase would be useful for the functional improvement of fish myofibrillar proteins by conjugation with AO.

Biodiversity and in situ abundance of gut microflora of abalone (Haliotis discus hannai) determined by culture-independent techniques
R Tanaka, M Ootsubo, T Sawabe, Y Ezura, K Tajima
AQUACULTURE, 241, 1-4, 453, 463, ELSEVIER SCIENCE BV, 2004年11月, ［査読有り］
英語, 研究論文（学術雑誌）, The compositions of bacterial communities in the gut of abalone were determined using the 16S rDNA clone library and fluorescence in situ hybridization (FISH). Sequencing of cloned 16S rDNA amplicons revealed a diverse community comprised Alpha-, Gamma- and Epsilonproteobacteria, and Mollicutes in the gut of artificial diet-fed abalone, and majority of Mollicutes, Fusobacteria, Alpha-and Gammaproteobacteria, in the gut of starved abalone. Biodiversity of gut bacterial community was rather high in artificial diet fed abalones than in Laminaria fed and starved animals. While, in situ abundance of the community composition determined by FISH revealed that 54% of 4',6-diamidino-2-phenylindole (DAPI)-stained bacteria were hybridized with a probe for Gammaprotcobacteria and 40% of DAPI-stained bacteria appeared to be of the Vibrio group. Alphaproteobacteria, which was frequent in clone libraries, was less abundant in artificial diet fed abalones determined by FISH. Our data show that the Vibrio group can be a dominant component in the gut microflora, of abalone. (C) 2004 Elsevier B.V. All rights reserved.

Molecular phylogeny of Neocalanus copepods in the subarctic Pacific Ocean, with notes on non-geographical genetic variations for Neocalanus cristatus
Marina Taniguchi, Toshinori Kanehisa, Tomoo Sawabe, Richard Christen, Tsutomu Ikeda
Journal of Plankton Research, 26, 10, 1249, 1255, 2004年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Monophylies of Neocalanus cristatus, Neocalanus plumchrus and Neocalanus flemingeri were revealed by nucleotide sequences of mitochondrial 16S rRNA gene (410 bp) but not of nuclear 18S rDNA gene (1802 bp). Intraspecific variations in mitochondrial 16S rRNA of N. cristatus collected from geographically distant regions were very low (<
0.5%).

Vibrio neonatus sp nov and Vibrio ezurae sp nov isolated from the Gut of Japanese Abalones
T Sawabe, K Hayashi, J Moriwaki, Y Fukui, FL Thompson, J Swings, R Christen
SYSTEMATIC AND APPLIED MICROBIOLOGY, 27, 5, 527, 534, URBAN & FISCHER VERLAG, 2004年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Five alginolytic, facultative anaerobic, non-motile bacteria were isolated from the gut of Japanese abalones (Haliotis discus discus, H. diversicolor diversicolor and H. diversicolor aquatilis). Phylogenetic analyses based on 16S rRNA gene and gap gene sequences indicated that these strains are closely related to V. balioticoli. DNA-DNA hybridizations, FAFLP fingerprintings, and phylogenies of gap and 16S rRNA gene sequences showed that the five strains represent two species different from all currently described vibrios. The names Vibrio neonatus sp. nov. (IAM 15060(T) = LMG 19973(T) = HDD3(_)1(T); Mol% G+C of DNA is 42.1-43.9), and Vibrio ezurae sp. nov. (IAM 15060(T) = LMG 19973(T) = HDD3-1(T); mol% G+C of DNA is 43.6-44.8) are proposed to encompass these new taxa. The two new species can be differentiated from V balioticoli on the basis of several features, including beta-galactosidase activity, assimilation of glycerol, D-mannose and D-gluconate.

Shewanella affinis sp nov., isolated from marine invertebrates
EP Ivanova, OI Nedashkovskaya, T Sawabe, NV Zhukova, GM Frolova, DV Nicolau, VV Mikhailov, JP Bowman
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 54, 1089, 1093, SOC GENERAL MICROBIOLOGY, 2004年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Four marine bacterial strains, designated KMM 3587(T), KMM 3586, KMM 3821 and KMM 3822, were isolated from the sipuncula Phascolosoma japonicum, a common inhabitant of Troitza Bay in the Gulf of Peter the Great (Sea of Japan region), and from an unidentified hydrocoral species collected in Makarov Bay (Iturup Islands), Kuril Islands, North-West Pacific Ocean. The strains were characterized to clarify their taxonomic position. 16S rRNA gene sequences of KMM 3587(T) and KMM 3586 indicated 99% similarity to Shewanella colwelliana. Despite such a high level of 16S rRNA gene sequence similarity, DNA-DNA hybridization experiments demonstrated only 45-52% binding with DNA of S. colwelliana ATCC 39565 T. The DNA G + C contents of the novel strains were 45 mol% and the shared level of DNA hybridization was conspecific (81-97%), indicating that they represent a single genospecies. The novel strains were mesophilic (able to grow at 10-34degreesC), neutrophilic and haemolytic, and able to degrade gelatin, casein and Tween 20, 40 and 80, but not starch, agar, elastin, alginate or chitin. The major fatty acids were i13: 0, i15:0, 16:0, 16:1omega7 and 17:1omega8 (68.9% of total). The major isoprenoid quinones were Q7 (47-62%) and Q8 (26-47%). Eicosapentaenoic acid was produced in minor amounts. Based on these data, the strains are assigned to a novel species, Shewanella affinis sp. nov. (type strain KMM 3587(T)=CIP 107703(T) = ATCC BAA-642(T)).

Vibrio gallicus sp nov., isolated from the gut of the French abalone Haliotis tuberculata
T Sawabe, K Hayashi, J Moriwaki, FL Thompson, J Swings, P Potin, R Christen, Y Ezura
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 54, 843, 846, SOC GENERAL MICROBIOLOGY, 2004年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Five alginolytic, facultatively anaerobic, non-motile bacteria were isolated from the gut of the abalone Haliotis tuberculata. Phylogenetic analyses based on 16S rDNA data indicated that these strains are related to Vibrio wodanis, Vibrio salmonicida, Vibrio logei and Vibrio fischeri (but with <97% 16S rRNA gene sequence similarity). DNA-DNA hybridization and fluorescence amplified fragment length polymorphism fingerprinting demonstrated that the five strains constituted a single species that was different from all currently known vibrios. The name Vibrio gallicus sp. nov. (type strain, CIP 107863(T) = LMG 21878(T) = HT2-1(T); DNA G + C content, 43.6-44.3 mol%) is proposed for this novel taxon. Several phenotypic features were disclosed that discriminated V. gallicus from other Vibrio species: V gallicus can be differentiated from Vibrio halioticoli on the basis of four traits (β-galactosidase test and assimilation of three carbon compounds) and from Vibrio superstes by 16 traits.

Sulfitobacter delicatus sp nov and Sulfitobacter dubius sp nov., respectively from a starfish (Stellaster equestris) and sea grass (Zostera marina)
EP Ivanova, NM Gorshkova, T Sawabe, NV Zhukova, K Hayashi, VV Kurilenko, Y Alexeeva, Buljan, V, DV Nicolau, VV Mikhailov, R Christen
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 54, 475, 480, SOC GENERAL MICROBIOLOGY, 2004年03月, ［査読有り］
英語, 研究論文（学術雑誌）, On the basis of data from phenotypic and genotypic characterization and analysis of 16S rRNA gene sequences, two novel species belonging to the genus Sulfitobacter are described. Strains KMM 3584(T), a pale-yellowish, non-motile strain isolated from a starfish (Stellaster equestris), and KMM 3554(T), which is motile by means of a single subpolar flagellum and was isolated from sea grass (Zostera marina), are marine, Gram-negative, aerobic, rod-shaped organisms. Both strains have the ability to degrade gelatin, but not casein, chitin, agar, DNA, Tween 80 or starch. Strain KMM 3584(T) decomposed alginate and grew at NaCl concentrations of 1-8% and temperatures of 12-37 degreesC, whereas strain KMM 3554(T) grew in 1-12% NaCl and at temperatures of 10-30 degreesC. The predominant fatty acid was 18:1 omega7, amounting to up to 80% of the total fatty acids. The other characteristic feature was the presence of 18:2 isomers. The DNA G + C contents of KMM 3584(T) and KMM 3554(T) were respectively 60(.)0 and 63(.)7 mol%. The level of DNA similarity between the two strains was 33%. DNA from KMM 3584(T) and KMM 3554(T) had hybridization values of 5-24% and 10-41%, respectively, with DNA from the type strains of Sulfitobacter pontiacus, Sulfitobacter brevis, Sulfitobacter mediterraneus and Staleya guttiformis. It is proposed that strains KMM3584(T) (= LMG 20554(T) ATCC BAA-321(T)) and KMM 3554(T) (= LMG 20555(T) = ATCC BAA-320(T)) represent two novel species, Sulfitobacter delicatus sp. nov. and Sulfitobacter dubius sp. nov., respectively.

Stereoisomers of marine bacterial phosphatidylglycerols
Hironori Fujishima, Toshiko Gamano, Yukako Taoka, Tomoo Sawabe, Yutaka Itabashi
Nippon Suisan Gakkaishi (Japanese Edition), 70, 2, 200, 202, 2004年03月, ［査読有り］
日本語, 研究論文（学術雑誌）

Vibrio superstes sp nov., isolated from the gut of Australian abalones Haliotis laevigata and Haliotis rubra
K Hayashi, J Moriwaki, T Sawabe, FL Thompson, J Swings, N Gudkovs, R Christen, Y Ezura
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 53, 1813, 1817, SOC GENERAL MICROBIOLOGY, 2003年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Five alginolytic, facultatively anaerobic, non-motile bacteria were isolated from the gut of abalones Haliotis laevigata and Haliotis rubra. Phylogenetic analyses based on 16S rDNA data indicated that these strains are related closely to Vibrio halioticoli (98 % 1 6S rDNA sequence similarity). DNA-DNA hybridization and fluorescent amplified fragment length polymorphism fingerprinting demonstrated that the five strains constituted a single species that was different from all currently known vibrios. The name Vibrio superstes sp. nov. (type strain, LMG 21323(T) = \AM 15009(T) = G3-29(T) ; DNA G + C content, 48(.)0-48(.)9 mol%) is proposed to encompass this novel taxon. Several phenotypic features were disclosed that discriminate V superstes from other Vibrio species: V. superstes sp. nov. and V. halioticoli can be differentiated on the basis of 17 traits (indole production, beta-galactosidase test and assimilation of 15 carbon compounds).

Gut microflora of abalone Haliotis discus hannai in culture changes coincident with a change in diet
R Tanaka, Sugimura, I, T Sawabe, M Yoshimizu, Y Ezura
FISHERIES SCIENCE, 69, 5, 951, 958, JAPANESE SOC FISHERIES SCIENCE, 2003年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Development of gut microflora in abalone Haliotis discus hannai cultured at two abalone farms in Japan was similar: (i) gut microflora of juvenile abalones fed on microalgae matched microflora cultured from seawater; and (ii) gut microflora changed coincident with the abalone switching food sources from microalgae to algal pellets. After abalone reached 4 months of age, the gut microflora was replaced by algal polysaccharide-degrading bacteria, which were almost entirely characterized as facultative anaerobes. Dominant species were alginolytic, non-motile fermenters (NMF) and Vibrio spp. The gut microflora seemed to be stable in abalone older than 1 year, with NMF bacteria dominating. Ninety-six percent of the NMF isolates were identified as Vibrio halioticoli by species-specific identification using the colony hybridization method. These results show that abalone H. discus hannai has a unique developmental process in which gut microflora shifts to alginate-degrading bacteria, especially V, halioticoli.

Stability and emulsion-forming ability of water-soluble fish myofibrillar protein prepared by conjugation with alginate oligosaccharide
R Sato, S Katayama, T Sawabe, H Saeki
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 51, 15, 4376, 4381, AMER CHEMICAL SOC, 2003年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Carp myofibrillar protein (Mf) was conjugated with alginate oligosaccharide (AO) through the Maillard reaction under low relative humidity, and the functional properties of the Mf-AO conjugate were investigated under different NaCl concentrations and pH levels. Mf became highly solubilized at lower NaCl concentrations by conjugation with AO, with a slight loss of available lysine. The thermal stability of Mf was effectively improved by conjugation with AO. Heat treatment at 80 degreesC for 2 h had no effect on the solubility of the Mf-AO conjugate attached to 227 mug/mg of AO regardless of the NaCl concentration and pH. Furthermore, the Mf-AO conjugate showed excellent emulsion-forming ability regardless of NaCl concentration. The improved functionalities of Mf by conjugation with AO remained even at a nearly isoelectric point. These results indicate that conjugation with AO through the Maillard reaction is an effective way to prepare high-functional food material from fish muscle protein.

Occurrence and diversity of mesophilic Shewanella strains isolated from the North-West Pacific Ocean
EP Ivanova, T Sawabe, NV Zhukova, NM Gorshkova, OI Nedashkovskaya, K Hayashi, GM Frolova, AF Sergeev, KG Pavel, VV Mikhailov, DV Nicolau
SYSTEMATIC AND APPLIED MICROBIOLOGY, 26, 2, 293, 301, URBAN & FISCHER VERLAG, 2003年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Although bacteria of the genus Shewanella belong to one of the readily cultivable groups of "Gammaproteobacteria", little is known about the occurrence and abundance of these microorganisms in the marine ecosystem. Studies revealed that of 654 isolates obtained from marine invertebrates (ophiuroid Amphiopholis kochii, sipuncula Phascolosoma japonicum, and holothurian Apostichopus japonicus, Cucumaria japonica), seawater and sediments of the North-West Pacific Ocean (i.e. the Sea of Japan and Iturup Is, Kurile Islands), 10.7% belonged to the genus Shewanella. The proportion of viable Shewanella species varied from 4% to 20% depending on the source of isolation. From the isolation study, representative strains of different phenotypes (from seventy presumptive Shewanella strains) were selected for detailed characterization using phenotypic, chemotaxonomic, and phylogenetic testing. 16S rDNA sequence-based phylogenetic analysis confirmed the results of tentative identification and placed the majority of these strains within only a few species of the genus Shewanella with 98-99% of 16S rDNA sequences identity mainly with S. japonica and S. colwelliana, suggesting that the strains studied might belong to these species. Numerically dominant strains of S. japonica were metabolically active and produced protemases (gelatinases, caseinases), lipases, amylases, agarases, and alginases. Shewanella strains studied demonstrated weak antimicrobial and antifungal activities that might be an indication of their passive role in the colonization on living and non-living surfaces.

Acetic acid production of Vibrio halioticoli from alginate: a possible role for establishment of abalone-V-halioticoli association
T Sawabe, N Setoguchi, S Inoue, R Tanaka, M Ootsubo, M Yoshimizu, Y Ezura
AQUACULTURE, 219, 1-4, 671, 679, ELSEVIER SCIENCE BV, 2003年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Acetic acid, which is converted from cellulose by means of the metabolism of their gut microbes, is an important oxidizable energy source and precursors of anabolism in ruminant animals and xylophagus insects. However, acetic acid production from algal polysaccharides by means of the metabolism of gut microbes of marine herbivorous invertebrates is not well studied. Abundance of Vibrio halioticoli, which is a dominant alginolytic gut microbe of abalone Haliotis discus hannai, in the gut of various marine herbivorous invertebrates and major fermentation products from alginate of these strains were investigated in this study. V halioticoli strains were detected from the gut of three species of the Japanese abalone, Haliotis discus discus, Haliotis diversicolor aquatilis, and Haliotis diversicolor diversicolor, and a South African abalone, Haliotis midae, with a range of 40-65% amongst these microflora. The bacterium was also found in the gut of a turban shell, Turbo cornutus, of which occupation rate was 16%. Furthermore, acetic acid and formic acid were detected as major fermentation products of alginate in these V halioticoli strains. It is suggested that the abundant populations of V halioticoli in the gut of Haliotis abalone may have a great role for converting alginate to acetic acid. (C) 2003 Elsevier Science B.V. All rights reserved.

Shewanella fidelis sp nov., isolated from sediments and sea water
EP Ivanova, T Sawabe, K Hayashi, NM Gorshkova, NV Zhukova, OI Nedashkovskaya, VV Mikhailov, DV Nicolau, R Christen
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 53, 577, 582, SOC GENERAL MICROBIOLOGY, 2003年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Two marine bacterial strains, KMM 3582(T) and KMM 3589, isolated respectively from sediments of the South China Sea and sea water of the Sea of Japan, have been characterized. Comparative 16S rDNA sequence-based phylogenetic analysis placed the two strains in a separate branch of the gamma-Proteobacteria within the members of the genus Shewanella. KMM 3582 T showed the highest similarity (97(.)1 and 97(.)4%, respectively) to Shewanella pealeana and Shewanella gelidimarina. The G + C contents of the DNAs of the two strains studied were 45(.)0 mol%. The level of DNA-DNA relatedness between the two strains was 82%, indicating that they represent a single genospecies. These organisms were slightly pinkish, Gram-negative, polarly flagellated, facultatively anaerobic, mesophilic (with temperature range from 4 to 30degreesC), neutrophilic: and haemolytic and were able to degrade alginate, gelatin and DNA. The novel organisms were susceptible to gentamicin, lincomycin, oleandomycin, streptomycin and polymyxin. The predominant fatty acids were characteristic for shewanellae: 13:0-i, 15:0-i, 16:0 and 16:1omega7. Eicosapentaenoic acid, 20:5omega3, was not detected. Phylogenetic evidence, together with phenotypic characteristics, showed that the two bacteria constitute a novel species of the genus Shewanella. The name Shewanella fidelis sp. nov. is proposed, with the type strain KIMM 3582(T) (=LMG 20551(T) =ATCC BAA-318(T)).

Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample
M Ootsubo, T Shimizu, R Tanaka, T Sawabe, K Tajima, Y Ezura
JOURNAL OF APPLIED MICROBIOLOGY, 95, 6, 1182, 1190, BLACKWELL PUBLISHING LTD, 2003年, ［査読有り］
英語, 研究論文（学術雑誌）, Aims: A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae-specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples.
Methods and Results: In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re-evaluated using cultured Escherichia coli. Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli, and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of x100 in ca 5 min. The total time for FISH with probe D following cultivation(FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g(-1)) and environmental water samples (above 1 cell ml(-1)).
Conclusions: Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method.
Significance and Impact of the Study: A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.

Pseudomonas extremorientalis sp nov., isolated from a drinking water reservoir
EP Ivanova, NM Gorshkova, T Sawabe, K Hayashi, NI Kalinovskaya, AM Lysenko, NV Zhukova, DV Nicolau, TA Kuznetsova, VV Mikhailov, R Christen
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 52, 2113, 2120, SOC GENERAL MICROBIOLOGY, 2002年11月, ［査読有り］
英語, 研究論文（学術雑誌）, On the basis of phenotypic and genotypic characteristics and 16S rDNA sequence analysis, a novel species belonging to the genus Pseudomonas sensu stricto was identified. The saprophytic, fluorescent bacterium, designated KMM 3447(T), was isolated from a drinking water reservoir near Vladivostok City, Russia. The novel organism was a Gram-negative, aerobic, rod-shaped bacterium that produced a cyclic depsipeptide with surface-active properties. It degraded casein, but did not degrade gelatin, starch, agar or Tween 80. The bacterium was also haemolytic. Growth of the novel bacterium occurred between 4 and 35 degreesC. The predominant cellular fatty acids of the novel pseudomonad were C-16:0, C16:1(n-7), C18:1(n-7) and C-17:0 cyclo; branched fatty acids were only found in trace amounts. The G+C content of the novel bacterium was 61.0 mol%. 16S rDNA sequence analysis indicated that the novel bacterium had a clear affiliation with Pseudomonas fluorescens and species closely related to this recognized pseudomonad. DNA-DNA hybridization experiments showed that the novel bacterium bound at low levels (27-53 %) with the DNA of the type strains of its nearest phylogenetic relatives, namely Pseudomonas tolaasii, Pseudomonas veronii, Pseudomonas orientalis and Pseudomonas rhodesiae, indicating that the novel bacterium represented a novel species within the genus Pseudomonas, for which the name Pseudomonas extremorientalis is proposed; the type strain is KMM 3447(T) (= LMG 19695(T)).

Pseudoalteromonas translucida sp nov and Pseudoalteromonas paragorgicola sp nov., and emended description of the genus
EP Ivanova, T Sawabe, AM Lysenko, NM Gorshkova, K Hayashi, NV Zhukova, DV Nicolau, R Christen, VV Mikhailov
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 52, 1759, 1766, SOC GENERAL MICROBIOLOGY, 2002年09月, ［査読有り］
英語, 研究論文（学術雑誌）, On the basis of phenotypic and genotypic characteristics and analysis of 16S rRNA sequences, two novel species belonging to the genus Pseudoalteromonas are described. A pale-orange-pigmented strain, KMM 3548(T), isolated from a sponge and a non-pigmented strain, KMM 520(T), isolated from sea water are marine, Gram-negative, aerobic, rod-shaped organisms. One of the strains, KMM 520(T), had bipolar flagella. Both strains had the ability to degrade gelatin, DNA and Tween 80 but not chitin or agar. Strain KMM 520(T) decomposed elastin and grew at NaCl concentrations of 1-8%, while strain KMM 3548T grew at 1-6% NaCl. The temperature range for both strains was 4-30degreesC. The DNA G+C contents were 46.3 (KMM 520(T)) and 41.1 mol % (KMM 3548(T)). The level of DNA relatedness between the two strains was 20%. DNA from strain KMM 520(T) showed 8-34% genetic relatedness and that of KMM 3548(T) showed 17-53% relatedness to the DNA of other type strains of the genus Pseudoalteromonas. 16S rRNA analysis indicated a clear affiliation of these novel bacteria with the genus Pseudoalteromonas. The type strains of the novel species are Pseudoalteromonas translucida sp. nov. KMM 520(T) (=LMG 19696(T)=ATCC BAA-315(T)) and Pseudoalteromonas paragorgicola sp. nov. KMM 3548(T) (=LMG 19694(T) = ATCC BAA-322(T)).

Fluorescent amplified fragment length polymorphism and repetitive extragenic palindrome-PCR fingerprinting reveal host-specific genetic diversity of Vibrio halioticoli-like strains isolated from the gut of Japanese abalone
T Sawabe, FL Thompson, J Heyrman, M Cnockaert, K Hayashi, R Tanaka, M Yoshimizu, B Hoste, J Swings, Y Ezura
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 68, 8, 4140, 4144, AMER SOC MICROBIOLOGY, 2002年08月, ［査読有り］
英語, 研究論文（学術雑誌）, When analyzed by fluorescent amplified fragment length polymorphism and repetitive extragenic palindrome-PCR fingerprinting, a total of 47 Vibrio halioticoli strains isolated from four Japanese abalone species and one turban shell species formed three clusters that roughly reflect the different species of host abalone from which they were isolated. The V. halioticoli isolates from turban shells were distributed evenly among the clusters. Representative isolates from two clusters were deemed separate species or subspecies by DNA-DNA hybridization.

Development of 16S rRNA targeted PCR for the identification of Vibrio spp., the causative bacteria of the disease in cultured sea urchin Strongylocentrotus intermedius occurring at low water temperatures
Y Sogabe, K Tajima, R Tanaka, T Sawabe, Y Ezura
NIPPON SUISAN GAKKAISHI, 68, 2, 201, 206, JAPANESE SOC FISHERIES SCIENCE, 2002年03月, ［査読有り］
日本語, 研究論文（学術雑誌）, The causative bacteria, Vibrio spp., have been isolated from the diseased sea urchin Strongylocentrotus intermedius at low water temperatures. To develop a specific identification method for the pathogens, species-specific nucleotide sequences in the 16S rRNA of Vibrio spp., were determined. We designed two PCR primers, DA2F and SK1F, based on the nucleotide sequences: DA2F forward primer (nucleotide numbers 456 to 476 in Escherichia coli 16S rRNA) for Date isolates and Sk1F for-ward primer (nucleotide numbers 455 to 478 in E. coli 16S rRNA) for Shiriuchi and Shikabe isolates.
The PCR product with about 1kbp was obtained in all causative bacteria from Date, Shiriuchi, Shikabe Breeding Centers and some of Vibrio reference strains by amplification with DA2F-1540R primer set, however, those from three Breeding Centers differed from the reference strains in the case of growth at 30degreesC. PCR amplification with SK1F-1540R primer set was able to differentiate the causative bacteria from Date and those from Shiriuchi and Shikabe. These results indicate that PCR amplification with two primers is useful for identification of Vibrio spp., which are the pathogens of cultured sea urchin.

Two species of culturable bacteria associated with degradation of brown algae Fucus evanescens
EP Ivanova, IY Bakunina, T Sawabe, K Hayashi, YV Alexeeva, NV Zhukova, DV Nicolau, TN Zvaygintseva, VV Mikhailov
MICROBIAL ECOLOGY, 43, 2, 242, 249, SPRINGER, 2002年03月, ［査読有り］
英語, 研究論文（学術雑誌）, The heterotrophic microbial enrichment community established during degradation of brown algae Fucus evanescens was characterized. A two-species bacterial community of marine culturable gamma-proteobacteria consisted of Pseudoalteromonas and Halomonas. The first member of the community, Pseudoalteromonas sp., was highly metabolically active, had bacteriolytic and hemolytic activities, produced proteinases (gelatinase and caseinase), lipases, DNases, and fucoidanhydrolases, laminaranases, alginases, pustulanases, beta-glucosiclases, beta-galactosidases, beta-N-acetylglucosaminidases, and beta-xylosidases. The second member of the community, Halomonas marina, produced only caseinase and DNase, and it did not hydrolyze algal polysaccharides. Both members of the studied bacterial community utilized a range of easily assimilable monosaccharides and other low molecular weight organic substances. The results provide an evidence of the complex metabolic interrelations between two members of this culturable community. One of them Pseudoalteromonas sp., most likely plays the major role in the initial stages of algal degradation; the other one, H. marina, resistant to the bacteriolytic activity of the former, is able to utilize the products of degradation of polysaccharides.

Identification of Vibrio halioticoli by colony hybridization with non-radioisotope labeled genomic DNA probe
R Tanaka, M Ootsubo, T Sawabe, K Tajima, J Vandenberghe, Y Ezura
FISHERIES SCIENCE, 68, 1, 227, 229, JAPANESE SOC FISHERIES SCIENCE, 2002年02月, ［査読有り］
英語, 研究論文（学術雑誌）

Pseudoalteromonas issachenkonii sp nov., a bacterium that degrades the thallus of the brown alga Fucus evanescens
EP Ivanova, T Sawabe, YV Alexeeva, AM Lysenko, NM Gorshkova, K Hayashi, NV Zukova, R Christen, VV Mikhailov
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 52, 229, 234, SOC GENERAL MICROBIOLOGY, 2002年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Eleven non-pigmented strains of Gram-negative, aerobic, marine bacteria with polar flagella were isolated from the thallus of the brown alga Fucus evanescens collected in the Kraternaya Bight of the Kurile Islands in the Pacific Ocean. These organisms were conspecific and exhibited high levels of genetic relatedness (up to 91 %). The G+C contents of the DNAs of these strains were 42.9-43.3 mol%. These halophilic bacteria had bacteriolytic, proteolytic and haemolytic activities and degraded algal polysaccharides, synthesizing a number of glycoside hydrolases (fucoidanases, laminaranases, alginases, agarases, pullulanases, beta-glucosidases, beta-galactosidases, beta-N- acetylglucosaminidases and beta-xylosidases). By 16S rDNA analysis, the bacteria were shown to belong to the genus Pseudoalteromonas, a member of the gamma-subclass of the Proteobacteria. DNA from the strains isolated from the brown alga showed 27-54 % genetic relatedness with respect to DNAs of other type strains of the genus Pseudoalteromonas. The phenotypic characteristics, together with the genetic evidence, indicate that this group of epiphytic bacteria represents a distinct species, Pseudoalteromonas issachenkonii sp. nov., for which the type strain is KMM 3549(T) (= LMG 19697(T)= CIP 106858(T)).

Pseudoalteromonas ruthenica sp nov., isolated from marine invertebrates
EP Ivanova, T Sawabe, AM Lysenko, NM Gorshkova, Svetashev, VI, DV Nicolau, N Yumoto, T Taguchi, S Yoshikawa, R Christen, VV Mikhailov
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 52, 235, 240, SOC GENERAL MICROBIOLOGY, 2002年01月, ［査読有り］
英語, 研究論文（学術雑誌）, On the basis of phenotypic and genotypic characteristics and analysis of 16S rRNA sequences, a novel species belonging to the genus Pseudoalteromonas is described. Two pale-orange-pigmented strains, KMM 300(T) and KMM 290, isolated respectively from a mussel, Crenomytilus grayanus, and a scallop, Patinopecten yessoensis, are marine, Gram-negative, aerobic, rod-shaped bacteria that produce a number of antimicrobial compounds. The strains are able to degrade gelatin, elastin, starch, DNA and Tween 80. Chitin and agar are not degraded. The isolates from marine invertebrates grew at NaCl concentrations of 1-9 % and a temperature range of 10-35 degreesC and did not utilize most of the wide range of carbohydrates tested, with the exception of D-glucose, cellobiose and sucrose. The DNA G+C content was 48.4-48.9 moll %. The level of DNA homology of the two strains was 98 %. DNA from the strains isolated from marine invertebrates showed 5-15 % genetic relatedness to the DNA of other type strains of the genus Pseudoalteromonas. 16S rRNA analysis indicated a clear affiliation of the novel bacteria to other species of the genus. The strains are assigned to a novel species, Pseudomonas ruthenica sp. nov., with the type strain KMM 300(T) (= LMG 19699(T) = CIP 106857(T)).

Pseudoalteromonas maricaloris sp nov., isolated from an Australian sponge, and reclassification of [Pseudoalteromonas aurantia] NCIMB 2033 as Pseudoalteromonas flavipulchra sp nov.
EP Ivanova, LS Shevchenko, TL Sawabe, AM Lysenko, Svetashev, VI, NM Gorshkova, M Satomi, R Christen, VV Mikhailov
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 52, 263, 271, SOC GENERAL MICROBIOLOGY, 2002年01月, ［査読有り］
英語, 研究論文（学術雑誌）, A marine, Gram-negative, aerobic bacterium that produced cytotoxic, lemon-yellow, chromopeptide pigments that inhibited the development of sea urchin eggs has been isolated from the Australian sponge Fascaplysinopsis reticulata Hentschel. The cells of the organism were rod-shaped with a single polar flagellum and they required NaCl for growth (0.5-10%) with optimum growth at 1-3% NaCl. The temperature for growth was 10-37 degreesC, with optimum growth at 25-30 degreesC. Growth occurred at pH values from 6.0 to 10.0, with optimum growth at pH 6.8-8.0. Major phospholipids were phosphatidylethanolamine, phosphatidylglycerol and lyso-phosphatidylethanolamine. Of 26 fatty acids with 11-19 carbon atoms that were detected, 16:1omega7, 16:0, 17:1omega8 and 18:1omega7 were predominant. The DNA G+C content was 38.9 mol%. All of these phenotypic and chemotaxonomic characters place the organism in the genus Pseudoalteromonas (Gauthier et al., 1995). These data are consistent with the phylogenetic analyses that confirmed that strain KMM 636(T) is a member of the Pseudoalteromonas cluster in the gamma-subclass of the Proteobacteria. DNA-DNA hybridization experiments revealed that the levels of relatedness between the DNA of the strain studied and DNAs of type strains of the species that clustered together (on the basis of 16S rDNA sequences) and [Pseudoalteromonas aurantia] NCIMB 2033 ranged from 19 to 35%, and that the DNA-DNA homology between [P. aurantia] NCIMB 2033 and other phylogenetically and/or phenotypically similar type strains ranged from 32 to 52%. According to the polyphasic evidence presented in this study, it is proposed that strain KMM 636(T) (= LMG 19692(T) = CIP 106859(T)) be classified as Pseudoalteromonas maricaloris sp. nov. and [P. aurantia] NCIMB 2033 be reclassified as Pseudoalteromonas flavipulchra NCIMB 2033(T) (= KMM 3630(T) = LMG 20361(T)) sp. nov.

Oligonucleotide probe for detecting Enterobacteriaceae by in situ hybridization
M Ootsubo, T Shimizu, R Tanaka, T Sawabe, K Tajima, M Yoshimizu, Y Ezura, T Ezaki, H Oyaizu
JOURNAL OF APPLIED MICROBIOLOGY, 93, 1, 60, 68, BLACKWELL PUBLISHING LTD, 2002年, ［査読有り］
英語, 研究論文（学術雑誌）, Aims: To develop oligonucleotide probes for visualizing bacteria belonging to Enterobacteriaceae.
Methods and Results: 24-mer oligonucleotide probe (probe D) was designed by comparison of 16S rDNA sequences of 35 species of Enterobacteriaceae, eight species of Vibrionaceae and six species of Pasteurellaceae. The sequence of the probe corresponding to the complementary sequence of a position 1251-1274 of Escherichia coli 16S rRNA was found to be a highly conserved region of 16S rDNA sequence in Enterobacteriaceae different from that of Vibrionaceae and Pasteurellaceae. The fluorescent dye-labelled probe was tested for the specificity by in situ hybridization and epifluorescence microscopy. Seventy-six out of 78 strains belonging to Enterobacteriaceae were visualized in an optimal hybridization condition. No bacterial strains belonging to Vibrionaceae (31 strains) and Gram-positive bacteria (three strains) were visualized.
Conclusions: In situ hybridization using probe D allows the detection of bacterial cells belonging to Enterobacteriaceae without false positive reaction.
Significance and Impact of the Study: In situ hybridization techniques using the probe D are potential tools for detecting Enterobacteriaceae in food and water samples.

Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase
T Sawabe, H Takahashi, Y Ezura, P Gacesa
CARBOHYDRATE RESEARCH, 335, 1, 11, 21, ELSEVIER SCI LTD, 2001年09月, ［査読有り］
英語, 研究論文（学術雑誌）, A gene (alyPEEC) encoding an alginate lyase of Pseudoalteromonas elyakovii IAM 14594 was cloned using the plasmid vector pUC118 and expressed in Escherichia coli. Sequencing of a 3.0kb fragment revealed a 1,197bp open reading frame encoding 398 amino acid residues. The calculated molecular mass and isoelectric point of the alyPEEC gene product are 43.2 kDa and pI 5.29. A region G(165) to V-194 in the AlyPEEC internal sequence is identical to the N-terminal amino acid sequence of the previously purified extracellular alginate lyase of P. elyakovii, and the calculated molecular mass (25.4 kDa) and isoelectric point (pI 4.78) of the region resembled those of the purified enzyme. Expression of enzymically-active alginate lyase from alyPEEC required growth of recombinant E. coli in LB broth containing 50% (v/v) artificial seawater (ASW). Alginate lyase activity with broad substrate specificity was detected in both 42 and 30 kDa products. Subcloning of the region G(165) to N-398 of AlyPEEC corresponding to the 30 kDa protein confirmed that this region of the alyPEEC gene encoded the active site of the enzyme. A region A(32) to G(164) corresponding to about 13 kDa of the N-terminal region of AlyPEEC showed about 30% identity to a putative chitin binding domain of Streptomyces chitinases, but did not exhibit any catalytic activity. (C) 2001 Elsevier Science Ltd. All rights reserved.

Hydrophobic cluster analysis and classification of sixteen bacterial alginate lyases
J Kraiwattanapong, T Ooi, S Kinoshita, Sugimura, I, T Sawabe, Y Ezura
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY, 16, 3, 219, 224, KLUWER ACADEMIC PUBL, 2000年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Sixteen alginate lyases whose primary sequences have been reported were compared, and classified into the following three groups on the basis of the identity of their primary sequences. Strong homology (> 50%): A-AlgL, A-AlgL*, P-AlgL, P-AlgL*, and AlgA; weak homology (> 20%): ALY, AlxM, P-Aly, K-Aly, AlyPG, AlgVGI, AlgVGII, and AlgVGIII; little homology (< 20%): ALYII, Al-III, and AlgVMI. Using hydrophobic cluster analysis (HCA), a secondary structure prediction method, the sixteen alginate lyases were placed into the following classes. Class 1: AlgA, A-AlgL, A-AlgL*, P-AlgL, and P-AlgL*; Class 2: AlgVMI and Al-III; Class 3: ALY and AlxM; Class 4A: ALYII, K-Aly, P-Aly, and AlyPG; Class 4B: AlgVGI and AlgVGII; Class 5: AlgVGIII, which is put in a class of its own, because it is unlike any of the other alginate lyases.

Isolation of Pseudoalteromonas elyakovii strains from spot-wounded fronds of Laminaria japonica
T Sawabe, M Narita, R Tanaka, M Onji, K Tajima, Y Ezura
NIPPON SUISAN GAKKAISHI, 66, 2, 249, 254, JAPANESE SOC FISHERIES SCIENCE, 2000年03月, ［査読有り］
日本語, 研究論文（学術雑誌）, Spot-disease of Laminaria fronds has been occasionally observed in coastal areas of Hokkaido Japan. A huge outbreak of the disease occurred in Laminaria japonica near the southeastern part of Hokkaido in 1998. Three bacterial strains showing Laminaria-frond degrading activity were isolated from the spot-wounded fronds of L. japonica collected at Minamikayabe, Hokkaido, Japan. All the strains were tentatively identified as Alteromonas strain, and two of them were identified as Pseudoalteromonas elyakovii, of which the species included an alginolytic marine bacterium isolated from a spot-disease of Laminaria japonica var, ochotensis occurring at Rishiri Island at 1985. The phenotypic and genotypic characterizations of the Laminaria-frond degrading bacteria were described.

Characteristics of virus like growth suppression agents against phytoplankton obtained from seawater at the mouth of Funka Bay, Hokkaido, Japan
M Onji, T Sawabe, Y Ezura
FISHERIES SCIENCE, 66, 1, 38, 43, JAPANESE SOC FISHERIES SCIENCE, 2000年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Eighteen samples of virus-like growth suppression agents against the marine phytoplankton Alexandrium catenella, Gymnodinium mikimotoi and Tetraselmis sp., obtained from the coastal waters at the mouth of Funka Bay, Hokkaido, Japan, from 1993 to 1995, were characterized on size estimation, heat stability, nuclease sensitivity, proteinase K sensitivity, stability under acidic condition, titration, diethyl ether sensitivity, and ultraviolet (UV) sensitivity. All agents were affected by heating at 50 degrees C for 30 min, exposure to acidic conditions below pH 5.0, passing through a 0.05 mu m filter, RNase treatment, and irradiation of UV dosage at 5 x 10(4) mu W/s per cm(2). The growth suppression effects of 11 agents from Tetraselmis sp, and A. catenella disappeared after proteinase K treatment, however, seven agents from G. mikimotoi were unaffected by this treatment. Furthermore, 11 agents against Tetraselmis sp. and A. catenella were collected from the bottom fraction by ultracentrifugation, while seven agents against G, mikimotoi were collected from the upper fraction, not from the precipitated fraction. These results suggest that at least two types exist in the virus-like agents showing growth suppression for phytoplankton.

Variation of size-fractionated Fe concentrations and Fe(III) hydroxide solubilities during a spring phytoplankton bloom in Funka Bay (Japan)
Kenshi Kuma, Akira Katsumoto, Naonobu Shiga, Tomoo Sawabe, Katsuhiko Matsunaga
Marine Chemistry, 71, 1-2, 111, 123, 2000年, ［査読有り］
英語, 研究論文（学術雑誌）, Vertical distributions of size-fractionated Fe concentrations (<
0.025-μm, 0.025-0.22-μm, <
0.22-μm and >
0.22-μm fractions) and Fe(III) hydroxide solubilities were studied during a spring phytoplankton bloom (February to April, 1995) in Funka Bay, Japan. 'Soluble Fe' (<
0.025-μm fraction) concentrations were approximately 70% of the <
0.22-μm Fe fraction with the exception of a few deeper samples. During the spring bloom, the vertical profiles of soluble Fe were variable (0.3-13 nM), probably resulting from temporal variations of atmospheric input, bottom sediment resuspension, decomposition of sinking organic matter and biological removal in this bay water. High 'colloidal Fe' (0.025-0.22-μm fraction) concentrations (5-8 nM) were found in subsurface water near the shelf sediment interface (50-80-m depths, bottom depth of 92 m) after the spring bloom. The vertical changes of Fe(III) hydroxide solubilities appeared to have a small variation with low values (0.2-0.3 nM) throughout the water column during intense vertical mixing before the bloom, and to increase slightly during the beginning of the bloom (0.3-0.4 nM). The highest Fe(III) solubilities (0.8-0.86 nM) were found in the surface waters (2-10-m depths) during and after the peak of bloom. The high Fe(III) solubilities may be due to higher concentrations, or a stronger affinity of natural organic Fe(III) chelators that were possibly released by phytoplankton or bacteria. (C) 2000 Published by Elsevier Science B.V.

Preparation of neoglycoprotein from carp myofibrillar protein and alginate oligosaccharide: Improved solubility in low ionic strength medium
R Sato, T Sawabe, H Kishimura, K Hayashi, H Saeki
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 48, 1, 17, 21, AMER CHEMICAL SOC, 2000年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Alginate oligosaccharide (AO) was conjugated with carp myofibrillar protein (Mf) by using the controlled Maillard reaction, and the change in the solubility of Mf in low ionic strength media as affected by the glycosylation was investigated. AO was prepared by degrading sodium alginate using alginate lyase, which was purified from the culture supernatant of Pseudoalteromonas elyakovii. When a lyophilized Mf and AO mixture was incubated at 40 degrees C and 65% relative humidity, the conjugation of AO was confirmed at myosin heavy chain, actin, and tropomyosin. When > 30 mu g/mg of AO was conjugated to Mf, the protein solubility in a low ionic strength medium was greatly improved without significant loss of available lysine. These results indicate that the conjugation with AO is a superior manner for improving the water solubility of Mf in view of its nutritional aspect.

Growth suppression of the marine phytoplankton Alexandrium catenella and Tetraselmis sp caused by ultrafiltrated seawater collected from coastal area on the mouth of Funka Bay
M Onji, T Sawabe, Y Ezura
FISHERIES SCIENCE, 65, 5, 687, 693, JAPAN SOC SCI FISHERIES SCI, 1999年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Filtrable pathogens infecting the phytoplankton Alexandrium catenella and Tetraselmis sp. were screened from coastal seawater on the mouth of Funka flay, Hokkaido, Japan from 1993 to 1994. Growth suppression against these phytoplankton species was observed in the seawater samples collected during September and October 1993. The growth of A. catenella was suppressed from 40 to 45%, and that of Tetraselmis sp. was suppressed from 20 to 30%. Re-inoculation of the culture filtrate of growth-suppressed phytoplankton after passing the culture through a 0.22-mu m filter also caused growth suppression of the fresh culture. However, these effects disappeared after several treatments including heating at 50 degrees C for 30 min, exposure to acidic conditions below pH 5.0, passing through a 0.05-mu m filter, and Proteinase K and RNase treatment. Cell free extracts of the growth-suppressed phytoplankton caused the same extent of growth suppression. Electron microscopic observation of A. catenella cells that were lead to the growth suppression revealed that the cells were severely damaged, whereas no virus-like particles or bacterial cells were observed. Growth suppression was observed in a fresh culture of A. catenella and an axenic culture of Gymnodinium mikimotoi by the growth suppressed Tetraselmis sp. culture filtrate, and the A. catenella culture filtrate affected the growth of Tetraselmis sp. and an axenic culture of G. mikimotoi. However, the growth suppression or inhibition was not observed in fresh cultures of Prorocentrum micans, P. minimum, A. tamarense, G. mikimotoi, Chattonella antiqua, C. marina, and Heterosigma akashiwo. These results suggested that unique filterable pathogens might be found in the seawater samples.

Taxonomical and serological studies on the causative bacteria of the disease of sea urchin Strongylocentrotus intermedius occurring at low water temperatures
K Takeuchi, K Tajima, MM Iqbal, T Sawabe, Y Ezura
FISHERIES SCIENCE, 65, 2, 264, 268, JAPAN SOC SCI FISHERIES SCI, 1999年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Identification of the causative bacteria Vibrio spp., which were isolated from diseased sea urchin Strongylocentrotus intermedius and their rearing cages at low seawater temperatures at Date, Shiriuchi and Shikabe Fisheries Breeding Centers in Hokkaido was undertaken based on their biochemical properties, DNA-DNA homology and serological analysis. All fifteen strains, five from each Breeding Center were almost identical in biochemical properties except indole production. However, the strains isolated at Date differed from the strains at Shiriuchi and Shikabe in DNA-DNA homology and analysis of thermostable and thermolabile antigenic compositions. These results indicated that the strains from Shiriuchi and Shikabe belonged to the same species but they were different from those of Date. Moreover, the causative Vibrio spp. differed in biochemical, genotypical and serological properties from any strains of Vibrio spp. isolated from the intestine of healthy sea urchin and any of type strains of the genus Vibrio.

Characterization of a facultatively psychrophilic bacterium, Vibrio rumoiensis sp. nov., that exhibits high catalase activity
Yumoto, I, H Iwata, T Sawabe, K Ueno, N Ichise, H Matsuyama, H Okuyama, K Kawasaki
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 65, 1, 67, 72, AMER SOC MICROBIOLOGY, 1999年01月, ［査読有り］
英語, 研究論文（学術雑誌）, A novel facultatively psychrophilic bacterium, strain S-1, which exhibits extraordinarily high catalase activity was isolated from the drain pool of a fish product processing plant that uses H2O2 as a bleaching and microbicidal agent. The catalase activity of the isolate was 1 or 2 orders of magnitude higher than those of Corynebacterium glutamicum, Staphylococcus aureus, Pseudomonas fluorescens, and five other species tested in this study. The strain seemed to possess only one kind of catalase, according to the results of polyacrylamide gel electrophoresis of the cell extract, The optimum temperature for catalase activity was about 30 degrees C, which was about 20 degrees C lower than that for bovine catalase activity. Electron microscopic observation revealed that the surface of the microorganism was covered by blebs, Although the isolate was nonflagellated, its taxonomic position on the basis of physiological and biochemical characteristics and analysis of 16S rRNA sequence and DNA-DNA relatedness data indicated that strain S-l is a new species belonging to the genus Vibrio. Accordingly, we propose the name Vibrio rumoiensis. The type strain is S-l (FERM P-14531).

Phenotypic and genotypic identification of motile aeromonads isolated from fish with epizootic ulcerative syndrome in Southeast Asian countries
MM Iqbal, K Tajima, T Sawabe, K Nakano, Y Ezura
FISH PATHOLOGY, 33, 4, 255, 263, JAPAN SOC FISH PATHOL DEPT FISHERIES-FAC AGR, 1998年10月, ［査読有り］
英語, 研究論文（学術雑誌）, The phenotypic properties and genotypic characteristics of 44 aeromonads isolated from fish affected by epizootic ulcerative syndrome (EUS) in Southeast Asia were investigated. Among the 13 A. hydrophila phenospecies 9 were genotypically identical to A. hydrophila (HGl) and 4 were A. veronii biotype sobria (HG8Y) or A. veronii biotype sobria-related genospecies. All the 6 A. veronii biotype sobria phenospecies were placed in the same genospecies, A. veronii biotype sobria. Of the 12 A. jandaei phenospecies, 5 were A. veronii biotype sobria or A. veronii biotype sobria-related genospecies and 7 were A. jandaei genospecies. Of 13 Aeromonas isolates unspeciated by phenotyping 9 were genotypically identical to A. hydrophila or very similar to A. hydrophila and 2 isolates were A. veronii biotype sobria or very similar to A. veronii biotype sobria; 2 isolates could not be identified to genospecies level. These results strongly suggest that Aeromonas species from fish affected by EUS could not correctly be identified to the species level using various published biochemical schemes; it needs genetic identification like DNA-DNA hybridization.

Pseudoalteromonas bacteriolytica sp. nov., a marine bacterium that is the causative agent of red spot disease of Laminaria japonica
T Sawabe, H Makino, M Tatsumi, K Nakano, K Tajima, MM Iqbal, Yumoto, I, Y Ezura, R Christen
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, 48, 769, 774, SOC GENERAL MICROBIOLOGY, 1998年07月, ［査読有り］
英語, 研究論文（学術雑誌）, An aerobic, polarly flagellated marine bacterium that produces a prodigiosin-like pigment was isolated from the red-spotted culture beds of Laminaria japonica. Five isolates had unique bacteriolytic activity for both Gram-positive and -negative bacteria, which had never been observed among Alteromonas or spot related species, The isolates were identified as the causative agent or reo disease of L. japonica seeds. The phenotypic features of the isolates were similar to these of Pseudoalteromonas rubra ATCC 29570(T), but they could be differentiated using 10 traits (growth at 37 degrees C, requirement for organic growth factors, bacteriolytic activity, utilization of sucrose, N-acetylglucosamine, fumarate, succinate, D-galactose, L-proline and acetate). The G+C content of DNAs from the isolates was 44-46 mol%. The isolates constitute a new species, distinct from the other Alteromonas and Pseudoalteromonas species, as shown by DNA-DNA hybridization experiments and phylogenetic clustering of 16S rRNA gene sequences, for which the name Pseudoalteromonas bacteriolytica sp. nov. (type strain = IAM 14595(T)) is proposed. A set of phenotypic features which differentiate this new species from closely related Pseudoalteromonas and Alteromonas species is provided.

Intracellular alginate-oligosaccharide degrading enzyme activity that is incapable of degrading intact sodium alginate from a marine bacterium Alteromonas sp.
T Sawabe, C Sawada, E Suzuki, Y Ezura
FISHERIES SCIENCE, 64, 2, 320, 324, JAPAN SOC SCI FISHERIES SCI, 1998年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Intracellular homo-and hetero-polymeric blocks degrading enzyme activity incapable of degrading intact sodium alginate was detected in Alteromonas sp. strain H-4. The enzyme activity for polyM and MG blocks was highest during the late log phase of the bacterium and was not induced by the addition of sodium alginate to the culture medium. The activity for MG random block was as high as that for polyM, but that for polyG block was half and that for sodium alginate was one fifth. At least 4 kinds of enzyme activities, a polyM specific, a MG-polyM specific, and two kinds of polyG specific enzymes, were detected from the crude intracellular fraction, but a trace spot for sodium alginate. Analysis of reaction products using a partially purified preparation of the enzyme indicated that the enzyme generated a saturated diuronate and an unsaturated polyuronide from polyM block. These results suggest that the intracellular enzymes can degrade only oligosaccharides generated from high molecular alginate by the extracellular alginate lyase and may have an important role in the alginate metabolism of the bacterium.

Bacillus horti sp. nov., a new Gram-negative alkaliphilic Bacillus
Yumoto, I, K Yamazaki, T Sawabe, K Nakano, K Kawasaki, Y Ezura, H Shinano
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, 48, 565, 571, SOC GENERAL MICROBIOLOGY, 1998年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Novel Cram-negative alkaliphilic strains were isolated from soil obtained from Atsuma, Hokkaido, Japan. The isolates were strictly aerobic rods that produced subterminally located ellipsoidal spores. Chemotaxonomic characteristics of the isolates included the presence of meso-diaminopimelic acid in the cell wall and a DNA G+C content of 40.2-40.9 mol%. The major isoprenoid quinone was menaquinone-7 and the cellular fatty acid profile consisted of a significant amount of 15.C branched-chain acids, iso-C-15:0 and anteiso-C-15:0. The growth rate was higher at ph 8-10 than at ph 7. Comparative sequence analysis of 16S rDNA of 14 alkaliphilic Bacillus strains indicates that the isolated strain has an equidistant relationship to three already defined rRNA groups of alkaliphilic Bacillus species. Based on the morphological and physiological characteristics, as well as phylogenetic position as determined by 16S rDNA analysis and DNA-DNA relatedness data, it is concluded that these isolates should be designated as a new species, for which the name Bacillus horti is proposed. The type strain is K13(T)(= JCM 9943(T)).

Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system
T Sawabe, Y Ezura, H Yamamoto
PLANT CELL REPORTS, 17, 2, 109, 112, SPRINGER VERLAG, 1997年12月, ［査読有り］
英語, 研究論文（学術雑誌）, A continuous-flow culture system was developed for culturing Laminaria japonica protoplasts. Protoplasts were settled on 5-mu m pore size nylon mesh fixed inside a 50-ml plastic syringe, and cultured in Provasoli's enriched seawater with iodine medium with a gentle upward flow generated by a peristaltic pump. In the culture system, 50% of the protoplasts regenerated their cell wall within 24 hours and almost all protoplasts regenerated a cell wall after 3 days culture. After cell wall regeneration, a number of cells divided and regenerated into sheet-shaped thalli. The thalli transferred to a tissue culture flask developed into sporophyte-like plantlets within 1 month. Plantlets then differentiated into blade, stipe, and holdfast, with a proper mucilage canal.

Detection and identification of infectious hematopoietic necrosis virus (IHNV) by reverse transcription (RT)-polymerase chain reaction (PCR)
T Yoshinaka, M Yoshimizu, T Sawabe, Y Ezura
FISHERIES SCIENCE, 63, 4, 592, 595, SPRINGER JAPAN KK, 1997年08月, ［査読有り］
英語, 研究論文（学術雑誌）, A method based on reverse transcription (RT)-polymerase chain reaction (PCR) was established for detection and identification of infectious hematopoietic necrosis virus (IHNV). A set of primers was prepared for amplification of a 510 bp nucleotide which encodes a region of IHNV nucleoprotein (N) gene. The PCR product was confirmed as the IHNV specific nucleotide by southern hybridization with an oligonucleotide probe synthesized from the N gene. The PCR was able to amplify the target sequence from five representative strains of IHNV from Japan and North America. There was no PCR product from five other fish rhabdoviruses. Viruses from ovarian fluid of masu salmon collected at Shibetsu River, Ichani River in Hokkaido and kidney tissue of rainbow trout in Aichi Prefecture were isolated and identified using the RT-PCR.

リボソーマルタンパク質遺伝子の塩基配列に基づくビブリオの分子系統
志水智美, 田仲真実, 美野さやか, 澤辺智雄, 日本水産学会大会講演要旨集, 2020, 2020年

沖縄トラフ深海底熱水活動域由来の新規好熱性Campylobacteria
深澤操, 美野さやか, 澤辺智雄, 中川聡, 高井研, 日本水産学会北海道支部大会講演要旨集, 2020 (CD-ROM), 2020年

水素生成マリンビブリオのギ酸水素リアーゼ(FHL)複合体遺伝子群の構造比較　　　　　　　　　　　　　　　
西川 紗代, 田仲 真実, 美野 さやか, 澤辺 智雄, 小椋 義俊, 林 哲也, 日本細菌学雑誌, 75, 1, 80, 80, 2020年01月
日本細菌学会, 日本語

Vibriosis in cultured marine fishes: a review
Nurliyana Mohamad, Mohammad Noor Azmai Amal, Ina Salwany Md Yasin, Mohd Zamri Saad, Nurrul Shaqinah Nasruddin, Nurhidayu Al-saari, Sayaka Mino, Tomoo Sawabe, Aquaculture, 512, 2019年10月15日
For more than a century, vibriosis affects various species of economically important cultured marine fishes around the globe. The knowledge of this bacterial disease on many species of cultured fish is still lacking, but progressing. This review focuses on updated fundamental knowledge related to vibriosis including the history, taxonomy, and various epidemiological aspects such as socio-economy, clinical signs, pathological changes, diagnosis, pathogenesis, transmission, risk factors and control measures of vibriosis. This review revealed a rising prevalence of vibriosis in aquaculture, concomitant with the rapid development of this industry worldwide. Yet, information on Vibrio infection in cultured fish, particularly on the Vibrio of non-medical importance, the influence of their virulence toxins to host cells, effects of global warming and the socio-economic impacts are still scarce, and need more profound studies. Moreover, comprehensive epidemiological information on vibriosis are quite limited in many Asian countries with tropical climate, limiting the progression in control and prevention aspects of the disease., Elsevier B.V., 英語, 書評論文,書評,文献紹介等

損傷菌 水産における損傷菌
澤辺 智雄, 里見 正隆, 日本防菌防黴学会誌, 47, 10, 423, 429, 2019年10月
生鮮水産物に存在する細菌種は陸上で生産される食品から分離される細菌種とは異なり、多くの細菌種は増殖に塩分を要求する好塩性細菌で、一部のものは35℃で増殖できない低温性細菌である。また、海洋性細菌には人工の培地ではコロニーを形成できない菌種やVBNC状態の細菌も存在しているため、一般的な培養法では十分な菌数が計数されないことがある。そのため、特殊な損傷菌が多数存在するからでは?との懸念がある。しかし、生鮮魚介類の主要な細菌種はグラム陰性の好気性菌または通性嫌気性菌であり、損傷機構や修復機能は大腸菌などとほぼ同じであると考えられる。本稿では、水産物に関連する微生物の特徴について解説するとともに、水産物の代表的な食中毒菌である腸炎ビブリオの損傷回復機構をRNA-Seqで解析した結果を紹介し、また、水産物で特徴的な食中毒菌であるヒスタミン生成菌の損傷状態でのヒスタミン生成機構についてもとりあげる。(著者抄録), 日本防菌防黴学会, 日本語

ニッケル輸送体遺伝子型は海洋ビブリオの水素産生能と相関する(Type of nickel transporter genes correlate with ability of hydrogen production in marine vibrios)　　　　　　　　　　　　　　　
松村 佑太, 美野 さやか, 丸山 史人, 小椋 義俊, 林 哲也, 黒川 顕, 澤辺 智雄, 日本細菌学雑誌, 73, 1, 72, 72, 2018年02月
日本細菌学会, 英語

水産物における損傷菌の発生と対策
山崎 浩司, 竹内 勘太郎, 山崎 耀平, 美野 さやか, 笠井 久会, 澤辺 桃子, 澤辺 智雄, 里見 正隆, 日本食品科学工学会誌, 65, 4, 197, 204, 2018年
<p>The control of food poisoning associated with fish and fish products is challenging, even in the developed world. Injured cells of bacterial pathogens, generated by various stressors during food processing, are considered to be an important risk factor in food poisoning incidents. It is important to accumulate more knowledge on the physiology, genetics, and molecular biology of injured bacterial cells related to fish and fish products, and also to apply this knowledge to the development of new technologies. Here, we introduce recent advances in the physiology and genetics of <i>Vibrio parahaemolyticus </i>and <i>Listeria monocytogenes </i>and discuss future directions of the risk control of pathogens in fisheries.</p>, 公益社団法人 日本食品科学工学会, 日本語, 書評論文,書評,文献紹介等

アルギン酸から水素を生成可能な新規ビブリオ属細菌の分類学的検討
田仲真実, 美野さやか, 土井秀高, 澤辺桃子, 澤辺智雄, 荒木利芳, マリンバイオテクノロジー学会大会講演要旨集, 19th, 89, 2017年06月03日
日本語

RNA‐Seqで探る腸炎ビブリオのストレス応答
竹内勘太郎, 美野さやか, 澤辺智雄, 澤辺桃子, 里見正隆, 日本水産学会大会講演要旨集, 2017, 84, 2017年03月26日
日本語

ナマコの消化管マイクロバイオーム:メタゲノム解析による成長格差の解明
山崎耀平, 美野さやか, 澤辺智雄, バイオサイエンスとインダストリー, 74, 6, 508‐509, 2016年11月10日
日本語

マリンビブリオを生物触媒としたアルギン酸の水素変換
天田愛梨, 松村祐太, AL‐SAARI Nurhidayu, 美野さやか, 澤辺智雄, 澤辺桃子, 日本水産学会大会講演要旨集, 2016, 33, 2016年09月08日
日本語

Vibrio tritonius AM2株のギ酸水素リアーゼ遺伝子群のトランスクリプトーム解析
水越草太, 松村祐太, AL‐SAARI Nurhidayu, 美野さやか, 澤辺智雄, 澤辺桃子, 日本水産学会大会講演要旨集, 2016, 33, 2016年09月08日
日本語

16SrRNAタイピング法を用いた幼児用簡易プール水の細菌群集構造解析　　　　　　　　　　　　　　　
澤辺 桃子, 須田 亙, 大島 健志朗, 服部 正平, 澤辺 智雄, 日本細菌学雑誌, 71, 1, 63, 63, 2016年02月
日本細菌学会, 日本語

深海底熱水活動域に普遍的に生息する化学合成独立栄養細菌の生物地理学的特徴の解明
美野さやか, 中川聡, 牧田寛子, 工藤桃李, 宮崎淳一, 稲垣史生, 加藤真悟, 布浦拓郎, 井町寛之, 和辻智郎, 高井研, 澤辺智雄, 日本微生物生態学会大会(Web), 31st, 183 (WEB ONLY), 2016年
日本語

ビブリオ科細菌が有するCRISPR/Casシステムの特徴
遠藤 祥子, 美野 さやか, 澤辺 智雄, 日本生物工学会大会講演要旨集, 平成27年度, 201, 201, 2015年09月
(公社)日本生物工学会, 日本語

水素生成マリンビブリオの網羅的発現解析
澤辺智雄, 水越草太, 美野さやか, 松村佑太, 澤辺桃子, マリンバイオテクノロジー学会大会講演要旨集, 17th, 96, 2015年05月30日
日本語

マナマコApostichopus japonicusの個体間微生物叢比較
山崎耀平, 美野さやか, 澤辺智雄, 須田亙, 大島健志朗, 服部正平, MEIRELLES P.M, THOMPSON F.L, マリンバイオテクノロジー学会大会講演要旨集, 17th, 99, 2015年05月30日
日本語

目指せ!細菌学の星☆2015 深海底熱水活動域における固有甲殻類血清の性状解析(We'll be the stars of bacteriology! 2015 Hemagglutination activity in the serum of deep-sea vent endemic crab)　　　　　　　　　　　　　　　
藤吉 奏, 福島 大介, 和辻 智郎, 美野 さやか, 澤辺 智雄, 澤山 茂樹, 高井 研, 中川 聡, 日本細菌学雑誌, 70, 1, 113, 113, 2015年02月
日本細菌学会, 英語

海洋細菌Jejuia pallidilutea 11shimoA1株の新規カロテノイド生合成系解析
高谷直己, 澤辺智雄, 宮下和夫, 細川雅史, マリンバイオテクノロジー学会大会講演要旨集, 17th, 2015年

深海底熱水活動域に生息する化学合成独立栄養細菌の集団遺伝学:遺伝的多様性と分布パターンの解明
美野さやか, 中川聡, 中川聡, 牧田寛子, 宮崎淳一, 加藤真悟, 和辻智郎, 井町寛之, 布浦拓郎, STEFAN Sievert, ANNE Godfroy, 高井研, 澤辺智雄, 日本微生物生態学会大会(Web), 30th, OA‐38 (WEB ONLY), 2015年
日本語

深海底熱水活動域に生息する巻貝類とその共生細菌の群集遺伝学的解析
村上竣一, 益崎庸介, 美野さやか, 澤辺智雄, 宮崎淳一, CHEN Chong, ROGERS Alex, COPLEY Jonathan, 高井研, 中川聡, 日本土壌微生物学会講演要旨集, 2014, 129, 2014年10月16日
日本語

海洋細菌が有するカロテノイド合成系の体系的理解:フラボバクテリアにおけるmyxol合成系の特徴
中西誠人, 高谷直己, 高峰, 宮下和夫, 細川雅史, 大熊盛也, 大島健志朗, 服部正平, 澤辺智雄, 日本水産学会大会講演要旨集, 2014, 34, 2014年09月19日
日本語

The famlily vibrionaceae
Bruno Gomez-Gil, Cristinane C. Thompson, Yuta Matsumura, Toko Sawabe, Tetsuya Iida, Richard Christen, Fabiano Thompson, Tomoo Sawabe, The Prokaryotes: Gammaproteobacteria, 9783642389221, 659, 747, 2014年07月01日
Vibrionaceae embraces the genera Vibrio (1854), Photobacterium (1889), Salinivibrio (1996), Enterovibrio (2002), Grimontia (2003), and Aliivibrio (2007). Totally 131 species are described currently. These described species are mainly marine origin, but important human pathogens, V. cholerae, V. parahaemolyticus, and V. vulnificus, are included. Many obligate fish/shellfish pathogens (e.g., V. anguillarum, P. damselae subsp. piscicida) are also included. Strains showing zoonotic features (between human and fish/shellfish) are known. On the contrary of pathogenic feature of some vibrio species/strains, A. fischeri are known to be mutual symbiont, and many vibrios show beneficial or commensal association against marine animal hosts. Vibrio species is defined as a group of strains forming rods with polar flagella enclosed in a sheath, facultative anaerobic metabolism, capable of fermenting d-glucose and growth at 20 °C. Primarily aquatic, most species are oxidase positive, reduce nitrate to nitrite, require Na+ for growth, and ferment d-fructose, maltose, and glycerol. Each vibrio species is further identified by an array of over 100 phenotypic tests
however, there is not an operational definition for genera within the vibrios. Vibrionaceae species are metabolically versatile
species showing gas production, nitrogen fixation, phototrophy, and nonmotile are increasing. Vibrionaceae species may be better defined on the basis of amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA), more recently by genome comparison. Strains of the same species (including the type strain) share more than 60 % mutual AFLP band pattern similarity and more than 95 % similarity in MLSA (using the loci rpoA, recA, pyrH, ftsA, topA, mreB, gyrB, and gapA). More importantly, strains of the same species and species of the same genus will form monophyletic groups on the basis of MLSA or genome comparison., Springer-Verlag Berlin Heidelberg, 英語

生産・流通・加工工程における体系的な危害要因の特性解明とリスク低減技術の開発〈かび毒・病原微生物〉第4章 水産物における病原微生物のリスク低減技術の開発(1)沿岸漁場および水揚げ場における腸炎ビブリオの動態解明
澤辺智雄, 澤辺桃子, 農林水産省農林水産技術会議事務局研究成果, 522, 156, 160, 2014年03月31日
日本語

O35-04 浅海性無脊椎動物の体腔液に共存する微生物の群集構造解析(口頭発表)
砂田 高志, 中川 聡, 澤辺 智雄, 日本微生物生態学会講演要旨集, 2014, 128, 128, 2014年
日本微生物生態学会, 日本語

1P-144 マリンビブリオを生物触媒としたギ酸塩の水素変換条件の最適化(代謝工学,一般講演)
天田 愛梨, 澤辺 智雄, 日本生物工学会大会講演要旨集, 66, 54, 54, 2014年
日本生物工学会, 日本語

1P-145 バイオ燃料生産性を向上させるマリンビブリオの代謝改変:海洋バイオマスのCBP に向けたVibrio halioticoliへのデンプン代謝能付与(代謝工学,一般講演)
杉谷 舞, 澤辺 智雄, 日本生物工学会大会講演要旨集, 66, 54, 54, 2014年
日本生物工学会, 日本語

2P-026 水素生成マリンビブリオの網羅的遺伝子発現解析:基質の還元度が中央代謝系に与える影響(遺伝子工学,一般講演)
水越 草太, 丸山 史人, 小椋 義俊, 林 哲也, 黒川 顕, 澤辺 智雄, 日本生物工学会大会講演要旨集, 66, 113, 113, 2014年
日本生物工学会, 日本語

深海底熱水活動域に生息する無脊椎動物の血リンパ中レクチンの探索と性状解析
藤吉奏, 和辻智郎, 美野さやか, 澤辺智雄, 澤山茂樹, 高井研, 中川聡, 中川聡, 日本土壌微生物学会講演要旨集, 2014, 2014年

Sulfurovum aggregans sp. nov., A hydrogenoxidizing, Thiosulfate-reducing chemolithoautotroph within the Epsilonproteobacteria isolated from a deep-sea hydrothermal vent chimney, And an emended description of the genus Sulfurovum
Sayaka Mino, Hideaki Kudo, Takayuki Arai, Tomoo Sawabe, Ken Takai, Satoshi Nakagawa, Satoshi Nakagawa, Satoshi Nakagawa, International Journal of Systematic and Evolutionary Microbiology, 64, 3195, 3201, 2014年01月01日
© 2014 IUMS. A novel mesophilic, strictly hydrogen-oxidizing, sulfur-, nitrate-and thiosulfate-reducing bacterium, designated strain Monchim33T, was isolated from a deep-sea hydrothermal vent chimney at the Central Indian Ridge. The non-motile, rod-shaped cells were Gram-stain-negative and non-sporulating. Growth was observed between 15 and 37 °C (optimum 33 °C; 3.2 h doubling time) and between pH 5.4 and 8.6 (optimum pH 6.0). The isolate was a strictly anaerobic chemolithoautotroph capable of using molecular hydrogen as the sole energy source and carbon dioxide as the sole carbon source. The G+C content of the genomic DNA was 42.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel isolate belonged to the genus Sulfurovum and was closely related to Sulfurovum sp. NBC37-1 and Sulfurovum lithotrophicum 42BKT (95.6 and 95.4% similarity, respectively). DNA-DNA hybridization demonstrated that the novel isolate could be differentiated genotypically from Sulfurovum sp. NBC37-1 and Sulfurovum lithotrophicum. On the basis of the molecular and physiological traits of the new isolate, the name Sulfurovum aggregans sp. nov. is proposed, with the type strain Monchim33T (=JCM 19824T=DSM 27205T).

深海底熱水活動域に生息する化学合成微生物の分布様式と集団構造の解明
美野さやか, 中川聡, 牧田寛子, 宮崎淳一, 和辻智郎, 渡部裕美, 西澤学, 中村謙太郎, 布浦拓郎, 井町寛之, 山本正浩, 高井研, 加藤真悟, 小島茂明, 島伸和, 石橋純一郎, 澤辺智雄, ブルーアース要旨集, 2014, 106, 2014年
日本語

海藻糖質の代謝過程におけるVibrio halioticoliの遺伝子発現解析
久我康太, 中川聡, 丸山史人, 小椋義俊, 林哲也, 黒川顕, 澤辺桃子, 澤辺智雄, 日本生物工学会大会講演要旨集, 65th, 134, 2013年08月25日
日本語

めざせ!細菌学の星 マリンビブリオの水素生産能の評価および実用化へのアプローチ(Young researchers workshop on bacteriology 2013 Characterization of hydrogen producing marine vibrios and the practical approach)　　　　　　　　　　　　　　　
佐藤 一道, 澤辺 智雄, 日本細菌学雑誌, 68, 1, 103, 103, 2013年02月
日本細菌学会, 英語

3P-167 Vibrio tritonius AM2^Tを用いた海藻糖質からの水素生産システムの構築(生物化学工学,一般講演)
佐藤 一道, 天田 愛梨, 中川 聡, 澤辺 智雄, 日本生物工学会大会講演要旨集, 65, 230, 230, 2013年
日本生物工学会, 日本語

海洋細菌Jejuia pallidilutea A1株の新規カロテノイド合成とストレス応答
高谷直己, 西田健太郎, 澤辺智雄, 宮下和夫, 細川雅史, マリンバイオテクノロジー学会大会講演要旨集, 15th, 2013年

深海底熱水活動域に棲息する大型無脊椎動物の血清中レクチンの探索と性状解析
藤吉奏, 中川聡, 美野さやか, 神藤彩加, 新井崇之, 澤辺智雄, 杉村誠, 和辻智郎, 山本正浩, 丸茂克美, ブルーアース要旨集, 2013, 2013年

Vibrio halioticoliの代謝改変:Zymomonas mobilis由来のピルビン酸脱炭酸酵素およびアルコール脱水素酵素IIの発現
猪原悠太郎, 中川聡, 澤辺智雄, 日本生物工学会大会講演要旨集, 65th, 2013年

深海底熱水活動域の固有甲殻類が有するレクチンの精製と性状解析
藤吉奏, 新井崇之, 美野さやか, 和辻智郎, 澤辺智雄, 中川聡, 日本糖質学会年会要旨集, 32nd, 2013年

食品の安全を確保するための評価・管理技術の開発 第1章 有害微生物検出技術の開発 3 高感度蛍光マイクロコロニー法による食中毒細菌の複数同時検出・定量システムの開発
山崎浩司, 澤辺智雄, 農林水産省農林水産技術会議事務局研究成果, 491, 32, 37, 2013年01月
日本語, 機関テクニカルレポート，技術報告書，プレプリント等

Updating the Vibrio clades defined by multilocus sequence phylogeny: Proposal of eight new clades, and the description of Vibrio tritonius sp. nov.
Tomoo Sawabe, Yoshitoshi Ogura, Yuta Matsumura, Gao Feng, A. K.M. Rohul Amin, Sayaka Mino, Satoshi Nakagawa, Toko Sawabe, Ramesh Kumar, Yohei Fukui, Masataka Satomi, Ryoji Matsushima, Fabiano L. Thompson, Bruno Gomez-Gil, Richard Christen, Fumito Maruyama, Ken Kurokawa, Tetsuya Hayashi, Frontiers in Microbiology, 4, 414, 1, 14, 2013年
To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera
Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on (1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis)
(2) clades amended since the 2007 proposal with recently described new species
(3) orphan clades of genomospecies F6 and F10
(4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3)
and (5) description of V. tritonius sp. nov., which is a member of the "Porteresiae" clade. © 2013 Sawabe, Ogura, Matsumura, Feng, Amin, Mino, Nakagawa, Sawabe, Kumar, Fukui, Satomi, Matsushima, Thompson, Gomez-Gil, Christen, Maruyama, Kurokawa and Hayashi., Frontiers Research Foundation, 英語

PK-05 深海底熱水活動域に棲息する無脊椎動物の血清中レクチンの性状解析(PK 相互作用・共生,ポスター発表)
藤吉 奏, 新井 崇之, 神藤 彩加, 美野 さやか, 和辻 智郎, 杉村 誠, 澤辺 智雄, 丸茂 克美, 中川 聡, 日本微生物生態学会講演要旨集, 28, 171, 171, 2012年
日本微生物生態学会, 英語

PK-04 深海底熱水活動域に棲息する化学合成独立栄養細菌の細胞表層に存在する糖脂質の構造比較(PK 相互作用・共生,ポスター発表)
神藤 彩加, 新井 崇之, 藤吉 奏, 美野 さやか, 和辻 智郎, 杉村 誠, 澤辺 智雄, 丸茂 克美, 中川 聡, 日本微生物生態学会講演要旨集, 28, 171, 171, 2012年
日本微生物生態学会, 英語

PK-03 深海性共生細菌とその自由生活型近縁種が有するリポ多糖の構造解析(PK 相互作用・共生,ポスター発表)
新井 崇之, 藤吉 奏, 神藤 彩加, 広瀬 知弘, YK09-13乗船研究者一同, 澤辺 智雄, 中川 聡, 日本微生物生態学会講演要旨集, 28, 171, 171, 2012年
日本微生物生態学会, 英語

ビブリオは線毛タンパク質PilAを介して海産魚腸内受容体に接着する
松吉亮, 徳永遼平, 松永尚之, 澤辺智雄, 沖野望, 伊東信, 日本水産学会大会講演要旨集, 2012, 2012年

深海底熱水活動域に固有の無脊椎動物が有する血清中レクチンの性状
藤吉奏, 新井崇之, 神藤彩加, 美野さやか, 和辻智郎, 杉村誠, 澤辺智雄, 丸茂克美, 中川聡, 日本糖質学会年会要旨集, 31st, 2012年

ベントスの腸内細菌叢
澤辺 智雄, 中川 聡, 澤辺 桃子, 日本水産學會誌 = Bulletin of the Japanese Society of Scientific Fisheries, 78, 4, 783, 783, 2012年
公益社団法人 日本水産学会, 日本語

PK-02 複数の深海底熱水活動域における固有巷貝とその共生細菌の群集遺伝学的解析(PK 相互作用・共生,ポスター発表)
益崎 庸介, 美野 さやか, 渡部 裕美, 宮崎 淳一, 布浦 拓郎, 小島 茂明, 澤辺 智雄, 中川 聡, 日本微生物生態学会講演要旨集, 28, 170, 170, 2012年
日本微生物生態学会, 英語

深海底熱水活動域に生息する化学合成微生物の遺伝学的多様性と群集構造の解明
美野さやか, 中川聡, 澤辺智雄, 宮崎淳一, 牧田寛子, 和辻智朗, 中村謙太郎, 加藤真悟, 小島茂明, 渡部裕美, 土岐知弘, 島伸和, 石橋純一郎, 布浦拓郎, 山本正浩, 高井研, ブルーアース要旨集, 2012, 185, 2012年
日本語

New frontier in Fisheries Sciences beyond the current microbial genome research
Yoshihiko Sako, Takashi Yoshida, Tomoo Sawabe, NIPPON SUISAN GAKKAISHI, 77, 2, 246, 246, 2011年03月
JAPANESE SOC FISHERIES SCIENCE, 日本語, その他

ゲノム情報を活用した病原ビブリオの分子疫学
澤辺 智雄, 日本水産學會誌 = Bulletin of the Japanese Society of Scientific Fisheries, 77, 2, 251, 251, 2011年03月
公益社団法人 日本水産学会, 日本語, その他

腸炎ビブリオの走性行動の可視化
澤辺 桃子, 坂牛 百合香, 澤辺 智雄, 函館短期大学紀要, 37, 41, 45, 2011年03月
函館短期大学, 日本語

インド洋の深海底熱水活動域に棲息する細胞内共生微生物のゲノム解析
中川聡, 島村繁, 高木善弘, 高井研, 澤辺智雄, ブルーアース要旨集, 2011, 2011年

PB-006 Vibrio tritoniusのアルコール脱水素酵素遺伝子の多様性(PB 生理/代謝/増殖,ポスター発表)
李 羨, 中川 聡, 丸山 史人, 小椋 義俊, 林 哲也, 黒川 顕, 澤辺 智雄, 日本微生物生態学会講演要旨集, 27, 85, 85, 2011年
日本微生物生態学会, 日本語

腸炎ビブリオによるカキの汚染動態解析
坂牛百合香, 中川聡, 澤辺智雄, 大橋智志, 矢野豊, 里見正隆, 日本水産学会大会講演要旨集, 2011, 2011年

はじめに
左子 芳彦, 吉田 天士, 澤辺 智雄, 日本水産学会誌, 77, 2, 2011年
The Japanese Society of Fisheries Science, 日本語

PA-043 深海底熱水活動域から分離・培養された新規好熱性Acidobacteriaの性状解析(PA 極限環境,ポスター発表)
和泉 裕志, 布浦 拓郎, 宮崎 征行, 美野 さやか, 土岐 知弘, 高井 研, 左子 芳彦, 澤辺 智雄, 中川 聡, 日本微生物生態学会講演要旨集, 27, 76, 76, 2011年
日本微生物生態学会, 日本語

ビブリオの多様性と進化
澤辺 智雄, 日本細菌学雑誌, 65, 2-3-4, 333, 342, 2010年12月
ビブリオは、コレラ菌、腸炎ビブリオ及びビブリオ・バルニフィカスというヒト病原性種を含むことから、病原微生物学から環境微生物学までにおける幅広い学術分野でモデル微生物として活発に研究されている細菌群である。コレラ菌の発見から156年を経過した今でも、新種のビブリオが発見され続けており、自然界におけるビブリオの種多様性は驚くほど高い。1965年にM.Veronによって作られたビブリオ科(Vibrionaceae)は、6属、103種が包含される巨大な分類群となっている。ビブリオで新種が記載され続ける背景には、株の遺伝的多様性を精密に検出する指紋鑑定法や個体識別法が取り入れられたことにある。また、個体識別の過程で得られる多座位の遺伝子配列を解析することにより、ビブリオの進化の系譜や種分化機構の推定も試みられるようになってきた。さらに、ビブリオをモデルとしたゲノム情報に基づく分類規範の構築も始まっている。本稿ではビブリオの分類の歴史を紐解きながら、ビブリオの多様性と進化に関する最新の成果をまとめて紹介する。(著者抄録), 日本細菌学会, 日本語

腸炎ビブリオの魚介類粘液に対する走性応答の可視化　　　　　　　　　　　　　　　
澤辺 桃子, 坂牛 百合香, 清水 陽子, 澤辺 智雄, 日本食品衛生学会学術講演会講演要旨集, 100回, 136, 136, 2010年09月
(公社)日本食品衛生学会, 日本語

培養併用蛍光in situハイブリダイゼーション(FISHFC)法によりミネラルウォーター中の飢餓緑膿菌を検出するための微小集落形成培養条件
澤辺 桃子, 大坪 雅史, 澤辺 智雄, 函館短期大学紀要, 36, 23, 28, 2010年03月
函館短期大学, 日本語

P-55 インド洋の深海底熱水活動域に棲息する細胎内共生微生物のゲノム解析(D.極限環境,ポスター発表)
中川 聡, 島村 繁, 高木 善弘, 高井 研, 澤辺 智雄, YK09-13 LEG2乗船研究者-同, 日本微生物生態学会講演要旨集, 26, 122, 122, 2010年
日本微生物生態学会, 日本語

P-49 深海底熱水噴出孔から分離された新規微生物の分類学的・生理学的特徴(D.極限環境,ポスター発表)
森 直哉, 中川 聡, GODFROY ANNE, 高井 研, 澤辺 智雄, 日本微生物生態学会講演要旨集, 26, 119, 119, 2010年
日本微生物生態学会, 日本語

P-69 浅海性無脊椎動物の体腔液から検出された微生物の分子系統学的性状(E.群集構造解析,ポスター発表)
相田 正典, 中川 聡, 澤辺 智雄, 日本微生物生態学会講演要旨集, 26, 129, 129, 2010年
日本微生物生態学会, 日本語

海洋好熱細菌Nitratiruptor sp.由来アルギン酸リアーゼの性状解析
井上晶, 安楽萌, 中川聡, 澤辺智雄, 尾島孝男, 日本水産学会大会講演要旨集, 2010, 2010年

GFP標識ビブリオ病原因菌Vibrio harveyiの糖脂質受容体への接着
清水耕平, 苣田慎一, 松永尚之, 座間宏太, 澤辺智雄, 沖野望, 伊東信, 日本水産学会大会講演要旨集, 2010, 2010年

エゾアワビ肝膵臓におけるアルギン酸代謝に関する研究
木本雄太, 熊谷祐也, 澤辺智雄, 田中啓之, 井上晶, 尾島孝男, 日本水産学会大会講演要旨集, 2010, 2010年

P-50 深海底に生息する化学合成微生物の群集遺伝学的構造解明へのアプローチ(D.極限環境,ポスター発表)
美野 さやか, 中川 聡, 牧田 寛子, 稲垣 史生, 山本 正浩, 布浦 拓郎, 中村 光一, GODFROY ANNE, 高井 研, 澤辺 智雄, 日本微生物生態学会講演要旨集, 26, 119, 119, 2010年
日本微生物生態学会, 日本語

培養併用蛍光in situハイブリダイゼーション法を用いた腸炎ビブリオの計数　　　　　　　　　　　　　　　
川西 優子, 澤辺 桃子, 大坪 雅史, 澤辺 智雄, 日本食品衛生学会学術講演会講演要旨集, 98回, 83, 83, 2009年10月
(公社)日本食品衛生学会, 日本語

GFP標識によるビブリオ属細菌と糖脂質受容体の接着の可視化　　　　　　　　　　　　　　　
清水 耕平, 苣田 慎一, 松永 尚之, 座間 宏太, 澤辺 智雄, 沖野 望, 伊東 信, 日本生化学会大会プログラム・講演要旨集, 82回, 4P, 066, 2009年09月
(公社)日本生化学会, 日本語

『海の微生物の利用-未知なる宝探し-』, 今田千秋著, 成山堂書店, ISBN978-4-425-88451-3, 1,680円
澤辺 智雄, 日本微生物生態学会誌, 24, 2, 77, 78, 2009年
日本微生物生態学会, 日本語

微生物モニタリングに関する新システムの開発
大坪 雅史, MASASHI OOTSUBO, 日本水産学会誌, 74, 2, 253, 254, 2008年03月15日
公益社団法人 日本水産学会, 日本語

Shewanella属菌のOXA-型カルバペネマーゼ保有状況に関する検討
石井 良和, 嵯峨 知生, 山本 啓之, 野中 里佐, 山口 惠三, 神山 裕一, 澤辺 智雄, 里見 正隆, 日本細菌学雑誌, 63, 1, 106, 106, 2008年02月
日本細菌学会, 日本語

11-220 海水温上昇傾向が海洋動物のVibrio harveyi感染症に与える影響(水圏生態系,研究発表)
福井 洋平, 澤辺 智雄, 日本微生物生態学会講演要旨集, 24, 168, 168, 2008年
日本微生物生態学会, 日本語

テラフォーミング : 環境微生物学の挑戦(学際シンポジウム報告)
澤辺 智雄, 宍戸 雅宏, 吉永 郁生, 日本微生物生態学会誌, 23, 2, 73, 74, 2008年
日本微生物生態学会, 日本語

『食中毒のリスクと人間社会』, 清水潮著, 幸書房, B6判, 225頁, 1,890円, ISBN978-4782103142
澤辺 智雄, 日本微生物生態学会誌, 23, 2, 75, 75, 2008年
日本微生物生態学会, 日本語

06-109 マナマコ付随性微生物の多様性(群集構造解析,研究発表)
榎本 雅基, 澤辺 智雄, 日本微生物生態学会講演要旨集, 24, 113, 113, 2008年
日本微生物生態学会, 日本語

10-199 培養併用in situ蛍光ハイブリダイゼーション法によるCampylobacter jejuni及びCampylobacter coliの迅速検出(環境衛生,研究発表)
澤辺 智雄, 吉澤 愛, 澤辺 桃子, 大坪 雅史, 伊藤 武, 日本微生物生態学会講演要旨集, 24, 158, 158, 2008年
日本微生物生態学会, 日本語

Specific detection of Listeria spp. using fluorescent in situ hybridization in combination with filter cultivation (FISHFC)　　　　　　　　　　　　　　　
Yamazaki, K, Fuchizawa, I, Kawai, Y, Sawabe, T, Ootsubo, M, Proceedings of the United States – Japan Cooperative Program in Natural Resources (UJNR) Food and Agriculture Panel 37th Annual Meeting, 60, 61, 2008年
英語, 記事・総説・解説・論説等（国際会議プロシーディングズ）

Real-time monitoring of microbes in aquatic environments and seafood
Tomoo Sawabe, NIPPON SUISAN GAKKAISHI, 73, 2, 290, 291, 2007年03月
JAPANESE SOC FISHERIES SCIENCE, 日本語, その他

PB-30 加熱損傷したEnterobacteriaceaeの培養併用FISH法による迅速検出(モニタリング,ポスターセッションB,ポスター発表)
大坪 雅史, 剣持 美帆, 宮原 則行, 山崎 浩司, 澤辺 智雄, 高橋 信行, 小高 秀正, 伊藤 武, 日本微生物生態学会講演要旨集, 23, 129, 129, 2007年
日本微生物生態学会, 日本語

PB-23 培養併用蛍光インサイチューハイブリダイゼーション法による腸炎ビブリオの検出(モニタリング,ポスターセッションB,ポスター発表)
澤辺 智雄, 吉澤 愛, 大坪 雅史, 日本微生物生態学会講演要旨集, 23, 126, 126, 2007年
日本微生物生態学会, 日本語

PB-24 培養併用蛍光インサイチューハイブリダイゼーション法による緑膿菌の迅速検出(モニタリング,ポスターセッションB,ポスター発表)
吉澤 愛, 大坪 雅史, 澤辺 智雄, 日本微生物生態学会講演要旨集, 23, 126, 126, 2007年
日本微生物生態学会, 日本語

南極舟底池および子鉢池における真核微生物群集
三宅絵理, 澤辺智雄, 大内真理子, 堀口健雄, 松崎雅広, 伊村智, 極域気水圏シンポジウムプログラム・講演要旨, 29th, 132, 2006年11月
日本語

C-10 腸内細菌科細菌の迅速計数をモデルとした培養併用FISH法の食品検査への適応範囲(食品衛生,口頭発表)
大坪 雅史, 剣持 美帆, 宮原 則行, 清水 健志, 澤辺 智雄, 山崎 浩司, 日本微生物生態学会講演要旨集, 21, 254, 254, 2005年
日本微生物生態学会, 日本語

アワビ消化管から分離されたVibrio halioticoli類縁種の遺伝的多様性を反映する遺伝子
沢辺智雄, 森脇潤, 田島研一, 絵面良男, 日本水産学会大会講演要旨集, 2004, 2004年

29-B-03 培養併用FISH法による食品および環境からの腸内細菌科細菌の迅速定量(食品衛生,一般講演)
大坪 雅史, 澤辺 智雄, 田島 研一, 日本微生物生態学会講演要旨集, 19, 136, 136, 2003年
日本微生物生態学会, 日本語

アワビ消化管内から分離された新規海洋細菌の分類
林香倫, 森脇潤, 沢辺智雄, 田島研一, 絵面良男, 日本水産学会大会講演要旨集, 2003, 2003年

アワビ消化管から分離されたVibrio halioticoliおよびその類似菌の16S rDNAとgap遺伝子に基づく系統解析
森脇潤, 沢辺智雄, 田島研一, 絵面良男, 日本水産学会北海道支部大会講演要旨集, 2002, 2002年

Distribution of Vibrio halioticoli around an Abalone-farming Center in Japan
Reiji Tanaka, Tomoo Sawabe, Mamoru Yoshimizu, Yoshio Ezura, Microbes and Environments, 17, 1, 6, 9, 2002年
Vibrio halioticoli is the dominant bacterial species in the gut of abalone, Haliotis discus hannai. The bacterium may contribute to the digestion of seaweed, and form a symbiotic association with the host. However, the process by which the bacterium colonizes the gut of abalone is unknown and intriguing given the non-motile nature of V. halioticoli. To clarify the colonization process, the distribution of the bacterium in seawater, as well as the fecal material and diet of abalone was investigated at an abalone farm. Viable bacterial counts of V. halioticoli were determined in each sample using a colony hybridization technique specifically designed for the bacterium. The count in seawater and culture seawater was 3 CFU/ml. The number of V. halioticoli in a diatom bed used as a diet for juvenile abalone was 7.9×102 CFU/g. Fractionation of the diatom bed sample using 0.2 and 8.0-μm filters indicated that the viable count was higher in the attached fraction (3.4×102 CFU/g
>
8.0 μm) than in free-living fraction (76 CFU/g
0.2 μm-8.0 μm). Furthermore, viable V. halioticoli was estimated to be 3.6×103 CFU/g in the fecal material of juvenile abalone which had ingested diatoms, as compared to 1.2×105 CFU/g in that of juveniles fed on artificial diet. These results suggest that V. halioticoli cells in seawater and/or on diatoms contribute to the colonization of the gut in abalone. © 2002, Japanese Society of Microbial Ecology &
The Japanese Society of Soil Microbiology. All rights reserved., 英語

Purification of A Virus-like Particle Causing Growth Suppression of Alexandrium catenella
Onji Masashi, Sawabe Tomoo, Ezura Yoshio, 北海道大学水産科学研究彙報, 52, 3, 135, 138, 2001年12月
北海道大学, 英語

Retrieval of the species Alteromonas tetraodonis Simidu et al. 1990 as Pseudoalteromonas tetraodonis comb. nov and emendation of description
EP Ivanova, LA Romanenko, MH Matte, GR Matte, AM Lysenko, U Simidu, K Kita-Tsukamoto, T Sawabe, MV Vysotskii, GM Frolova, Mikhailov, V, R Christen, RR Colwell, INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 51, 1071, 1078, 2001年05月
A polyphasic taxonomy study was undertaken of three strains of Pseudoalteromonas haloplanktis subsp. tetraodonis (Simidu et ah 1990) Gauthier et al. 1995. DNA was prepared from each of the strains and genomic relatedness was measured by DNA-DNA hybridization. Strains KMM 458(T) and IAM 14160(T) shared 99 % genetic relatedness, but were only 48-49 % related to the type strain of Pseudoalteromonas haloplanktis subsp. haloplanktis, IAM 12915(T). The third strain, P, haloplanktis subsp. tetraodonis A-M, showed 83 % genetic similarity with P. haloplanktis subsp, haloplanktis IAM 12915(T) and 32 % with KMM 458(T). From these results, it is concluded that strains KMM 458(T) and IAM 14160(T) comprise a separate species, originally described as Alteromonas tetraodonis, whereas strain A-M belongs to the species Pseudoalteromonas haloplanktis. Based on phenotypic and chemotaxonomic data, genomic fingerprint patterns, DNA-DNA hybridization data and phylogenetic analysis of 16S rRNA, it is proposed that the species Alteromonas tetraodonis be retrieved and recognized as Pseudoalteromonas tetraodonis comb. nov. (type strain IAM 14160T = KMM 458T)., SOC GENERAL MICROBIOLOGY, 英語

Shewanella japonica sp nov.
EP Ivanova, T Sawabe, NM Gorshkova, Svetashev, VI, VV Mikhailov, DV Nicolau, R Christen, INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 51, 1027, 1033, 2001年05月
Two strains of agar-digesting bacteria, KMM 3299(T) and KMM 3300, respectively isolated from sea water and the mussel Protothaca jedoensis, have been characterized. Based on sequencing of the 16S rRNA gene, KMM 3299T showed the highest similarity (93-95 %) to members of the genus Shewanella. The G+C contents of the DNAs of these strains were 43-44 mol%. The level of DNA homology between the two strains was conspecific (95 %), indicating that they represent a distinct genospecies. These organisms were non-pigmented, Gramnegative, polarly flagellated, facultatively anaerobic, mesophilic, neutrophilic and able to degrade a wide range of high molecular mass polymers, including alginate, carrageenan, laminaran and agar. The novel organisms were susceptible to gentamycin, carbenicillin, lincomycin and oleandomycin. The predominant cellular fatty acids were i-15:0, 16:0, 16:1(n-7), 18:1(n-7). Eicosapentaenoic acid, 20:5(n-3), was detected in the two isolates at levels of 1-8 %, depending on the temperature of cultivation. Phylogenetic evidence, together with phenotypic characteristics, showed that the two isolates studied constitute a novel species of the genus Shewanella. The name Shewanella japonica is proposed; the type strain is KMM 3299(T)(= LMG 19691(T) = CIP 106860(T))., SOC GENERAL MICROBIOLOGY, 英語

Identification of Vibrio halioticoli using 16s rDNA PCR/RFLP (restriction fragment length polymorphism) analysis
Reiji Tanaka, Tomoo Sawabe, Kenichi Tajima, Johan Vandenberghe, Yoshio Ezura, Fisheries Science, 67, 1, 185, 187, 2001年02月
英語

An Evaluation of Viable Staining Dyes Suitable for Marine Phytoplankton
Onji Masashi, Sawabe Tomoo, Ezura Yoshio, 北海道大學水産學部研究彙報, 51, 3, 153, 157, 2000年12月
北海道大学, 英語

環境に調和した水産業と微生物利用
澤辺 智雄, 農林水産技術研究ジャーナル, 23, 11, 36, 43, 2000年11月01日
農林水産技術情報協会, 日本語

Properties of novel alginolytic marine bacteria and their applications for fisheries science
T Sawabe, NIPPON SUISAN GAKKAISHI, 66, 4, 615, 618, 2000年07月
JAPANESE SOC FISHERIES SCIENCE, 日本語, 書評論文,書評,文献紹介等

アルギン酸分解性海洋細菌とその応用に関する研究
澤辺 智雄, 絵面 良男, Tomoo Sawabe, 日本水産学会誌 = Bulletin of the Japanese Society of Scientific Fisheries, 66, 3, 368, 368, 2000年05月15日
日本語

C-115 異体類の腸内細菌叢と抗ウイルス活性を有する細菌の分離(水圏生態系-4、水処理生態系-3,ポスター発表)
吉水 守, 葛西 砂織, 澤辺 智雄, 絵面 良男, 日本微生物生態学会講演要旨集, 16, 107, 107, 2000年
日本微生物生態学会, 日本語

C-214 各種海産無脊椎動物消化管でのVibrio halioticoliの分布(土壌生態系-4、消化管生態系,ポスター発表)
澤辺 智雄, 瀬戸口 央, 田中 礼士, 吉水 守, 絵面 良男, 日本微生物生態学会講演要旨集, 16, 150, 150, 2000年
日本微生物生態学会, 日本語

海洋細菌の分類と同定 (総特集 海洋微生物) -- (1章 海洋微生物の分類,同定)
絵面 良男, 田島 研一, 澤辺 智雄, 号外海洋, 23, 12, 17, 2000年
海洋出版, 日本語

In situ polymerase chain reaction visualization of Vibrio halioticoli using alginate lyase gene AlyVG2
Sugimura, I, T Sawabe, Y Ezura, MARINE BIOTECHNOLOGY, 2, 1, 74, 79, 2000年01月
A prokaryotic in situ polymerase chain reaction (PI-PCR) technique was applied to visualize Vibrio halioticoli cells using alginate lyase gene alyVG2 as a target gene. Prior to PI-PCR, a primer set, VG2-OS3, for specific amplification of an approximately 1.0-kb fragment from V. halioticoli genomic DNA was developed with amplified fragments from V. pelagius and V. fischeri DNAs as reference strains. One-stage PI-PCR using the primer set, digoxigenin-labeled dUTP, and indirect alkaline-phosphatase-linked fluorescence detection technique (HNPP/Fast Red TR as a substrate) failed to differentiate V. halioticoli IAM14596(T) cells from ATCC25916(T) cells of the closely related species V. pelagius. However, two-stage PI-PCR adding the extension and digoxigenin-labeling step of the amplified fragment into the first amplification stage allowed us to differentiate V. halioticoli cells from V. pelagius cells., SPRINGER VERLAG, 英語

Cloning and sequence analysis of Vibrio halioticoli genes encoding three types of polyguluronate lyase
Sugimura, I, T Sawabe, Y Ezura, MARINE BIOTECHNOLOGY, 2, 1, 65, 73, 2000年01月
The alginate lyase-coding genes of Vibrio halioticoli IAM 14596(T), which was isolated from the gut of the abalone Haliotis discus hannai, were cloned using plasmid vector pUC 18, and expressed in Escherichia coli. Three alginate lyase-positive clones, pVHB, pVHC, and pVHE, were obtained, and all clones expressed the enzyme activity specific for polyguluronate. Three genes, alyVG1, alyVG2, and alyVG3, encoding polyguluronate lyase were sequenced: alyVG1 from pVHB was composed of a 1056-bp open reading frame (ORF) encoding 352 amino acid residues; alyVG2 gene from pVHC was composed of a 993-bp ORF encoding 331 amino acid residues; and alyVG3 gene from pVHE was composed of a 705-bp ORF encoding 235 amino acid residues. Comparison of nucleotide and deduced amino acid sequences among AlyVG1, AlyVG2, and AlyVG3 revealed low homologies. The identity value between AlyVG1 and AlyVG2 was 18.7%, and that between AlyVG2 and AlyVG3 was 17.0%. A higher identity value (26.0%) was observed between AlyVG1 and AlyVG3. Sequence comparison among known polyguluronate lyases including AlyVG1, AlyVG2, and AlyVG3 also did not reveal an identical region in these sequences. However, AlyVG1 showed the highest identity value (36.2%) and the highest similarity (73.3%) to AlyA from Klebsiella pneumoniae. A consensus region comprising nine amino acid (YFKAGXYXQ) in the carboxy-terminal region previously reported by Mallisard and colleagues was observed only in AlyVG1 and AlyVG2., SPRINGER VERLAG, 英語

Assignment of Alteromonas elyakovii KMM 162(T) and five strains isolated from spot-wounded fronds of Laminaria japonica to Pseudoalteromonas elyakovii comb. nov and the extended description of the species
T Sawabe, R Tanaka, MM Iqbal, K Tajima, Y Ezura, EP Ivanova, R Christen, INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 50, 265, 271, 2000年01月
A marine bacterium, Alteromonas elyakovii KMM 162T, which was described recently, and five strains isolated from spot-wounded fronds of Laminaria japonica have been subjected to phylogenetic analysis, and geno- and phenotypic characterization. The phenotypic features of Pseudoalteromonas elyakovii strains were closely related to that of Pseudoalteromonas espejiana IAM 12640(T), but utilization of three carbon compounds (D-mannose, L-tyrosine and trehalose) distinguished both species. The G+C content of Pseudoalteromonas elyakovii was between 38.5 and 38.9 mol%. Pseudoalteromonas elyakovii KMM 162(T) and the five Laminaria isolates constitute a single species different from any other Alteromonas and Pseudoalteromonas species as revealed by DNA-DNA hybridization data, especially Pseudoalteromonas distincta KMM 638(T) (52.4%), Pseudoalteromonas citrea KMM 216 (49.5 %), Pseudoalteromonas carrageenovora NCIMB 302(T) (46.9 %) and Pseudoalteromonas espejiana IAM 12640T (29.9 %). All the data indicated that Alteromonas elyakovii KMM 162T should be reclassified as Pseudoalteromonas elyakovii and five strains isolated from Laminaria japonica have to be included in the species. Pseudoalteromonas elyakovii comb. nov. (type strain, KMM 162(T) = ATCC 700519(T)) is proposed and a set of phenotypic features which differentiate the Pseudoalteromonas species is described., SOC GENERAL MICROBIOLOGY, 英語

Bacillus horti sp. nov., a new Gram-negative alkaliphilic bacillus (vol 48, pg 565, 1998)
Yumoto, I, K Yamazaki, T Sawabe, K Nakano, K Kawasaki, Y Ezura, H Shinano, INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, 49, 1951, 1951, 1999年10月
SOC GENERAL MICROBIOLOGY, 英語, その他

下北半島沿岸域の細菌相調査
小幡 智美, 澤辺 智雄, 田中 礼士, 谷内 喜一, 生地 暢, 田島 研一, 絵面 良男, 北海道大學水産學部研究彙報, 50, 2, 115, 122, 1999年08月
北海道大学, 日本語

ハナサキガニ幼生の大量死に関する細菌学的研究 : 原因菌の性状と病原性
渡辺 研一, 吉水 守, 尾花 博幸, 鎌田 研一, 杉澤 輝文, 澤辺 智雄, 絵面 良男, 北海道大學水産學部研究彙報, 50, 1, 19, 32, 1999年03月
北海道大学, 日本語

Pseudoalteromonas bacteriolytica sp. nov., a marine bacterium that is the causative agent of red spot disease of Laminaria japonica
澤辺 智雄, Makino H., Tatsumi M., Nakano K., Tajima K., Iqbal MM, Yumoto I., Ezura Y., Christen R., International Journal of Systematic Bacteriology, 48, 48, 769, 774, 1998年11月
An aerobic, polarly flagellated marine bacterium that produces a prodigiosin-like pigment was isolated from the red-spotted culture beds of Laminaria japonica. Five isolates had unique bacteriolytic activity for both Gram-positive and -negative bacteria, which had never been observed among Alteromonas or related species. The isolates were identified as the causative agent of red spot disease of L. japonica seeds. The phenotypic features of the isolates were similar to these of Pseudoalteromonas rubra ATCC 29570T, but they could be differentiated using 10 traits (growth at 37 degrees C, requirement for organic growth factors, bacteriolytic activity, utilization of sucrose, N- acetylglucosamine, fumarate, succinate, D-galactose, L-proline and acetate). The G+C content of DNAs from the isolates was 44-46 mol%. The isolates constitute a new species, distinct from the other Alteromonas and Pseudoalteromonas species, as shown by DNA-DNA hybridization experiments and phylogenetic clustering of 16S rRNA gene sequences, for which the name Pseudoalteromonas bacteriolytica sp. nov. (type strain = IAM 14595T) is proposed. A set of phenotypic features which differentiate this new species from closely related Pseudoalteromonas and Alteromonas species is provided., International Union of Microbiological Societies, 英語

Vibrio halioticoli sp. nov., a non-motile alginolytic marine bacterium isolated from the gut of the abalone Haliotis discus hannai
T Sawabe, Sugimura, I, M Ohtsuka, K Nakano, K Tajima, Y Ezura, R Christen, INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, 48, 573, 580, 1998年04月
Six alginolytic, facultatively anaerobic, non-motile marine bacteria were isolated from the gut of abalone Haliotis discus hannai. DNA-DNA hybridization data showed that the six strains constituted a single genospecies. Phylogenetic analyses of 16S rDNA sequences indicated that the isolates should be assigned to the genus Vibrio. The phenotypic features of the isolates were closely related to Vibrio fischeri and Vibrio pelagius biovar I, but 13 traits (motility, luminescence, alginase production, lipase production, lysine decarboxylase, indole production, growth in 1 and 6% NaCl and assimilation of five carbon compounds) distinguished these strains from V. fischeri, and 17 traits (motility, growth at 37 degrees C, lipase production, indole production, growth in 1 and 6% NaCl, acid from sucrose and D-sorbitol, and assimilation of nine carbon compounds) distinguished these strains from V. pelagius. The G+C content of the isolates was 41.6-43.1 mol%. According to DNA-DNA hybridization data and 16S rDNA phylogenetic analyses, it was concluded that the six isolates constitute a new species different from any other Vibrio species. The name Vibrio halioticoli sp. nov. (type strain IAM 14596(T)) is proposed. A set of phenotypic features which enables differentiation of the new species from other species of the Vibrionaceae family is described., SOC GENERAL MICROBIOLOGY, 英語

海産食藻動物消化管由来Vibrio属細菌のin situ PCR法による検出--生態解明への応用の可能性 (総特集 バイオマ-カ-による海洋微生物群集の解析)
沢辺 智雄, 杉村 逸郎, 絵面 良男, 海洋, 29, 12, 732, 737, 1997年12月
海洋出版, 日本語

Novel alginate lyases from marine bacterium Alteromonas sp. strain H-4
T Sawabe, M Ohtsuka, Y Ezura, CARBOHYDRATE RESEARCH, 304, 1, 69, 76, 1997年10月
A bacterium Alteromonas sp. strain H-4 isolated from Laminaria fronds produced extra- and intra-cellular alginate lyases and utilized alginate as its sole carbon source. An extracellular alginate lyase was purified from the culture supernatant of the strain and its substrate specificity was characterized. The estimated molecular mass of the enzyme was 32 kDa and the isoelectric point was 4.7. Both polyM and polyG block degrading activities were observed using the substrate-containing gel overlay technique after isoelectric focusing of the enzyme. By analyzing the reaction products from the polyM block, polyG block, MG random block and intact alginate, three major peaks containing unsaturated tri-uronide through octa-uronide were detected for each substrate. The results indicate that the enzyme of Alteromonas sp. H-4 can degrade both polyM and polyG blocks with a K-m in mg/mL 20-times higher for the polyM block. (C) 1997 Elsevier Science Ltd., ELSEVIER SCI LTD, 英語

4つの新アルギン酸リアーゼの疎水クラスター解析による分類
クライワタナポン ジンタナ, 大井 俊彦, 木下 晋一, 杉村 逸郎, 澤辺 智雄, 絵面 良男, 日本生物工学会大会講演要旨集, 9, 8, 8, 1997年
日本生物工学会, 英語

Regeneration from Laminaria japonica Areschoug (Laminariales, Phaeophyceae) protoplasts isolated with bacterial alginase
T Sawabe, Y Ezura, PLANT CELL REPORTS, 15, 12, 892, 895, 1996年09月
The conditions for effective isolation of viable protoplasts from Laminaria japonica with an alginase produced by marine bacterium Alteromonas sp. and a commercially available cellulase were investigated. The highest yields of viable protoplasts (7.9 similar to 10.4x10(6) cells g(-1) FW) were obtained with a hypertonic solution containing 50% seawater, 25 mM MgCl2, 5 mM HEPES buffer system, and 0.5 M mannitol. Protoplasts were not obtained from thalli of L. japonica when an abalone alginase (abalone acetone powder; AAP: Sigma) was used instead of the bacterial alginase. The isolated protoplasts were cultured in an PESI medium at 5 degrees C. Complete cell wall formation was observed within 7 days, and dividing cells were first observed in a 9-day-old culture. Some protoplasts regenerated into sheet-shaped thalli and rhizoid structures mere also observed on some thalli after 30 to 40 days in culture. This is the first report of protoplast regeneration into plantlets of L. japonica Areschoug (Laminariales, Phaeophyceae)., SPRINGER VERLAG, 英語

細菌由来アルギナーゼを用いて単離したプロトプラストの再生　　　　　　　　　　　　　　　
プラントセルリポート, 15, 892, 1996年

ALGINATE DEGRADATION BY BACTERIA ISOLATED FROM THE GUT OF SEA-URCHINS AND ABALONES
T SAWABE, Y ODA, Y SHIOMI, Y EZURA, MICROBIAL ECOLOGY, 30, 2, 193, 202, 1995年09月
Degradation of alginate and its constituents, polymannuronate (polyM) and polyguluronate (polyG), by gut bacteria isolated from sea urchins and abalones in the northern part of Japan, were investigated. Bacterial counts in the guts of sea urchin S. intermedius, were 10(5) to 10(8) CFU/g, and in abalone H. discus hannai, counts ranged from 10(6) to 10(9) CFU/g. More than 80% of total 600 isolates were found to have alginolytic activity. The alginolytic bacteria were predominantly fermentative, but some differences were observed in their substrate specificity as well as between the flora in the gut of sea urchins and the abalones. Seventy percent of the alginolytic bacteria from the sea urchins showed no degrading preference for polyM or polyG blocks, and were able to degrade both the substrates simultaneously. Most of the alginolytic bacteria (96.6%) from sea urchins belonged to the genus Vibrio. The majority of alginolytic bacteria (68.0% on average) from abalones only degraded polyG and they were predominantly non-motile fermenters. From these results, it appeared that a different type of association exists between alginolytic gut microflora and the marine algal feeders with respect to the level of contribution by bacteria to the host's digestion of alginate., SPRINGER, 英語

赤潮藻類Gymnodinium mikimotoiの増殖に及ぼす田辺湾分離細菌の影響　　　　　　　　　　　　　　　
北海道大学水産研究彙報, 46, 2, 39, 46, 1995年

ウニ、アワビ消化管から分離した細菌のアルギン酸分解性　　　　　　　　　　　　　　　
30, 193, 202, 1995年

Effect of marine bactera isolated from Tanabe Bay on the growth of marine red tide dinoflagellate,Gymnodinium mikimotoi　　　　　　　　　　　　　　　
46, 2, 39, 46, 1995年

349 海洋細菌Alteromonas sp.H-4株の産生する菌体外アルギン酸リアーゼの基質特異性
澤辺 智雄, 小田 恭弘, 絵面 良男, 日本生物工学会大会講演要旨集, 6, 93, 93, 1994年
日本生物工学会, 日本語

APPLICATION OF AN ALGINATE LYASE FROM ALTEROMONAS SP FOR ISOLATION OF PROTOPLASTS FROM A BROWN-ALGAE LAMINARIA-JAPONICA
T SAWABE, Y EZURA, T KIMURA, NIPPON SUISAN GAKKAISHI, 59, 4, 705, 709, 1993年04月
An alginate lyase from Alteromonas sp. H-4 was used for the preparation of protoplast from Young sporophytes of Laminaria japonica. Optimum conditions for the isolation of protoplasts were as follows: a) fine pieces of the sporophytes chopped by a razor were incubated in an enzyme mixture for 3 to 5 h at 15-degrees-C with shaking, b) the composition of the enzyme mixture was purified alginate lyase (30 U/ml) with cellulase Onozuka R-10 (1.5%) in a hypertonic solution (0.7 m mannitol, 25 mM MgCl2, and 5 mM HEPES in 50% seawater, pH 7.8). Yields of protoplast were 10(6)-10(7) cells/g (sporophytes) and protoplast viability was more than 75%. Protoplasts were brown-greenish and spherical with a diameter of 10-25 mum. The purity of alginate lyase from the strain H-4 scarcely influenced the yield of protoplast, but purified enzyme produced the highest viability of cells., JAPANESE SOC FISHERIES SCIENCE, 英語

マコンブプロトプラストの単離への┣DBAlteromonas(/)-┫DB sp産生アルギン酸リアーゼの利用
Tomoo Sawabe, Yoshio Ezura, Takahisa Kimura, 日本水産学会誌, 59, 4, 705, 709, 1993年
An alginate lyase from Alteromonas sp H-4 was used for the preparation of protoplast from young sporophytes of Laminaria japonica. Optimum conditions for the isolation of protoplasts were as follows: a) fine pieces of the sporophytes chopped by a razor were incubated in an enzyme mixture for 3 to 5h at 15° with shaking, b) the composition of the enzyme mixture was purified alginate lyase (30U/ml) with cellulase Onozuka R-10 (1.5%) in a hypertonic solution (0.7M man nitol, 25mM MgCl2, and 5mM HEPES in 50% seawater pH7.8). Yields of protoplast were 106- 107 cells/g (sporophytes) and protoplast viability was more than 75% Protoplasts were browngreenish and spherical with a diameter of 10-25µm. The purity of alginate lyase from the strain H-4 scarcely influenced the yield of protoplast, but purified enzyme produced the highest viability of cells. © 1993, The Japanese Society of Fisheries Science. All rights reserved., 英語

PURIFICATION AND CHARACTERIZATION OF AN ALGINATE LYASE FROM MARINE ALTEROMONAS-SP
T SAWABE, Y EZURA, T KIMURA, NIPPON SUISAN GAKKAISHI, 58, 3, 521, 527, 1992年03月
An alginate lyase from the culture medium of Alteromonas sp. H-4 was purified by ultrafiltration, gel filtration, and anion-exchange chromatography and was characterized. A molecular weight of the purified enzyme was estimated as 32,000 by sodium dodecyl sulfate-polyacrylamide gelectrophoresis (SDS-PAGE). Optimum pH and temperature of the enzyme activity were 7.5 and 30-degrees-C, respectively. The enzyme was unstable on heating and in acidic solution. The alginate lyase required 50 mm MgCl2 or MgSO4, 0.5 m NaCl, and 0.2 m KCl for the maximum activity., JAPAN SOC SCI FISHERIES TOKYO UNIV FISHERIES, 英語

海洋性┣DBAlteromonas(/)-┫DB sp.の産生するアルギン酸リアーゼの精製と性質
澤辺 智雄, 絵面 良男, 木村 喬久, 日本水産学会誌, 58, 3, 521, 527, 1992年

リシリコンブ穴あき病葉体から分離されたアルギン酸分解菌の性状
日本水産学会誌, 58,141-145, 1992年

CHARACTERIZATION OF AN ALGINOLYTIC MARINE BACTERIUM FROM DECAYING RISHIRI-KOMBU LAMINARIA-JAPONICA VAR OCHOTENSIS
T SAWABE, Y EZURA, T KIMURA, NIPPON SUISAN GAKKAISHI, 58, 1, 141, 145, 1992年01月
An alginolytic marine bacterium, H-4, was isolated from decaying thalli of Laminaria japonica var. ochotensis. Strain H-4 is a gram-negative polarly flagellated rod, which oxidatively produces acid from glucose, is oxidase positive and requires seawater for growth. DNA base composition measured from melting point of DNA is 39.8 mol % G + C. According to these characteristics, the strain was identified as genus Alteromonas. Alteromonas sp. H-4 resembles A. espejiana but differs in DNA base composition and some properties and could not be identified to any species in the genus Alteromonas.
The productivity of extracellular alginate lyase by Alteromonas sp. H-4 increased with seawater concentration and decreased with casitone concentration in the media. Its productivity was observed in a medium without alginate. The bacterium produced a maximum amonut of alginate lyase in liquid media containing 75 % seawater, 0.5 % casitone, and 0. 1 % sodium alginate after 96 h incubation with shaking at 25-degrees-C., JAPAN SOC SCI FISHERIES TOKYO UNIV FISHERIES, 英語

水圏微生物学の基礎
濵﨑 恒二, 木暮 一啓, 澤 辺智雄, 澤辺 桃子, 鈴木 聡, 砂村 倫成, 永田 俊, 春田 伸, 福田 秀樹, 美野 さやか, 和田 実, 濵﨑 恒二, 木暮 一啓
恒星社厚生閣, 2015年09月25日, 4769915683, 280, ［共著］

The Prokaryotes 4th edition　　　　　　　　　　　　　　　
B. Gomez-Gil, C.C. Thompson, Y. Matsumura, T. Sawabe, T. Iida, R. Christen, F.T. Thompson, T. Sawabe, Family Vibrionaceae
Springer, 2014年, ［分担執筆］

海藻バイオ燃料　　　　　　　　　　　　　　　
澤辺 智雄, マリンビブリオを活用した海藻からのエタノール生産
CMC出版, 2011年, ［分担執筆］

The Biology of Vibrios　　　　　　　　　　　　　　　
澤辺 智雄, The mutual partnership between Vibrio halioticoli and abalones
ASM Press, 2006年, ［分担執筆］

The biology of vibrios
Thompson, Fabiano Lopes, Austin, B. (Brian), Swings, J. G., American Society for Microbiology
ASM Press, 2006年, 1555813658, xiii, 423 p., 英語

養殖コンブの疾病調査　　　　　　　　　　　　　　　
2000年

Research on disease of Laminaria　　　　　　　　　　　　　　　
2000年

海洋バイオ水素生成技術の革新に向けたマリンビブリオの分子育種基盤の構築
科学研究費助成事業 基盤研究(B)
2019年04月01日 - 2023年03月31日
澤辺 智雄, 美野 さやか
化石燃料代替エネルギー生産技術開発は，学術的・社会的要請が高い地球規模課題である。海洋バイオマスのエネルギー変換技術の開発もその一つであるが，アルギン酸などの難燃料化成分が多く，その完全変換にはさらなる技術革新が必要である。我々が見いだした新規マリンビブリオは，Hyf複合体を核とするギ酸水素リアーゼ（FHL）複合体を介した独特の水素代謝を示し，海藻由来の種々の糖を水素化する。しかし，マリンビブリオ触媒の水素生成能を高める分子育種基盤の構築は発展途上であり，海洋バイオ水素生産性向上のボトルネックでもある。Hyf複合体の分子レベルで特徴を理解するためには，マリンビブリオは恰好の生物材料であるため，本研究では，マリンビブリオが有する活性型Hyf複合体の特徴を理解し，多彩な海洋バイオマスからのより効率的な水素生成が可能な海洋微生物触媒の構築にフィードバックさせる知見を得ることを目的に研究を進めた。その結果以下の成果を得た。
①バイオ水素生成マリンビブリオ全種の完全ゲノムの取得
水素生成能が観察されているほぼ全種を含む17種のビブリオ科細菌の完全ゲノムを取得した。遺伝子構造に基づき，主に3タイプに分かれることを明らかにした。
②水素生成マリンビブリオ触媒の能力比較
上記17種のうち，水素生成が確認できる12種について，グルコースを基質とした場合の水素生成プロファイルを調べたところ，ギ酸の再取り込み量が水素生成と相関することを明らかにした。
日本学術振興会, 基盤研究(B), 北海道大学, 19H03041

マナマコの資源増産に向けたパイオニア微生物資源の獲得と利用
科学研究費助成事業 挑戦的研究(萌芽)
2019年06月28日 - 2021年03月31日
澤辺 智雄, 美野 さやか
生物は絶えず環境の微生物から接触を受け，その親和性の時間軸の長さに応じて，共生や寄生といった宿主－微生物間相互作用にまで，その関係性は緊密化する。宿主―微生物間の相互作用は，生物が誕生した時から開始され，“パイオニア微生物”と呼ばれる微生物がその役割を担うが，生物進化上重要性の高い海洋無脊椎動物由来のパイオニア微生物探査は未知の研究領域である。パイオニア微生物は，宿主が生まれて初めて自身の上皮組織に定着し，細胞あるいは分子レベルで刺激しあい，成長や免疫系の成熟に寄与し，健全性の基盤を築くものと考えられている。本研究では，知見が少ない海洋動物のパイオニア微生物研究の創生を目的とした。目的の達成に向け，マナマコをモデルとした海洋無脊椎動物の宿主の生理状態とそれに付随する微生物群の構造機能のダイナミックな変化を明らかにするため，以下の研究を随時進展させる。1)マナマコ・ホロビオントの全容解明とパイオニア微生物の発掘、2) 易培養性パイオニア微生物とそのゲノム情報コレクションの整備、3) 難培養性パイオニア微生物の取得、4）パイオニア微生物の宿主への影響把握
本年度は，この中で， 1)マナマコ・ホロビオントの全容解明とパイオニア微生物の発掘，および2) 易培養性パイオニア微生物とそのゲノム情報コレクションの整備，を進めた。その結果，受精卵から着底幼生にいたる6つの成長段階の遺伝子情報を取得し，ある時期から菌叢が変化し始めることが示唆された。また，約250株の易培養株を単離し，これらは約30属40種以上から構成されていた。さらに，ナマコ株の比較のため，ウニの培養株の取得も進め，MALDI-TOF-MASSデータベースの整備を開始した。
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 19K22262

海洋バイオ水素生成技術の革新に向けたマリンビブリオの分子育種基盤の創成
科学研究費助成事業 基盤研究(B)
2016年04月01日 - 2020年03月31日
澤辺 智雄, 美野 さやか
水素社会の形成に向けて水素生成技術基盤の強固化が求められている。研究代表者が見いだした新規マリンビブリオは，海藻に含まれるユニークな炭水化物群を効率よく水素化するため，日本発の海洋生物資源を活用した水素生産技術となり得る。しかし，海洋微生物
を活用したバイオ水素変換技術の基盤，特にマリンビブリオの水素生成能を高める分子育種技術は未成熟であり，水素生産性の向上にはボトルネックとなっている。そこで，本研究では，水素生成能の高いマンビブリオが有する水素生成分子マシナリーの細胞生理学的
特性と遺伝子発現制御の解明および分子育種の進展を目的とした。特に，本年度は，Vibrio tritonius のHyfの特性の解明とアルギン酸から水素を生成可能な新規ビブリオの比較ゲノム解析を進め以下の結果を得た。
1.V.tritonius AM2T株の種々のNaCl濃度下における水素生成キネティクスを測定し，0.5%から1.0% NaCl存在下で水素生成能は高いものの，海水存在下において水素生成が可能である細菌であるとの特徴付けを行った。
2.アルギン酸から水素を生成するV. aphrogenesでは，アルギン酸を基質とした場合の水素生成モル収率がD-グルコースを基質とした場合のそれとほぼ同程度であることを明らかにした。
3. V. ahrogenesと単系統性が強く示唆される全6種の完全ゲノムを取得し，比較ゲノムを行い，アルギン酸から水素を生成する代謝系に関連する遺伝子を保有しているのは，V. aphrogenesのみであることを明らかにした。
日本学術振興会, 基盤研究(B), 北海道大学, 16H04976

海藻バイオマスの効果的物質変換に資するマリンビブリオ触媒の一貫バイオプロセス化
科学研究費助成事業 挑戦的萌芽研究
2016年04月01日 - 2018年03月31日
澤辺 智雄, 美野 さやか
より効率的に海藻原料をバイオ燃料へと生物変換するために，多彩な糖質で構成される海洋バイオマスの効率的利用に適応可能な複数基質の糖化と発酵を統合（一貫バイオプロセス（CBP）化）した生物触媒の開発を行い，①アルギン酸分解性マリンビブリオを核としてポリウロン酸，糖アルコール，グルカンを利用可能な海洋細菌触媒および②増殖能が高いマリンビブリオを核としてガラクトースからもエタノール生産性の高い株を得た。
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 16K14978

バイオ燃料生産性を飛躍させる光エネルギー駆動型マリンビブリオファクトリーの創成
科学研究費助成事業 挑戦的萌芽研究
2014年04月01日 - 2016年03月31日
澤辺 智雄, 細川 雅史
より効率的に海藻原料をバイオ燃料へと生物変換するために，「光エネルギー」を効果的に利用する生物触媒の開発を目的として，①Nonlabens sediminisが有するプロテオロドプシン遺伝子の発現、②新規カロテノイド生合成遺伝子群の収集とクローニングを行った。また，マリンビブリオで適応可能な効率な遺伝子改変システムの開発に向け，マリンビブリオにおけるCRISPR/Casシステムの特徴を調べた。
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 26660168

マリンビブリオを核とした海洋バイオ水素生産技術のビッグバン
科学研究費助成事業 基盤研究(B)
2013年04月01日 - 2016年03月31日
澤辺 智雄, 細川 雅史, 中川 聡
高い水素生成能を有するマリンビブリオを生物触媒として，①連続的かつ安定的なバイオ水素生産を達成するための連続培養系の構築，および②発酵残滓の高付加価値化を検討し，①マンニトールを基質として70日間にわたり連続的に水素の生成が可能な培養系の構築，②2種類のマリンビブリオ生物触媒を利用した，アルギン酸‐水素生成システムの構築，および③種々の新規・希少カロテノイド生合成遺伝子資源の獲得に成功した。
日本学術振興会, 基盤研究(B), 北海道大学, 研究代表者, 競争的資金, 25292122

バイオマス燃料生産に適する最少遺伝子セットを持つマリンビブリオセルの創成
科学研究費助成事業 挑戦的萌芽研究
2011年 - 2013年
澤辺 智雄, 細川 雅史, 中川 聡
再生可能な新エネルギーの開発に向けて、海洋バイオマス由来のバイオ燃料の開発は技術基盤の成熟が待たれている。本研究では、海藻糖質を基質にバイオエタノール生産能を示すマリンビブリオに着目し、トランスクリプトーム、代謝産物解析、および合成生物学的代謝改変技術を導入し、バイオマス燃料生産に適する最少の遺伝子セットを持つビブリオセルの創成を行った。
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 研究代表者, 競争的資金, 23658172

深海生物が身にまとう磁性硫化ナノメタル:金属濃縮機構の網羅解析
科学研究費助成事業 挑戦的萌芽研究
2011年 - 2012年
中川 聡, 高井 研, 澤辺 智雄, 鈴木 庸平
本研究は、インド洋の深海底熱水活動域に棲息する固有巻貝とその共生微生物の環境応答機構や生物間相互作用を発現遺伝子およびタンパク質の両レベルで網羅的に解明することを目的としている。これまでの研究において、共生微生物およびホスト生物の両者について、次世代シーケンサーを用いたメタトランスクリプトーム解析を行ない、それらの環境応答機構をmRNAレベルで網羅的に解析することに成功した。
日本学術振興会, 挑戦的萌芽研究, 北海道大学, 23658154

マリンビブリオを活用した海藻糖質のバイオエタノール変換技術プラットフォームの構築
科学研究費助成事業 基盤研究(B)
2009年 - 2012年
澤辺 智雄, 尾島 孝男, 中川 聡
再生可能な新エネルギーの開発に向けて、海洋バイオマス由来のバイオ燃料の開発は技術基盤の成熟が待たれている。本研究では、海藻糖質を基質にバイオエタノール生産能を示す2種類のマリンビブリオに着目し、これら海洋細菌が有する海藻糖質の発酵代謝系の再構成、各酵素遺伝子の機能解析及びそれら遺伝子群の網羅的解析を実行し、マリンビブリオを活用した海藻糖質のバイオエタノール変換技術プラットフォームの構築を進めた。
日本学術振興会, 基盤研究(B), 北海道大学, 研究代表者, 競争的資金, 21380129

深海底熱水活動域における化学合成微生物の共生機構を分子的に理解する
科学研究費助成事業 若手研究(A)
2008年 - 2010年
中川 聡, 高井 研, 牧田 寛子, 澤辺 智雄
本研究は、深海底熱水孔環境において共生関係にある微生物-大型生物の相互作用・相互認識機構を分子レベルで解明することを目的としている。特に生物間の相互認識に関わる生体分子「糖鎖」に注目し研究を進めてきた。これまでの研究において、深海底熱水孔環境に生息する共生微生物およびそのホスト生物の両者について、糖鎖の発現解析・プロファイリング・構造解析を行い、両者の相互認識・相互作用に寄与する可能性の高い特異な新奇糖鎖を見いだすことに成功した。
日本学術振興会, 若手研究(A), 北海道大学, 連携研究者, 競争的資金, 20687003

アワビの海藻多糖代謝機構の解明
科学研究費助成事業 基盤研究(B)
2007年 - 2010年
尾島 孝男, 井上 晶, 田中 啓之, 澤辺 智雄
アワビやアメフラシなどの食藻性腹足類は、アルギン酸やラミラナン、マンナン、キシラン、セルロースなどの海藻多糖を特異的に分解する酵素をもつ。これらの酵素によって分解された海藻多糖は、腹足類の解糖系やTCA回路により代謝される。本研究では、腹足類の海藻多糖代謝機構に関する理解を深めるために、消化液に含まれる一連の多糖代謝系酵素、特にアルギン酸、ラミナラン、マンナンの分解に関連する酵素の性状を解析した。
日本学術振興会, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 19380117

安全で安心な牡蠣の周年出荷に向けた研究
科学研究費助成事業 基盤研究(B)
2005年 - 2008年
吉水 守, 田島 研一, 西澤 豊彦, 澤辺 智雄, 笠井 久会
20℃以上に調温した0.3mg/Lの塩素を含む電解海水あるいは中圧紫外線処理海水を用い、V. alginolyticus優勢キートセロスを給餌しながら浄化すれば、カキが痩せることなく、大腸菌、ノロウイルスおよびV. parahaemolyticusを排除できると考えられる。さらに安全性を確保するために2,000気圧の高静水圧をかけて脱殻を行えば、安全性はより向上する。
日本学術振興会, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 17380110

大河川の影響を受ける亜寒帯沿岸域における基礎生産過程のダイナミクス
科学研究費助成事業 基盤研究(B)
2003年 - 2005年
工藤 勲, 門谷 茂, 齋藤 誠一, 澤辺 智雄
わが国を代表する一級河川である十勝川とその流入する十勝河口域から沿岸域にかけて基礎生産過程の理解に必要な化学成分、生物化学パラメータの測定を行った。研究期間中に得られた知見は以下のとおりである。
1.十勝川の水量は、4月から雪解けによる増水期を迎え、通常時の10倍程度増加した。この高い水量は、6月下旬まで継続した。河川水中には、植物プランクトンの増殖に必須の窒素、珪素、鉄等の栄養塩を豊富に含んでいた。上流から下流および支流の水質解析より、この栄養塩の起源として、森林、農業・酪農、都市排水などが考えられた。
2.沿岸醐の調査から十勝川の影響が、河口から10km四方、深度20m程度まで確認された。河口に近接した地点では塩分の低下に伴う栄養塩の増加が確認された。十勝川から沿岸域に一年間でもたらされる窒素態栄養塩の総量は、7.83x10^8molと見積もられた。この窒素量は、植物プランクトンの炭素生産量(基礎生産量)に換算して62,000トンであり、これは十勝川の影響を受けている沿岸域における基礎生産量を上回る量である。つまり、この海域における基礎生産量は、河川からの栄養塩によって主に支えられていること、さらに栄養塩の循環過程を経ることにより、より広範囲の海域における基礎生産過程に大きな影響を与えていることが明らかになった。
3.基礎生産の行方の一つとして近年注目されている微生物ループについて、従属栄養バクテリアは、河口域で豊富に存在していた。これは従属栄養バクテリアの成長に必要な溶存有機物が河川を通じて海域に供給されているためと考えられた。また、従属栄養バクテリアの死亡要因としてウイルスによる溶菌が8割以上を占めていることが明らかになった。
日本学術振興会, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 15310019

牡蠣の小型球形ウイルス(SRSV)浄化法に関する研究
科学研究費助成事業 基盤研究(B)
2002年 - 2004年
吉水 守, 田島 研一, 西澤 豊彦, 澤辺 智雄
牡蠣の汚染による衛生上の問題に関しては、腸チフス等18世紀から報告があり、我が国でも1924年熊本県不知火での規模な腸チフスの発生が知られている。1950年に『食品衛生検査指針』により貝類の衛生基準が定められた。牡蠣は1日当たり2トンもの海水を吸入し、餌料生物を接種し、同時に細菌やウイルスを濃縮する。しかし清浄海水で飼育すると蓄積した細菌やウイルスを放出する。いかにして、清浄海水を確保するかに関しては、坂井(1953,1954)、河端(1953)の研究により、現在の浄化法の基礎が築かれ、広島・宮城県から全国に普及していった。
平成9年5月31日付けの食品衛生法の一部改正により、食中毒原因物質として新たに小型球形ウイルスとその他のウイルスが追加された。小型球形ウイルス(SRSV;現在、ノロウイルス)は電子顕微鏡像での形態が類似する直径27〜38nの球形ウイルスの総称であり、1972年に米国オハイオ州で起きた非細菌性集団胃腸炎の患者糞便より発見されたNorwalkウイルスがその原型である。ノロウイルスは培養細胞や実験動物を使用して増殖させることが困難であり、現在行われている紫外線やオゾンを用いた循環型浄化装置では、ウイルスが不活化されていてもRT-PCR法では陽性となり、製品の出荷ができない。本研究は、牡蛎のノロウイルス浄化法を培養可能なネコカリシウイルスを代替えウイルスとして検討したものであり、得られた成果は以下のとおりである。1.電解海水を用いることにより牡蠣の大腸菌浄化が可能であることを示した。2.ネコカリシウイルス(FCV)を用いた場合、FCVは紫外線に抵抗性を示したが、海水電解水に高い感受性を示した。3.FCVは高水温下で不安定であったが、低水温下では安定であった。3.FCVは半数以上の牡蠣の消化管内容物で不活化された。4.FCVは牡蠣の脱殻条件、40℃・800気圧で90%以上不活化された。これらを組み合わせることによりカキのノロウイルス浄化は可能となると考えられる。今後はノロウイルスの感染性を評価する系を作る必要性があると改めて認識された。
日本学術振興会, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 14360102

漁獲から加工場に至る水産物のHACCP対応用海水殺菌装置の開発
科学研究費助成事業 基盤研究(B)
2001年 - 2004年
吉水 守, 澤辺 智雄, 上西 敏夫, 絵面 良男
PL法の施行に伴う製造者責任の明確化や、HACCP方式による衛生管理が世界的なスタンダードになりつつあるなど、近年食品産業全体を取り巻く環境は急速に変化し、「品質・安全」に対する取り組みは各分野でますます強化されている。現代社会における水産物の取り扱いは、大量生産、大量流通、大量消費を特徴とし、食中毒など人間に及ぼす危害は、ほんの僅かな過失であってもその規模は著しく拡大する恐れがある。また、流通経路が複雑なために、原因の特定が困難を極め、その間消費者の不安が増幅し、風評被害によりその影響が関連産業全体におよぶ恐れがあるなど、その対策に当たっては、業界や地域全体の取り組みなくしては、万全を期すことは困難である。
水産加工場ではHACCP対応に向けた施設の改善が進んでいるが、水産物の漁獲から加工に至るまでの過程は、対策が不十分である。刺身や寿司といった生・非加熱で食べる食材としての魚を地面に置くことに、何の違和感をも抱かない現状を考えると、産地における衛生意識の啓蒙と一貫した品質管理について緊急に取り組む必要があると考える。特に、加工場に搬入されるまでの、漁獲-漁港-産地市場での衛生的な取り扱いが重要であり、漁船の船体洗浄や船倉に入れる海水、漁港で用いる海水、産地市場で使用する海水の衛生管理が重要な課題と考える。
魚介類の飼育用水および飼育排水の効果的な殺菌法とその装置の開発を行い、海水を直接電気分解する装置の導入により大量の海水を有効にかつ安価に殺菌できることを明らかにした。漁獲から産地市場までの衛生管理を目的に、試験的に海水電気分解装置を漁船、漁港および産地市場に導入し、殺菌効果に関する基礎的知見の集積を行い装置の開発を行った。漁獲物の衛生管理を行うに際し、漁船の船倉に低温の殺菌海水を入れておくことは極めて有効な手法だった。また、電解海水は漁港の岸壁あるいは選別台や魚を入れるパレット、魚箱を洗浄する海水にも適していた。さらに産地市場の床や魚を載せる台あるいはパレットの洗浄にも有効であり、海水電解装置は漁港内の海水を殺菌するに十分な効果を示し、洗浄・除菌効果が実証された。
日本学術振興会, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 13556027

水圏生物におけるD型リン脂質の分析法,分布および生理機能
科学研究費助成事業 基盤研究(B)
2001年 - 2003年
板橋 豊, 安藤 靖浩, 澤辺 智雄
本研究では非天然型の構造を有するグリセロ脂質(リン脂質,糖脂質,トリアシルグリセロール)海洋生物における分布,生合成,生物機能を解明することを目的として行われたものである。得られた成果は以下のように要約される。
(1)非天然型グリセロ脂質を分析するためのキラルHPLC,逆相HPLC及びHPLC/質量分析法(ESI/MS)を確立した。
(2)海洋細菌と好熱性細菌を含む約70種の細菌のリン脂質を本研究で開発した方法を用いて分析した結果,約70%の菌株から,非天然型(R, R)配置の構造を有するホスファチジルグリセロール(PG)が検出され(総PG中1〜15%の割合で存在),R, R型PGが細菌に広く分布することが明らかとなった。
(3)非天然型PGの立体構造に及ぼす培養温度の影響を検討した結果,温度の上昇につれて,R, R型PGの割合は著しく増加することを突き止めた。このことから,細菌はPGの脂肪酸組成ばかりでなく,PGの立体構造をも変えて環境の変化に適応していることが明確となった。
(4)PG異性体の合成に関わる酵素(ホスホリパーゼD)の立体特異性を明らかにした。
(5)逆相HPLC/ESI-MSを駆使して,グリセロ脂質海中の位置異性体を詳細に検討した結果,海藻(オゴノリなどの紅藻)のグリセロ糖脂質(モノガラクトシルジアシルグリセロール,MGDG)中に,アシル基の結合位置の逆転した異性体(reverse isomers)の存在することを初めて見出した。このことから,MGDGの前駆体である1,2-ジアシルグリセロールは2つの異なる経路で合成されることが明確となった。
(6)魚類や哺乳類のリン脂質からも正常な構造とは異なるreverse isomerが少量検出された。代謝異常や病気との関連性について言及した。
日本学術振興会, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 13460088

アワビ消化管内細菌叢の人工制御に関する研究
科学研究費助成事業 若手研究(B)
2001年 - 2002年
澤辺 智雄
本年度の成果は以下の通り。
1.アワビ消化管内細菌叢の抗生物質添加飼料を用いた人工的置換
Vibrio halioticoliは、ペニシリン系、セファロスポリン系、およびクロラムフェニコール(CP)に高い感受性を示した。この中で最小発育阻止濃度の低いベンジルペニシリン(PCG)とCPをそれぞれ0.5-50mg/50gおよび1.5-150mg/50g飼料となるように混合したアワビ配合飼料を1ヶ月間エゾアワビに給餌し、アワビ消化管からのV. halioticoliの除去を試みた。その結果、50mg PCGおよび150mg CP添加飼料給餌区でV. halioticoliの比率は30%から0.04%に減少し、総菌数は10^8cells/9(消化管)から2桁減少した。しかし、生菌数は減少せず10^6CFU/g(消化管)を維持していたが、飼育試験終了時にはPolaribacter sp.が優占し、細菌叢は変化した。次に、アワビ消化管内細菌叢の完全除去を目指し、PCG-CP添加飼料給餌区で残存したPolaribacter sp.に高い感受性を示すセフォタキシム(CTX)を添加したPCG-CP-CTX添加飼料を調製し、飼育試験を試みたが、総菌数および生菌数を著しく減少させる条件を見いだすまでには至らなかった。
2.緑色蛍光タンパク質(GFP)標識V. halioticoliにおけるGFP発現条件の検討
GFP標識V. halioticoli株について細胞単位でのGFP蛍光発現条件の至適化やGFP発現細胞の検出限界時間などを検討した。その結果、GFP発現細胞の比率は培養温度15℃で高く、また、嫌気培養条件(ガスパック法)下においては、好気条件下よりも比率は10%低下するものの、GFP発現細胞は観察された。さらに、15℃-好気条件下でGFPを発現させた細胞は、15-25℃下では滅菌海水中で4日以内までは正確に検出できた。このGFP標識株を用いたトレーサー実験が今後の課題となった。
日本学術振興会, 若手研究(B), 北海道大学, 研究代表者, 競争的資金, 13760134

ウニ病原細菌Flexibacter SP.の生存戦略に関する研究
科学研究費助成事業 基盤研究(C)
2000年 - 2002年
田島 研一, 澤辺 智雄
エゾバフンウニ斑点病原因菌Flexibacter sp.の低水温環境における生残性、VBNC状態にある本菌の検出法およびVBNC状態からの蘇生を検討した。得られた主なる成果は以下の通りである。
1.本病原因菌は、75%ASW中5℃で保持すると、約15日でコロニー形成能を消失した。この状態の菌体は、VBNC状態にあることが明らかになった。VBNC状態移行菌体は、短桿状または球状に近い形態を呈し、ウニに対する病原性は消失していた。
2.VBNC細胞を特異検出するためのプライマーセットを、16S rRNAの塩基配列から作成した。本プライマーセットは、原因菌およびそのVBNC細胞以外には増幅産物が見られなかったことから、原因菌の特異検出の可能性を示唆した。
3.VBNC状態からの蘇生を検討した。塩化第二鉄溶液を0.0017%(約60μM鉄濃度)の割合で加えた75%ASWの系とウニ成分を添加した75%ASWの系で5℃から25℃への温度のシフトアップにより蘇生がみられた。ウニ成分添加75%ASWの系では、鉄添加の系では鉄無添加の系でよりもより長いVBNC状態からの蘇生がみられた。蘇生細胞は、主なる性状、元株抗血清との凝集性、ウニに対する病原性、細胞形態などの点で元細胞と一致した性状を示した。
以上の結果から、本病原因菌は、低水温期にVBNC状態となりウニ個体表面などに付着して生残し、高水温期にVBNC状態から蘇生して、再び本病を発生せしめる可能性が強く示唆された。
日本学術振興会, 基盤研究(C), 北海道大学, 連携研究者, 競争的資金, 12660159

アルギン酸オリゴ糖修飾による魚類筋原線維タンパク質の機能性改変
科学研究費助成事業 基盤研究(C)
2000年 - 2001年
佐伯 宏樹, 澤辺 智雄, 中村 宗一郎
魚類筋原線維タンパク質(Mf)分子中にメイラード反応を利用してアルギン酸オリゴ糖(AO:平均重合度6)を導入し,ネオグリコプロテインを調製した。そして,AO修飾によってMfの諸機能(低イオン強度溶媒に対する溶解性,熱安定性,乳化能,消化性)がどのように変化するか検討した。また,AO修飾したMfの食品衛生学的安全性について調査した。得られた結果を以下に示す。
(1)AO修飾によってMfの低イオン強度溶媒に対する溶解性は著しく向上し,0.05M-0.5Mの広範な塩濃度下では塩濃度に依存せずに溶解するようになった。
(2)AO修飾によってMfの熱安定性は飛躍的に改善された。すなわち,AO結合量が200μg/mgを越えると,Mfは80℃で2h加熱しても全く不溶化しなかった。なお,この熱安定性の改善はpH4.5<で確認できた。
(3)Mf-AOはAO結合量の増加に伴って優れた乳化活性、乳化安定性を呈し,その機能改変効果は加熱処理によっても損なわれなかった。
(4)水溶化,熱安定性,乳化能の改変が達成できたAO結合量の範囲では,Mfの消化性の低下は起こらなかった。
(5)レック・アッセイとエイムス・テストによってMf-AOの変異原性試験をおこなったところ,変異原性の存在は確認できなかった。さらにラットを用いた急性毒性試験では,Mf-AOに顕著な毒性は認められなかった。
以上,本研究の結果から,AO修飾が食品素材としての魚類筋肉タンパク質の機能を改変する極めて有効な手段であることが確認できた。
日本学術振興会, 基盤研究(C), 北海道大学, 連携研究者, 競争的資金, 12660182

海洋細菌の遺伝子産物大量発現系の開発
科学研究費助成事業 奨励研究(A)
1999年 - 2000年
澤辺 智雄
本年度は、海洋細菌由来のプラスミッドの探索とそれらのプラスミッドを用いた形質転換の可能性について検討し、以下の結果を得た。
1.海洋細菌由来新規プラスミッドベクターの探索
海水試料から分離した100株の海洋細菌からプラスミッド様核酸を保持するの菌株の探索を行い、5菌株がプラスミッド様核酸を有していた。前年度の成果により、3菌株のプラスミッド保持菌株を見いだしたが、本研究課題で計8菌株が得られた。これら8菌株のプラスミド様核酸に抗生物質耐性マーカー遺伝子が存在するか否かを調べるために、エレクトロポレーション法により、大腸菌JM109株の形質転換を試みた。その結果、OM918-2-6株およびH8株のプラスミッドが、大腸菌内で複製可能で、しかもアンピシリン耐性遺伝子がコードされている可能性が示唆された。
2.Pseudoalteromonas elyakoviiのアルギン酸分解酵素遺伝子を高発現する海洋細菌宿主の探索
P.elyakoviiからクローン化したアルギン酸分解酵素遺伝子alyPEECを挿入した3種類のプラスミッド(pTPB24、pTPB31、pCD11)を用い、2種のアルギン酸非分解性海洋細菌P.haloplanktis IAM12915^TおよびP.tetraodonis IAM14610^Tをエレクトロポレーション法により、形質転換することを試みたが、いずれのプラスミッドとも両海洋細菌で複製しえなかった。
本研究で見いだされたアンピシリン耐性プラスミッドの遺伝子地図の作成とこれらのプラスミッドを安定して保持、複製できる海洋細菌宿主の探索が急務となった。
日本学術振興会, 奨励研究(A), 北海道大学, 研究代表者, 競争的資金, 11760129

食藻動物消化管内細菌の栄養共生に関する研究
科学研究費補助金(基盤研究(B))
1997年 - 2000年
絵面 良男, 澤辺 智雄, 吉水 守, 田島 研一
海洋性食藻性動物とその消化管内細菌との栄養共生関係の解明を目指し、それら消化管内細菌の生理・生態学的意義の解明を試みた。得られた成果は以下の通りである。1.養殖エゾアワビの食性の変化と消化管内細菌叢の変化が密接に関連していることを明らかにした。特に、消化管内で優勢な非運動性アルギン酸分解性はVibrio属の新種であり、V.halioticoliと命名した。2.V.halioticoliを特異的に検出同定できる3種類の特異検出手法((1)16s rDNA制限酵素断片長多型(RFLP)解析、(2)コロニー交雑法、(3)in situ PCR)を開発した。3.前項の特異検出手法を利用し、1)本菌がアワビ養殖施設周辺の海水に分布し、餌料微細藻類に付着して稚アワビの消化管に取り込まれた後、餌料の変化に伴いアワビ消化管内が本菌の増殖しやすい環境に変化し、本菌が優勢となること、および2)日本沿岸に生息するクロアワビ、トコブシ、フクトコブシおよびサザエのみから本菌が検出されることを明らかにした。また、アワビ・サザエ由来V.halioticoli菌株のフィンガープリンティング解析により、本菌種の株間で宿主特異的な遺伝的変化が生じている可能性を示した。4.V.halioticoli IAM14596^T株から4種のアルギン酸分解酵素遺伝子のクローン化に成功し、本菌が複数のアルギン酸分解酵素を産生している可能性を示した。5.150mg/50g(飼料)のクロラムフェニコールおよび50mg/50g(飼料)のペニシリンを添加した餌料を投餌することで、エゾアワビ消化管内でのV.halioticoliの存在比率を0.1%以下に減少させることができた。また、V.halioticoliによるアルギン酸塩の発酵主産物が酢酸および蟻酸であることを明らかにした。
文部科学省, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 09460081

種苗生産施設における用水および排水殺菌処理システムの開発
科学研究費補助金(基盤研究(B))
1997年 - 2000年
吉水 守, 渡辺 研一, 三村 元, 上西 敏夫, 澤辺 智雄, 絵面 良男
本研究は、種苗生産施設における飼育用水および飼育排水の効果的殺菌法を開発することを目的とした。特に飼育排水は量が多く、申請時の殺菌法では技術的にもコスト的にも不可能であった。まず飼育用水を対象に低圧および中圧紫外線ランプによる殺菌効果を、次いでオゾン処理による海水の殺菌効果を検討した。飼育排水については紫外線およびオゾン処理に加え、海水を電気分解して得られる次亜塩素酸を用いた殺菌法を検討した。得られた成果は以下のとおりである。1.魚類病原微生物は紫外線感受性から2グループに分けられ、市販の殺菌灯の線量(10^4μW・sec/cm^2)で殺菌されるグループと、中圧ランプ(10^5μW・sec/cm^2)を要するグループに分けられた。2.オゾン殺菌の場合も上記同様、病原体の感受性が異なった。オゾンあるいはオキシダント量で0.5mg/L、1分間の処理で十分な殺菌効果が得られた。淡水では瀑気処理によりオゾンを除去できたが、海水では生成されるオキシダントが魚毒性を示すため、飼育用水として使用するには活性炭等により除去する必要であった。そのまま使用すれば、消毒薬としての利用が可能であった。3.海水をはじめ食塩溶液を電気分解すると、次亜塩素酸が生成され、殺菌効果が認められた。0.5〜1.0mg/Lで1分間処理すれば飼育排水の殺菌が可能であった。本装置は構造が簡単で、残留塩素の処理法を解決すれば、大量の水を殺菌することができ、有機物除去装置との併用で、一般飼育排水の殺菌が可能となった。これらの成果を総合し、種苗生産施設の飼育用水および飼育排水の殺菌に適した殺菌法と殺菌装置を選択し、殺菌処理システムの開発を行った。現在、いくつかの施設では良好な成績が得られている。
文部科学省, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 09556041

海産食藻動物の腸内細菌のアルギン酸分解特異性と栄養生理学的役割に関する研究
科学研究費補助金(奨励研究(A))
1996年 - 1996年
澤辺 智雄
本年度に得られた成果は以下の通りである。I.ウニ・アワビ消化管由来アルギン酸分解菌の種構成と分解特異性エゾバフンウニ消化管から分離したアルギン酸分解菌はその一般性状からVibrio属と同定されたものが大半を占めていたが、代表株についてDNA-DNA相同性を測定した結果、少なくとも2菌種以上が混在していることが明らかとなった。一方、エゾアワビ消化管から分離したアルギン酸分解菌は非運動性のVibrio属類似細菌が主体をなしていた。これらの菌群は16SrDNA塩基配列による分子系統解析およびアルギン酸分解性Vibrio属標準株とのDNA-DNA相同性を測定した結果、Vivrio属の新種であることが明らかとなった。また、ウニ消化管由来アルギン酸分解菌のアルギン酸分解特異性はアルギン酸を構成するpolyMおよびpolyGいずれのホモポリマーとも分解する菌株が30%以上を占めているのに対し、アワビ分離株ではpolyGブロックに対する分解性の強い菌株が30-70%占めていた。ウニ自体はアルギン酸分解酵素を分泌しないことから、消化管内細菌がアルギン酸分解の大部分を担っていると考えられた。一方、アワビはpolyM特異的な分解酵素を分泌することから、アワビ消化酵素で分解されにくい部分を分解する消化管内細菌が定住していることが示唆された。II.アルギン酸分解酵素の特性ウニ由来アルギン酸分解菌代表株Ud10株は基質特異性を示さないNaCl要求性の強い酵素を誘導的に産生していた。一方、アワビ由来アルギン酸分解菌A431株はpolyG特異的分解酵素以外にも、基質特異性の異なる6種類以上の酵素を産生しており、その中でpolyMに強い特異性を示す分解活性画分およびpolyGに強い特異性を示す分解活性画分の部分精製物について酵素化学的性状を調べた。それぞれの画分の至適反応温度は40℃および30℃と異なっていたが、至適反応温度(pH7.5)および塩類の要求性(100mM NaCl)は同様の性質を示した。
文部科学省, 奨励研究(A), 北海道大学, 研究代表者, 競争的資金, 08760172

海洋における溶存鉄濃度を支配する因子としての天然有機リガンドの存在とその起源
科学研究費補助金(基盤研究(C))
1996年 - 1996年
久万 健志, 志賀 直信, 澤辺 智雄
1、(1)紫外線を照射して有機物を除去した海水の含水酸化鉄の溶解度(20℃)は、天然海水に比べ著しく低く、その値は【less than or equal】0.1nMであった。(2)含水酸化鉄の溶解度の高い沿岸水(天然有機リガンドを含む)と紫外線を照射して得られた溶解度の低い沿岸水(有機リガンドを除去した)における沿岸性植物プランクトン(珪藻)の鉄添加増殖実験から、紫外線を照射した海水ではその増殖速度は遅くなる。このことは、天然海水に存在する有機リガンドが溶存鉄濃度を高め、より生物に使われやすい溶存形態になっていると思われる。2、(1)北海道噴火湾でのスプリングブルーム前の鉛直混合期(2月上旬)には、海水における含水酸化鉄の溶解度は低く、ほぼ一定値(0.2-0.3nM)を示した。しかしながら、ブルーム最盛期(3月中旬)には20m以浅で最大値(0.7-0.8nM)に達し、ブルーム終了後においても高い溶解度が維持された。これは、植物プランクトン、バクテリア等から放出された有機リガンドによるものと推察され、藻類による鉄の取り込みに重要な影響を与えていると考えられる。しかしながら、溶解度とクロロフィル-a濃度とは必ずしも相関性がなく、すべての植物プランクトンが有機リガンドを放出しているとは考えられない。(2)ブルーム前後における珪藻類優占種の変動は、ブルーム前から最盛期(2月〜3月中旬)にかけてThalassiosira nordenskioldiiが最も優占し、最盛期以降(3月中旬以降)にChaetoceros compressumやChaetoceros concavicorneに遷移した。このことは、ある種の植物プランクトンが有機リガンドを放出している可能性を示している。またある種の海産性バクテリアにもシドロフォアのような有機リガンドを放出することが確認された。以上の結果から、沿岸海水には3価鉄と溶存有機鉄錯体を形成する有機リガンドが存在しており、沿岸域での生物生産に重要な役割を果たしていると
文部科学省, 基盤研究(C), 北海道大学, 連携研究者, 競争的資金, 08640617

魚類ウィルス病の遺伝子診断法の開発とその応用に関する研究
科学研究費補助金(一般研究(B), 基盤研究(B))
1994年 - 1996年
吉水 守, 西澤 豊彦, 沢辺 智雄, 田島 研一, 絵面 良男
各種有用魚介類の種苗生産技術が確立され、生産が軌道に乗る過程においてウイルス性の疾病が発生し大きな障害の一つとなっている。このウイルス病対策の確立が増養殖事業における緊急かつ重要な課題である。本研究では海産魚の種苗生産で問題となっているウイルス性神経壊死症(VNN)およびサケ科魚類の伝染性造血器壊死症(IHN)を対象にPCRによる遺伝子診断法の確立を試みた。得られた成果は以下のとおりである。1.シマアジのウイルス性神経壊死症原因ウイルス(SJNNV)の外被タンパク質をコードしているRNA2遺伝子の塩基配列をもとに、本ウイルス検出用RT-PCRを検討し、海産魚のウイルス性神経壊死症の遺伝子診断法を確立した。2.伝染性造血器壊死症ウイルス(IHNV)のNタンパク質をコードしているRNA遺伝子の塩基配列をもとに、本ウイルス検出用RT-PCRを検討し、本法による診断法を確立し、分離株の迅速同定への応用を可能にした。3.伝染性造血器壊死症ウイルス(IHNV)の分離に最適な細胞の選抜と、ウイルス性神経壊死症原因ウイルスに感受性を示す細胞の検索を行い、RTE-2細胞およびSBK-2細胞を選抜し得た。4.本PCRを用いた採卵親魚の生殖産物の検査結果による親魚選別法が有効であることを明らかにし、魚病被害の軽減に貢献した。5.不顕性感染魚あるいはキャリヤ-の検出法として、遺伝子組換え大腸菌が産生する外被タンパク質を抗原としたELISA法を確立し、PCRによる遺伝子診断法と併用することによりウイルス病被害の軽減を計ることができた。6.ウイルス性神経壊死症原因ウイルスのRNA2遺伝子の可変領域の塩基配列の比較から、本病原因ウイルスの進化速度を求め、宿主である種苗の移動との関連性を考察した。7.IHNVに対するプライマーとIPNVに対するプライマーを同時に用い、最適条件下でRT-PCRを行うことにより、同時に両ウイルスの検出が可能になった。
文部科学省, 一般研究(B), 基盤研究(B), 北海道大学, 競争的資金, 06454095

海産藻類に感染するウイルスの探索とその生態に関する研究
科学研究費補助金(奨励研究(A))
1995年 - 1995年
澤辺 智雄
本年度に得られた成果は以下の通りである。1.海産微細藻類感染ウイルスの探索と特性北海道噴火湾沿岸海水から海産微細藻類の増殖に影響を与えるウイルスの探索を試み、濃縮した海水の0.45μmろ過性画分に渦鞭毛藻、Alexandrium catenella、Gymnodinium mikimotoiおよびProrocentrum sp.の3藻類の増殖を抑制する現象を見出した。A.catenellaおよびG.mikimotoiに対し増殖抑制効果を示すろ過性因子は、今年度(1995年)と昨年度の9月〜10月に採水した海水に認められ、その抑制効果は40〜90%の範囲であった。また、Prorocentrum sp.に対し増殖抑制効果を示すろ過性因子は1993年9月〜10月に採水した海水に見い出され、その抑制効果は20〜30%であったが、今年度は観察されなかった。いずれの藻類増殖抑制因子とも孔径0.45μmおよび孔径0.22μmのフィルターを通過させた後でも抑制効果は持続した。同様の増殖抑制因子は調査を行なった3年間とも9月〜10月にのみ見出され、季節変動が観察された。Prorocentrum sp.、A.catenellaおよびG.mikimotoiの各増殖抑制因子は0.22μm以上の孔径のフィルターを増殖抑制効果の低減なしに通過したが、孔径0.05μmフィルターでのろ過では増殖抑制効果が消失した。さらに、1993年の海水試料から検出されたProrocentrum sp.、A.catenellaの増殖抑制因子は50℃、30分の加熱およびRNase処理により増殖抑制効果が消失した。また、超遠心沈降画分のネガティブ染色像の観察により長さ約400nm、幅約30nmの屈曲棒状のウイルス様構造体が観察された。これらの特性から、本藻類増殖抑制因子が植物ウイルスに多く見られる屈曲棒状のRNAウイルスである可能性が示唆された。II.海産大型藻類感染ウイルス培養系の構築マコンブプロトプラストの連続培養系を考案し、プロトプラストの再生率を向上させることができた。この培養系はマコンブ感染ウイルスの分離に応用が期待される。
文部科学省, 奨励研究(A), 北海道大学, 研究代表者, 競争的資金, 07856018

海洋細菌由来のアルギン酸分解酵素を利用した褐藻細胞のプロトプラスト化に関する研究
科学研究費補助金(奨励研究(A))
1993年 - 1993年
澤辺 智雄
海洋細菌Alteromonas sp.H-4株の培養上清から精製したアルギン酸分解酵素を5mM HEPES-25mM MgCl_2-50%天然海水-0.7Mマンニトールに対し透析し、アルギン酸分解活性を30U/mlに調整し、1.5%Cellulase Onozuka R-10(Yakult本社)を添加したものを細胞壁分解酵素液とし、マコンブ芽胞体からプロトプラストの単離を試みた。15℃で反応を行った結果、約1時間後からプロトプラストの遊離が認められ、反応3時間後には藻体1g当たり4.5×10^6cells、5時間後には1.3×10^7cellsのプロトプラストが得られた。得られたプロトプラストは球形、黄金色で10〜25mumの大きさであった。また、ニュートラルレッド染色法によりほとんどの細胞が生存していることが確認された。さらにプロトプラストの収量と生存率を高める反応条件を検討した結果、プロトプラストの単離にはアルギン酸分解酵素の他にセルラーゼが必須であり、高張液の組成は海水濃度が50%、MgCl_2濃度は50〜100mM、緩衝液成分は5mMHEPES、マンニトールの濃度は0.5Mとした場合が至適であった。本条件下(MgCl_2濃度は25mM、反応3時間)で得られたプロトプラストの収量は5.2×10^6cells/g、生存率は98%であった。対照として市販のアワビアセトンパウダー(Sigma社)をアルギン酸分解酵素源とし、同一条件下で反応を行った場合ではプロトプラストの単離が認められず、Alteromonas sp.の産生するアルギン酸分解酵素の有効性が明らかとなった。また、マコンブプロトプラストの培養を試み、培養液への抗生物質の添加はプロトプラストの再生に悪影響を及ぼすことが明らかとなった。上記の至適培養条件下で単離したプロトプラストをProvasoliのESI培地で5℃、35001x、12h:12h明暗周期の条件下で培養を行ったところ、培養2〜3日目に細胞壁が再生し、6日目から分裂細胞が観察され始め、その後さらに繰り返し分裂した細胞も認められた。本結果からマコンブプロトプラスト培養系の確立が進展すると期待される。
文部科学省, 奨励研究(A), 北海道大学, 研究代表者, 競争的資金, 05760144

水産無脊椎動物の腸内細菌に関する研究　　　　　　　　　　　　　　　
競争的資金

海洋細菌の産生するアルギン酸分解酵素に関する研究　　　　　　　　　　　　　　　
競争的資金

Studies on intestinal microflora of marine invertebrates　　　　　　　　　　　　　　　
競争的資金

Studies on alginate degrading enzymes produced by marin bacteria.　　　　　　　　　　　　　　　
競争的資金