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A novel marine bacterial strain, designated JAMH 043T, was isolated from cold-seep sediment in Sagami Bay, Japan. Cells were Gram-stain-negative, rod-shaped, non-motile and aerobic chemo-organotrophs. The isolate grew optimally at 25 °C, at pH 7.0–7.5 and with 3 % (w/v) NaCl. The major respiratory quinone was ubiquinone-10 (Q-10). The predominant fatty acid was C18 : 1ω7c. On the basis of 16S rRNA gene sequence analysis, the isolated strain was closely affiliated to members of the genus Thalassobius in the class Alphaproteobacteria, and 16S rRNA gene sequence similarity of the novel isolate with the type strain of its closest related species, Thalassobius aestuarii JC2049T, was 98.4 %. The DNA G+C content of the novel strain was 58.0 mol%. The hybridization values for DNA–DNA relatedness between strain JAMH 043T and reference strains belonging to the genus Thalassobius were less than 14.1 ± 2.2 %. Based on differences in taxonomic characteristics, the isolated strain represents a novel species of the genus Thalassobius, for which the name Thalassobius abyssi sp. nov. is proposed. The type strain is JAMH 043T ( = JCM 30900T = DSM 100673T).

A novel marine bacterial strain designated JAMH 011Twas isolated from the cold-seep sediment in Sagami Bay, Japan. Cells were Gram-stain-negative, rod-shaped, non-spore-forming, aerobic chemo-organotrophs and motile by means of a single polar flagellum. Growth occurred at temperatures below 31 °C, with the optimum at 25 °C. The major respiratory quinone was Q-10. The predominant fatty acid was C18 : 1ω7c. On the basis of 16S rRNA gene sequence analysis, the isolated strain was closely affiliated with members of the genusShimiain the classAlphaproteobacteria, and the 16S rRNA gene sequence similarity of the novel isolate with the type strain of the closest related species,Shimia haliotisWM35T, was 98.1 %. The DNA G+C content of the novel strain was 57.3 mol%. The hybridization values for DNA–DNA relatedness between strain JAMH 011Tand reference strains belonging to the genusShimiawere less than 9.4 ± 0.7 %. Based on differences in taxonomic characteristics, the isolated strain represents a novel species of the genusShimia,for which the nameShimia sagamensissp. nov. is proposed. The type strain is JAMH 011T( = JCM 30583T = DSM 29734T)

A novel Gram-positive-staining, strictly aerobic and heterotrophic bacterium, designated strain LL-002T, was isolated from organics- and methane-rich seafloor sediment at a depth of 100 m in Kagoshima Bay, Kagoshima, Japan. Colonies were lustreless and translucent white in colour. The temperature, pH and salt concentration ranges for growth were 10–30 °C, pH 6.0–6.5 and 0–1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain LL-002T belongs to the genus Aneurinibacillus of the family Paenibacillaceae. 16S rRNA gene sequence similarities between strain LL-002T and the type strains of species of the genus Aneurinibacillus were 92.8–95.7 %; the highest sequence identity was with the type strain of Aneurinibacillus migulanus. The DNA G+C content of strain LL-002T was 46.2 mol%. MK-7 was the predominant menaquinone. The predominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0, and the cell-wall peptidoglycan contained meso-diaminopimelic acid and glutamic acid, glycine and alanine in addition to muramic acid and glucosamine. The peptidoglycan type was A1γ. In DNA–DNA hybridization assays between strain LL-002T and the type strains of the other species of the genus Aneurinibacillus, the level of hybridization was 6.3–30.1 %. On the basis of its biological features and the 16S rRNA gene sequence comparison presented here, strain LL-002T is considered to represent a novel species of the genus Aneurinibacillus, for which the name Aneurinibacillus tyrosinisolvens sp. nov. is proposed; the type strain is LL-002T ( = NBRC 110097T = CECT 8536T).

A moderately halophilic, slightly acidophilic, aerobic bacterium, designated strain YTM-1T, was isolated from the body surface of Malacocottus gibber. Cells were Gram-stain-negative, short rods or cocci, approximately 0.9–1.1 µm long and 1.0–1.8 µm wide. Strain YTM-1T was able to grow with 1–30 % NaCl (optimum, 7.5–10 %, w/v), at 4–30 °C (optimum, 20–25 °C) and at pH 3.8–9.5 (optimum, pH 5.0–5.5). Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain YTM-1T belonged to the genus Salinisphaera with low similarity values to the type strains of recognized species of this genus (T consisted of diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, three unknown phospholipids and one unknown lipid. The predominant isoprenoid quinone was Q-8. The major fatty acids were C19 : 0ω8c cyclo, C18 : 1ω7c, C16 : 1ω5c and C16 : 0. The DNA G+C content of strain YTM-1T was 67.3 mol%. These phylogenetic, physiological and chemotaxonomic data indicated that strain YTM-1T represents a novel species of the genus Salinisphaera , for which the name Salinisphaera japonica sp. nov. is proposed. The type strain is YTM-1T ( = JCM 18087T = CECT 8012T). An emended description of the genus Salinisphaera is also proposed.

To investigate novel extremozymes encoded by sequenced metagenes from a microbial community in an extreme environment, we have characterized a recombinant glycosyl hydrolase (rGH) from an uncultured bacterium within the order Chloroflexi. rGH formed insoluble bodies in an Escherichia coli protein expression system. The protein was partially dissolved by a surfactant and was enzymatically characterized. The MW of the monomeric peptide was ~62 kDa, and it formed a homodimers in buffer. It was optimally active at 65 °C and from pH 4 to 8. rGH showed hydrolytic activity for α-1,1, α-1,2 and α-1,6 linkages, including isomaltose, but not α-1,4 and β-linkages.

A novel, facultatively anaerobic bacterium (strain JAM-BA0501T) was isolated from a deep subseafloor sediment sample at a depth of 247 m below seafloor off the Shimokita Peninsula of Japan in the north-western Pacific Ocean (Site C9001, 1180 m water depth). Cells of strain JAM-BA0501T were Gram-negative, filamentous, non-spore-forming and motile on solid medium by gliding. Phylogenetic analysis based on the 16S rRNA gene sequence of strain JAM-BA0501T indicated a distant relationship to strains representing genera within the order Bacteroidales , such as Alkaliflexus imshenetskii Z-7010T (91.1 % similarity), Marinilabilia salmonicolor ATCC 19041T (86.2 %) and Anaerophaga thermohalophila Fru22T (89.3 %). The new isolate produced isoprenoid quinones with menaquinone MK-7 as the major component, and the predominant fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The DNA G+C content of the isolate was 42.9 mol%. Based on its taxonomic distinctiveness, strain JAM-BA0501T is considered to represent a novel species of a new genus within the family Marinilabiliaceae , for which the name Geofilum rubicundum gen. nov., sp. nov. is proposed. The type strain of Geofilum rubicundum is JAM-BA0501T ( = JCM 15548T = NCIMB 14482T).

"A meta-enzyme approach" is proposed as an ecological enzymatic method to explore the potential functions of microbial communities in extreme environments such as the deep marine subsurface. We evaluated a variety of extra-cellular enzyme activities of sediment slurries and isolates from a deep subseafloor sediment core. Using the new deep-sea drilling vessel "Chikyu", we obtained 365 m of core sediments that contained approximately 2% organic matter and considerable amounts of methane from offshore the Shimokita Peninsula in Japan at a water depth of 1,180 m. In the extra-sediment fraction of the slurry samples, phosphatase, esterase, and catalase activities were detected consistently throughout the core sediments down to the deepest slurry sample from 342.5 m below seafloor (mbsf). Detectable enzyme activities predicted the existence of a sizable population of viable aerobic microorganisms even in deep subseafloor habitats. The subsequent quantitative cultivation using solid media represented remarkably high numbers of aerobic, heterotrophic microbial populations (e.g., maximally 4.4x10(7) cells cm(-3) at 342.5 mbsf). Analysis of 16S rRNA gene sequences revealed that the predominant cultivated microbial components were affiliated with the genera Bacillus, Shewanella, Pseudoalteromonas, Halomonas, Pseudomonas, Paracoccus, Rhodococcus, Microbacterium, and Flexibacteracea. Many of the predominant and scarce isolates produced a variety of extra-cellular enzymes such as proteases, amylases, lipases, chitinases, phosphatases, and deoxyribonucleases. Our results indicate that microbes in the deep subseafloor environment off Shimokita are metabolically active and that the cultivable populations may have a great potential in biotechnology.

A novel Gram-stain-negative, aerobic, heterotrophic, stalked and capsulated bacterium with potential denitrification ability, designated strain TAR-002T, was isolated from deep seafloor sediment in Japan. Colonies lacked lustre, and were viscous and translucent white. The ranges of temperature, pH and salt concentration for growth were 8–30 °C, pH 6.0–10.0 and 1–3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain TAR-002T belongs to the genus Brevundimonas of the class Alphaproteobacteria . Levels of similarity between the 16S rRNA gene sequence of strain TAR-002T and those of the type strains of species of the genus Brevundimonas were 93.5–98.9 %; the most closely related species was Brevundimonas basaltis . In DNA–DNA hybridization assays between strain TAR-002T and its phylogenetic neighbours, Brevundimonas lenta DS-18T, B. basaltis J22T, Brevundimonas subvibrioides ATCC 15264T and Brevundimonas alba DSM 4736T, mean hybridization levels were 6.4–27.7 %. The G+C content of strain TAR-002T was 70.3 mol%. Q-10 was the major respiratory isoprenoid quinone. The major fatty acids were C18 : 1ω7c and C16 : 0, and the presence of 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d-glucopyranuronosyl]glycerol (DGL) indicates the affiliation of strain TAR-002T with the genus Brevundimonas . On the basis of biological characteristics and 16S rRNA gene sequence comparisons, strain TAR-002T is considered to represent a novel species of the genus Brevundimonas , for which the name Brevundimonas denitrificans sp. nov. is proposed; the type strain is TAR-002T ( = NBRC 110107T = CECT 8537T).

Two marine bacteria, designated strains MBE#61T and MBE#74T, were isolated from a piece of sunken bamboo in the marine environment in Japan. Both of these strains were Gram-stain-negative, but had different cell shapes: MBE#61T was spiral, whereas MBE#74T was rod-shaped. The temperature, pH and salt concentration ranges for growth of strain MBE#61T were 4–38 °C (optimal at 32 °C), pH 4.5–11.0 (optimal at pH 7.0–8.0) and 1–11 % (optimal at 2 %) NaCl, whereas those of strain MBE#74T were 4–36 °C (optimal at 30 °C), pH 4.0–10.5 (optimal at pH 7.0–8.0) and 1–12 % (optimal at 4 %) NaCl. Phylogenetic analysis based on partial 16S rRNA gene sequences revealed that both strains belong to the genus Thalassospira within the class Alphaproteobacteria . Similarity between the 16S rRNA gene sequence of strain MBE#61T and those of the type strains of species of the genus Thalassospira was 97.5–99.0 %, and that of strain MBE#74T was 96.9–98.6 %; these two isolates were most closely related to Thalassospira lucentensis QMT2T. However, the DNA–DNA hybridization values between T. lucentensis QMT2T and strain MBE#61T or MBE#74T were only 16.0 % and 7.1 %, respectively. The DNA G+C content of strain MBE#61T was 54.4 mol%, and that of strain MBE#74T was 55.9 mol%. The predominant isoprenoid quinone of the two strains was Q-10 (MBE#61T, 97.3 %; MBE#74T, 93.5 %). The major cellular fatty acids of strain MBE#61T were C18 : 1ω7c (31.1 %), summed feature 3 comprising C16 : 0ω7c/iso-C15 : 0 2-OH (26.1 %) and C16 : 0 (20.9 %); those of strain MBE#74T were C16 : 0 (26.2 %), C17 : 0 cyclo (19.9 %) and C18 : 1ω7c (12.1 %). On the basis of these results, strain MBE#61T and strain MBE#74T are considered to represent novel species of the genus Thalassospira , for which names Thalassospira alkalitolerans sp. nov. and Thalassospira mesophila sp. nov. are proposed. The type strains are MBE#61T ( = JCM 18968T = CECT 8273T) and MBE#74T ( = JCM 18969T = CECT 8274T), respectively. An emended description of the genus Thalassospira is also proposed.

A Gram-stain-positive, non-motile, non-spore-forming, rod-shaped bacterium, designated strain K-1T, was isolated from soil at a sucrose refinery in Japan. The strain grew at 9–37 °C (optimum, 30 °C) and at pH 6–11 (optimum, pH 7.0). Phylogenetic analysis based on the full-length 16S rRNA gene sequence of strain K-1T revealed that it was a member of the genus Microbacterium . High 16S rRNA gene sequence similarities were found between strains K-1T and both Microbacterium pumilum NBRC 101279T (99.7 %) and Microbacterium deminutum NRRL B-24453T (99.5 %). However, the DNA–DNA hybridization values between strain K-1T and M. pumilum NBRC 101279T and M. deminutum NRRL B-24453T were only 12 % and 10 %, respectively. The DNA G+C content of strain K-1T was 73 mol%. The major fatty acids of strain K-1T were anteiso-C15 : 0 and anteiso-C17 : 0, and the major menaquinones were MK-12 and MK-13. The diamino acid in the cell-wall peptidoglycan was lysine. On the basis of these results, strain K-1T is considered to represent a novel species of the genus Microbacterium , for which the name Microbacterium saccharophilum sp. nov. is proposed. The type strain is K-1T ( = NBRC 108778T = NCIMB 14782T).

A novel Gram-negative, aerobic, psychrotolerant, alkali-tolerant, heterotrophic and dimorphic prosthecate bacterium, designated strain TAR-001T, was isolated from deep-sea floor sediment in Japan. Cells of this strain had a dimorphic life cycle and developed an adhesive stalk at a site not coincident with the centre of the cell pole, and the other type of cell, a swarm cell, had a polar flagellum. Colonies were glossy, viscous and yellowish-white in colour. The temperature, pH and salt concentration range for growth were 2–41 °C, pH 6.5–10.0 and 1–4 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain TAR-001T belongs to the family Caulobacteraceae of the class Alphaproteobacteria , and lies between the genus Brevundimonas and the genus Caulobacter . Levels of similarity between the 16S rRNA gene sequence of strain TAR-001T and those of the type strains of Brevundimonas species were 93.3–95.7 %; highest sequence similarity was with the type strain of Brevundimonas diminuta . Levels of sequence similarity between those of the type strains of Caulobacter species were 94.9–96.0 %; highest sequence similarity was with the type strain of Caulobacter mirabilis . The G+C content of strain TAR-001T was 67.6 mol%. Q-10 was the major respiratory isoprenoid quinone. The major fatty acids were C18 : 1ω7c and C16 : 0, and the presence of 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d-glucopyranuronosyl]glycerol suggests strain TAR-001T is more closely to the genus Brevundimonas than to the genus Caulobacter . The mean DNA–DNA hybridization levels between strain TAR-001T and the type strains of two species of the genus Brevundimonas were higher than that of the genus Caulobacter . On the basis of polyphasic biological features and the 16S rRNA gene sequence comparison presented here, strain TAR-001T is considered to represent a novel species of the genus Brevundimonas , for which the name Brevundimonas abyssalis sp. nov. is proposed; the type strain is TAR-001T ( = JCM 18150T = CECT 8073T).

A novel filamentous bacterium, designated strain JIR-001T, was isolated from hemipelagic sediment in deep seawater. This strain was non-motile, Gram-positive, aerobic, heterotrophic and thermophilic; colonies were of infinite form and ivory coloured with wrinkles between the centre and the edge of the colony on ISP2 medium. The isolate grew aerobically at 55–73 °C with the formation of aerial mycelia; spores were produced singly along the aerial mycelium. These morphological features show some similarities to those of the type strains of some species belonging to the family Thermoactinomycetaceae . Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain JIR-001T belongs to the family Thermoactinomycetaceae within the class Bacilli . Similarity levels between the 16S rRNA gene sequence of strain JIR-001T and those of the type strains of Thermoactinomycetaceae species were 85.5–93.5 %; highest sequence similarity was with Melghirimyces algeriensis NariEXT. In the DNA–DNA hybridization assays between strain JIR-001T and its phylogenetic neighbours the mean hybridization levels with Melghirimyces algeriensis NariEXT, Planifilum fimeticola H0165T, Planifilum fulgidum 500275T and Planifilum yunnanense LA5T were 5.3–7.5, 2.3–4.7, 2.1–4.8 and 2.5–4.9 %, respectively. The DNA G+C content of strain JIR-001T was 55.1 mol%. The major fatty acids were iso-C15 : 0, iso-C17 : 0, iso-C16 : 0 and C16 : 0. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, glucolipid, phosphatidylserine, an amino-group containing phospholipid, an unknown phospholipid and two unknown lipids. The predominant menaquinone was MK-7 and the cell-wall peptidoglycan contained meso-diaminopimelic acid, glutamic acid and alanine. On the basis of phenotypic characteristics and 16S rRNA gene sequence comparisons, strain JIR-001T is considered to represent a novel species in a new genus of the family Thermoactinomycetaceae , for which the name Polycladomyces abyssicola gen. nov., sp. nov. is proposed. The type strain of Polycladomyces abyssicola is JIR-001T ( = JCM 18147T = CECT 8074T).

A Gram-stain-negative, aerobic, heterotrophic and salt-tolerant bacterium, designated strain LL-001T, was isolated from a deep subseafloor sediment in Japanese waters. Cells were non-motile rods and colonies were smooth, convex, circular and vermilion. The conditions for growth were 15–35 °C, pH 5.5–7.5 and 1–8 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain LL-001T belonged to the genus Loktanella within the family Rhodobacteraceae of the class Alphaproteobacteria . 16S rRNA gene sequence similarity between strain LL-001T and members of the genus Loktanella was 94.5–98.5 %; the highest sequence similarity was with Loktanella hongkongensis UST950701-009PT. DNA–DNA relatedness between strain LL-001T and L. hongkongensis UST950701-009PT was 41.5–43.6 %. The DNA G+C content of strain LL-001T was 69.3 mol%. On the basis of biochemical features and 16S rRNA gene sequence comparison, strain LL-001T is considered to represent a novel species of the genus Loktanella , for which the name Loktanella cinnabarina sp. nov. is proposed. The type strain is LL-001T ( = JCM 18161T = CECT 8072T). The description of the genus Loktanella is also emended.

The aim of this cross-sectional, nationwide study in Japan was to develop a support skill scale for insulin therapy (IT-SSS) and to evaluate its validity and reliability. The sample consisted of 1604 nurses at 123 hospitals throughout the country. The factor validity, known-group validity, convergent validity, discriminant validity and internal consistency of IT-SSS were assessed. IT-SSS consisted of 26 minimum and 25 standard support skills. They included 4 subscales for minimum skills: management strategy for hypoglycemia, education about insulin injection technique, individual assessment and support about insulin rejection, and collaboration with medical professionals and patient/family. Three subscales for standard skills: apprehensions concerning the will and emotion of the patient, management for blood glucose control, and coordination in insulin management. Cronbach's alpha coefficient was between 0.75 and 0.90, suggesting strong internal consistency. Multitrait analysis showed that convergent validity was complete, and discriminant validity was found to be almost complete in both minimum and standard skill scales (scaling success rates of 97.6% and 98.7% across all subgroups, respectively). Known group analysis clearly showed that specialist nurses have significantly higher skills than general nurses. These findings indicate that IT-SSS has a reasonable factor validity, convergent validity, discriminant validity, known group validity, and internal consistency.

To explore a novel glycolipid, we performed biochemical reactions using a recombinant α-glucosidase from Geobacillus sp. which shows excellent transglycosylation reaction to hydroxyl groups in a variety of compounds. Two different glycolipids (GL-1 and GL-2) were prepared from ricinoleic acid using a recombinant α-glucosidase from Geobacillus sp. The molecular structure of GL-1 was confirmed as 12-O-α-D-glucopyranosyl-9-hexadecenoic acid by 1D and 2D NMR analyses. According to MALDI-TOF/MS, GL-1 and GL-2 showed single major peaks at m/z 483.82 and 645.97, respectively. The peaks corresponded to the [M + Na](+) ions of the glycolipids. GL-2 was estimated as 12-O-α-D-glucopyranosyl-(4'-O-α-glucopyranosyl)-9-hexadecenoic acid. Light polarization microscopy revealed that GL-2 easily formed self-assembled vesicles in aqueous solution.